Triazole-Modified Nucleic Acids for the Application in Bioorganic and Medicinal Chemistry

Home , Automated synthesis, Bioorganic chemistry, Chemical synthesis, Convergent synthesis, Phosphoramidate, Ribonucleoside

biomedicines

Review Triazole-Modiﬁed Nucleic Acids for the Application in Bioorganic and Medicinal Chemistry

Dagmara Baraniak * and Jerzy Boryski

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-61-852-8503

Abstract: This review covers studies which exploit triazole-modified nucleic acids in the range of chemistry and biology to medicine. The 1,2,3-triazole unit, which is obtained via click chemistry approach, shows valuable and unique properties. For example, it does not occur in nature, constitutes an additional pharmacophore with attractive properties being resistant to hydrolysis and other reactions at physiological pH, exhibits biological activity (i.e., antibacterial, antitumor, and antiviral), and can be considered as a rigid mimetic of amide linkage. Herein, it is presented a whole area of useful artificial compounds, from the clickable monomers and dimers to modified oligonucleotides, in the field of nucleic acids sciences. Such modifications of internucleotide linkages are designed to increase the hybridization binding affinity toward native DNA or RNA, to enhance resistance to nucleases, and to improve ability to penetrate cell membranes. The insertion of an artificial backbone is used for understanding effects of chemically modified oligonucleotides, and their potential usefulness in therapeutic applications. We describe the state-of-the-art knowledge on their implications for synthetic genes and other large modified DNA and RNA constructs including non-coding RNAs.

Citation: Baraniak, D.; Boryski, J. Keywords: click chemistry; 1,2,3-triazoles; backbone modifications; triazole-linkage; clickable nucle- Triazole-Modified Nucleic Acids for osides and nucleotides; triazole-modified oligonucleotides; (TL)DNA; (TL)RNA; (TL)LNA; (TL)BNA; the Application in Bioorganic and (TL)PNA; (TL)quadruplexes; non-coding RNA; synthetic genes Medicinal Chemistry. Biomedicines 2021, 9, 628. https://doi.org/ 10.3390/biomedicines9060628

Academic Editor: Shaker A. Mousa 1. Introduction 1.1. A Brief Introduction to DNA and RNA World Received: 29 April 2021 The natural nucleic acids, DNA and RNA, are exquisitely suited to store and propagate Accepted: 26 May 2021 genetic information [1,2]. Nucleic acids are unique among biopolymers, because of their Published: 31 May 2021 high-water solubility together with denaturing and refolding abilities [3]. DNA is an enormously intricate yet simple molecule. Its beauty is hidden in a stable double-stranded Publisher’s Note: MDPI stays neutral helix, that is guarded in the cell nucleus for storing genetic material and because of with regard to jurisdictional claims in its self-replication abilities. In turn, remarkable RNA most often is a single-stranded published maps and institutional affil- molecule, but it could adopt different structures and functions in the cell. It can mediate iations. the heritable storage, recognize specific targets (aptamers), and even catalyze reactions [4]. RNA molecules are generally less stable and are quickly degraded by intracellular enzymes, hence the great need for chemical modifications. Thus, understanding and cognition of their capabilities such as direct protein synthesis and modulation of gene expression allow Copyright: © 2021 by the authors. us to understand how life works [5]. Likewise, it is worth to notice that in all DNA and Licensee MDPI, Basel, Switzerland. RNA technologies the Watson–Crick base pairs are responsible for the information content This article is an open access article of all living systems [6–9]. Nowadays, RNA has become a focus of attention because it distributed under the terms and turned out that various RNAs are valuable tools in molecular biology and medicine. These conditions of the Creative Commons molecules, including all classes of ncRNAs, are used in the design and development of Attribution (CC BY) license (https:// drugs for fighting cancers, viruses, and other diseases at the nucleic acids level. creativecommons.org/licenses/by/ 4.0/).

Biomedicines 2021, 9, 628. https://doi.org/10.3390/biomedicines9060628 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 628 2 of 34

Biomedicines 2021, 9, 628 2 of 35 development of drugs for fighting cancers, viruses, and other diseases at the nucleic acids level.

1.2.1.2. Non-CodingNon-Coding RNARNA InIn the the world world of of science, science, the the key key role role for for regulating regulating life life processes processes is ascribed is ascribed to proteins. to pro- 0 Sinceteins. the Since genetic the genetic information information about them about isencoded them is inencoded 2 -deoxyribonucleic in 2′-deoxyribonucleic acid (DNA), acid it found(DNA), itself it found on the itself podium on the of podium fame. An of unappreciatedfame. An unappreciated ribonucleic ribonucleic acid (RNA) acid stood (RNA) in itsstood shade—a in its shade—a silent hero silent with hero many with faces. many From faces. the From central the dogma central of dogma molecular of molecular biology, itbiology, can be seenit can that be seen the ﬂow that ofthe genetic flow of information, genetic information, which takes which place takes from place DNA from through DNA RNAthrough to protein, RNA to involves protein, ainvolves molecule a ofmolecu RNA,le and of RNA, exactly and the exactly messenger the messenger RNA (mRNA). RNA It(mRNA). follows thatIt follows the mRNA that the molecule mRNA ismolecule coding RNAis coding in transcription, RNA in transcription, because it because rewrites it informationrewrites information from DNA from about DNA proteins. about proteins. A further A stagefurther is stage yet involved: is yet involved: ribosomal ribosomal RNA (rRNA)RNA (rRNA) and transfer and transfer RNA (tRNA). RNA (tRNA). The ﬁrst The one first acts one as aacts scaffold, as a scaffold, and the secondand the one second is a courierone is a in courier the delivery in the of delivery amino acidsof amino that areacids linked that intoare linked long chains into long to form chains a protein. to form So, a inprotein. translation, So, in thesetranslation, two RNAs these belong two RNAs to the belong group to of the ncRNAs group as of they ncRNAs do not as encodethey do any not proteinencode sequenceany protein [10 ,sequence11]. We know [10,11]. that We the know proteins-coding that the proteins-coding genes constitute genes only constitute 2–3% of theonly entire 2–3% human of the entire genome human and thegenome remaining and the 97–98% remaining (wrongly 97–98% called (wrongly “junk” called DNA) “junk” [12], theyDNA) are [12], sequences they are responsible sequences responsible for the formation for the offormation a numerous of a numerous and diverse and group diverse of ncRNA molecules (Figure1)[13,14]. group of ncRNA molecules (Figure 1) [13,14].

RNA Transcript

Coding RNA Non-coding RNA mRNA ncRNA

long ncRNA small ncRNA 200 nt 200 nt

linear: rRNA, asRNA, tRNA, snRNA, snoRNA, ceRNA, eRNA, pRNA, sdRNA, rasiRNA, scaRNA gsRNA, lincRNA, lncRNA, NAT miRNA, siRNA, piRNA, circular: circRNA tiRNA, stRNA, endo-siRNA

FigureFigure 1.1.Diagram Diagram presentingpresenting thethe RNARNA world. world.

MoreMore thanthan 40%40% ofof thethe humanhuman genomegenome isis transcribedtranscribed asas ncRNA,ncRNA, whatwhat causescauses thatthat chemicalchemical diversity diversity in in RNAs RNAs is is huge.huge. TheThe above-mentionedabove-mentioned rRNA rRNA and and tRNA tRNA are are molecules molecules thatthat occur occur in in all all cells, cells, because because they they control control the the basic basic processes, processes, therefore therefore minor minor disturbances disturb- inances the functionin the function can cause can seriouscause serious diseases. diseas Thees. fact The is fact that is the that RNA the worldRNA world is rich—it is rich— can beit can more beindependent, more independent, which which is evidenced is evidenced in the in discovery the discovery of ribozymes, of ribozymes, and thatand hasthat becomehas become a milestone a milestone in molecular in molecular biology biology [15 [15].]. It hasIt has been been proved proved that that RNA RNA molecules molecules areare capablecapable ofof carryingcarrying outout thethe reaction—editing reaction—editingmRNA, mRNA,modifying modifyingimmature immaturetRNA, tRNA,or or participatingparticipating inin thethe synthesissynthesis ofof ribosomes.ribosomes. ButBut theythey cancan alsoalso actact asas diagnosticdiagnostic tools,tools, e.g.e.g.,, smallsmall nuclear nuclear RNA RNA (snRNA) (snRNA) or or small small nucleolar nucleolar RNA RNA (snoRNA) (snoRNA) [16 ,[16,17].17]. In turn,In turn, regulatory regula- microRNAstory microRNAs (miRNAs) (miRNAs) control control gene expression, gene expression, and are and also are involved also involved in brain in development brain devel- andopment nerve and cell nerve specialization cell specialization [18]. Disorders [18]. Di ofsorders these molecules of these molecules lead to the lead development to the devel- of tumors,opment including of tumors, brain including gliomas brain [19,20 gliomas], gastric [19,20], cancer [gastric21], or breastcancer cancer[21], or [22 breast]. Therefore, cancer research[22]. Therefore, is ongoing research to develop is ongoing anti-cancer to deve strategieslop anti-cancer based onstrategies miRNAs based [23], one.g. miRNAs, preparation[23], e.g. of, synthetic preparation oligonucleotides of synthetic oligonucleotides complementary complementary to these miRNA to molecules, these miRNA of which mol- overexpressionecules, of which causes overexpression brain gliomas causes [24 ,brain25]. Summarizing gliomas [24,25]. and Summarizing “encoding a non-coding”and “encod- RNAs,ing a non-coding” they are thought RNAs, to they contribute are thought in structural to contribute functions—(a) in structural they buildfunctions—(a) a framework they for proteins with which they occur in a complex, e.g., rRNA; (b) form antisense threads (“guides”)—complementary to target RNAs, form Watson–Crick pairs with them, e.g., miRNA, siRNA, piRNA; (c) catalytic (“ribozymes”)—they catalyze biochemical reactions, Biomedicines 2021, 9, 628 3 of 34

Biomedicines 2021, 9, 628 build a framework for proteins with which they occur in a complex, e.g., rRNA; (b)3 ofform 35 antisense threads (“guides”)—complementary to target RNAs, form Watson–Crick pairs with them, e.g., miRNA, siRNA, piRNA; (c) catalytic (“ribozymes”)—they catalyze biochemical reactions, e.g., hammerhead, RNaseP; (d) regulatory functions—regulate gene e.g., hammerhead, RNaseP; (d) regulatory functions—regulate gene expression, e.g., ri- expression, e.g., riboswitches; (e) control epigenetic phenomena [26,27]. In the treatment boswitches; (e) control epigenetic phenomena [26,27]. In the treatment of human diseases, of human diseases, miRNA are regulators of endogenous gene expression, and thus bi- miRNA are regulators of endogenous gene expression, and thus biomarkers in cancer [28]. omarkers in cancer [28]. In turn, siRNAs are directed against oncogenes, but also in the In turn, siRNAs are directed against oncogenes, but also in the treatment of viral diseases (HIV,treatment HCV, of inﬂuenza viral diseases virus). (HIV, We see HCV, that theinflue hugenza functional virus). We diversity see that ofthe these huge molecules functional allowsdiversity us to of better these understand molecules geneallows expression, us to better create understand new therapeutic gene expression, strategies andcreate better new developtherapeutic diagnostic strategies methods and better for many develop diseases diagnostic [29,30]. methods for many diseases [29,30].

1.3.1.3. Click Click Chemistry Chemistry TheThe concept concept of of organic organic chemistry, chemistry, introduced introduced by by Sharpless Sharpless and and Meldal, Meldal, named namedclick click chemistrychemistry, lies, lies in in using using simple simple and and efficient efficient reactions reactions designed designed to to bind bind various various compounds compounds inin an an easy easy way way [[31,32].31,32]. SimpleSimple reaction conditions conditions and and readily readily available available reagents reagents lead lead to tothe the formation formation of ofstable stable products, products, which which very very often often exhibit exhibit some some uniq uniqueue biological biological prop- properties.erties. Among Among the theclick click reactions, reactions, the themost most important important role role is played is played by bythe the catalyzed catalyzed 1,3- 1,3-dipolardipolar Huisgen Huisgen cycloaddition cycloaddition involving involving organic organic azides azides and and terminal terminal alkynes, alkynes, and andlead- leadinging to products to products of 1,2,3-triazole of 1,2,3-triazole structures structures (Figure (Figure 2). Click2). Click chemistry, chemistry, especially especially the Huis- the Huisgengen reaction reaction catalyzed catalyzed by by copper(I) copper(I) or or ruth ruthenium(II)enium(II) ions ions (in (in short: CuAACCuAAC oror RuAAC, RuAAC, respectively),respectively), arouses arouses great great interest interest among among scientists scientists due due to to its its simplicity simplicity and and convenient convenient conditionsconditions [ 33[33–35].–35]. In In recent recent years, years, hundreds hundreds of of scientific scientific works works describing describing applications applications ofof click click chemistrychemistry havehave been published, including including extensive extensive research research papers. papers. For For us, us, the theworks works in the in thefield field of biological of biological chemistry chemistry are of are particular of particular importance, importance, pertaining pertaining to syn- tothesis synthesis of small of small molecules—clickable molecules—clickable nucleosides nucleosides and nucleotides and nucleotides [36–40], [36 dimers–40], dimers deriva- derivatives—canonicaltives—canonical [41,42] [41 and,42] andnon-canonical non-canonical [43] [linked43] linked dinucleosides, dinucleosides, and and modified modified oli- oligodeoxynucleotidesgodeoxynucleotides [44–46]. [44–46]. Among Among the the discusseddiscussed topics,topics, otherother importantimportant applications applications cancan be be listed, listed, such such as: as: receiving receiving fluorescent fluorescent markers markers [47 [47–54],–54], ligand ligand design design [55 ],[55], crosslinking crosslink- duplexesing duplexes [56–58 [56–58],], labeling labeling [59–63 [59–63],], conjugates conjug [64ates–66 [64–66],], surface surface modification modification [67], artificial[67], arti- molecularficial molecular machines machines [68], nanomaterials, [68], nanomaterials, and nanostructures and nanostructures [69–71 ].[69–71].

FigureFigure 2. 2. TheThe comparison of of the the natural natural internucleotide internucleotide lin linkagekage with with a 1,4- a 1,4- or 1,5-disubstituted or 1,5-disubstituted 1,2,3- 1,2,3-triazoletriazole linker. linker.

ExploitingExploiting the the CuAAC CuAAC and and RuAAC RuAAC tools tools also also opens opens access access to to complex complex architecture architecture inin biomedicinal biomedicinal science, science, forfor example:example: drug discovery [72–75], [72–75], triazole-linked triazole-linked analogues analogues of ofoligonucleotides oligonucleotides [76], [76 and], and therapeutic therapeutic nucleic nucleic acids acids (“clicking” (“clicking” genes) genes) [77–79] [77 with–79] differ- with differentent arrangement arrangement of the of the triazole triazole linker linker [80] [80, ],and and enables enables gene editingediting byby CRISP-Cas9 CRISP-Cas9 methodmethod [ 81[81–84].–84]. AnAn additional additional impact impact on on the the science science is is described described in in works works related related to to aptamers aptamers and and chimericchimeric biomolecules biomolecules [ 85[85–90],–90], peptides peptides and and proteins proteins [91 [91–100],–100], antibody–oligonucleotide antibody–oligonucleotide conjugates [101–106], vaccines [107], or building blocks of viruses [108]. There are many reviews discussing modiﬁed nucleic acids as therapeutic agents [109–111]. This presents how great is the need for oligonucleotides with improved properties including enhanced Biomedicines 2021, 9, 628 4 of 35

recognition and binding to RNA, duplex DNA, and proteins. This results in the development of new potent antisense and antigen agents [112–114]; however, only extremely accurate chemistry and biochemistry can make genes and even genomes. Every scientist knows how it can be frustrating because of the cost of synthetic genes. Additionally, the standard chemical approach to synthesize oligonucleotides, namely the phosphoramidite chemistry cycle, is limited to around 100-base building blocks; beyond that length they are very difficult to make [115]. Therefore, people all over the world are trying to find a way to make them more affordable and on a larger scale. Herein, the development of various chemical methods can come to aid the nucleic acid synthesis. One of them is so-called click chemistry, that assembles modified nucleoside and nucleotide building blocks using entirely chemical methods [116]. Here, we explore recent discoveries in the synthesis and functional research of triazole-modified oligonucleotides with a view towards therapeutic applications. Using click reaction that creates a triazole, we receive a stable linker replacing the internucleotide bond [117–119]. With the need for better gene synthesis, all innovative ideas attract a great deal of attention [120–123].

2. Triazole-Modified Oligonucleotides Chemical modifications of oligonucleotides (ONs) came to prominence in the early 1990s with the advent of the antisense approach to control gene expression [124,125]. An- tisense strategy as a part of controlling gene expression is a fast-growing research area. Naturally occurring oligonucleotides are unsuitable for that purpose because of the low stability towards nucleolytic degradation and poor cell penetration due to their polyanionic structure. These problems could be overcome by the chemical modification of oligonucleotides. ONs can be used in antisense or triple helix therapy to inhibit RNA translation or DNA transcription. One of the most radical modifications brought to the familiar backbone is the complete substitution of the phosphodiester bridge, to achieve stronger affinity for the nucleic acid target, and/or enhanced resistance to nucleases, or improve membrane permeability and cellular uptake. Here, the catalyzed Huisgen reaction appears promising as a way to generate non-natural oligonucleotides, that cannot be biologically degraded but can be used as biological tools binding to native DNA/RNA. Thus, oligonucleotide analogues with triazole internucleotide linkages continue to attract significant attention on medical applications for gene silencing such as antisense, antigen [126,127], and siRNA [128–132] interference.

2.1. (TL)DNA Skeleton DNA is the molecule of life, because the information about the structure and function- ing of the organism is encoded in it. Therefore, its structure and possibilities of “improvement” have always intrigued scientists all over the world (Figure3). DNA usually forms a duplex, in other words double-stranded structure. Some elements of nucleic acids can take unusual structures, e.g., in the case of an excess of guanine base. Then, a four-strand nucleic acid structure called G-quadruplex is formed, characterized by high plasticity compared to the standard double-stranded form, which translates into their important biological functions. Biomedicines 2021, 9, 628 5 of 35 BiomedicinesBiomedicines 2021 2021, ,9 9, ,628 628 55 ofof 3434

Figure 3. Triazole DNA template. FigureFigure 3. 3.Triazole Triazole DNA DNA template. template.

StudiesStudies of of the the properties properties of of modifiedmodified modified oligonucleotidesolig oligonucleotidesonucleotides led led to to the the development development ofof un-un- naturalnatural oligomers oligomers withwith with hugehuge huge therapeutictherapeutic therapeutic potential,potentia potential,l, especiallyespecially especially forfor for thethe the treatmenttreatment treatment ofof of diseasesdiseases diseases characterizedcharacterized by byby the thethe expression expressionexpression of of unwantedof unwantedunwanted genes. genes.genes. The The mostThe mostmost promising promisingpromising of such ofof analogues suchsuch ana-ana- arelogueslogues those areare exhibiting thosethose exhibitingexhibiting high binding highhighaffinity bindingbinding toward afaffinityfinity native towardtoward DNA/RNA, nativenative DNA/RNA,DNA/RNA, since they may sincesince prove theythey tomaymay be prove efficientprove to to antisensebe be efficient efficient or antisense antisense siRNA agents or or siRN siRN [133AA ].agents agents That is[133]. [133]. why That That a comprehensive is is why why a a comprehensive comprehensive analysis of theanalysisanalysis relation of of the the between relation relation the between between length the ofthe triazolelength length of internucleotideof triazole triazole internucleotide internucleotide linkage and linkage linkage duplex and and stability duplex duplex isstabilitystability essential isis essential [essential34,76,134 [34,76,134,135].,[34,76,134,135].135]. Therefore, Therefore,Therefore, it is logical itit isthatis logicallogical researchers thatthat researchersresearchers turned their turnedturned attention theirtheir towardattentionattention the toward toward possible the the benefitspossible possible ofbenefits benefits innovative of of i nnovativeinnovative and new and and synthetic new new synthetic synthetic approaches, approaches, approaches, such as such such the CuAACasas the the CuAAC CuAAC methods methods methods [33]. [33]. [33]. TheThe first firstfirst works worksworks which which describe describe describe the the the replacement replacement replacement of of ofthe the the phosphodiester phosphodiester phosphodiester linkage linkage linkage in in oli- oli- in oligonucleotidesgonucleotidesgonucleotides byby by heterocyclesheterocycles heterocycles haha haveveve beenbeen been reportedreported reported byby by vonvon von MattMatt etet al. al.al. [136,137].[[136,137].136,137]. This ThisThis re-re- searchsearch presentspresents presents synthesis synthesis synthesis of of of thymidine thymidine thymidine dinucleoside dinucleoside dinucleoside analogues analogues analogues with with with imidazole imidazole imidazole and and and triazole tria- tria- modifiedzolezole modified modified backbones backbones backbones before before before the era the the ofera era click of of click click chemistry chemistry chemistry (Figure (Figure (Figure4). Authors 4). 4). Authors Authors used used used a thermal a a ther- ther- cycloadditionmalmal cycloaddition cycloaddition to generate to to generate generate T-T T-T dimersT-T dimers dimers wherein wherein wherein triazole triazole triazole units units units were were were inserted inserted inserted (analogues (analogues (analogues of typesofof types types3 and 3 3 and and4). The4 4).). The The two two communicationstwo communications communications described described described the detailed th thee detailed detailed chemical chemical chemical synthesis synthesis synthesis of these of of compoundsthesethese compoundscompounds with an withwith overview anan overviewoverview of the introduction ofof thethe inintroductiontroduction into oligodeoxyribonucleotides intointo oligodeoxyribonucleotidesoligodeoxyribonucleotides strand in astrandstrand DNA/RNA in in a a DNA/RNA DNA/RNA heteroduplex heteroduplex heteroduplex and evaluation and and evaluation evaluation of the influence of of the the ofinfluence influence polarity of andof polarity polarity basicity and and of theba- ba- modifiedsicitysicity of of the the backbone modified modified structure backbone backbone on structure RNAstructure binding on on RNA RNA affinity, binding binding as well affinity, affinity, as thermodynamic as as well well as as thermody- thermody- properties.namicnamic Only properties. properties. dimer 3 Only couldOnly dimer dimer oligomerize, 3 3 could could while oligomerize, oligomerize, all attempts while while to all all incorporate attempts attempts to theto incorporate incorporate dimer 4 were the the completelydimerdimer 44 werewere unsuccessful. completelycompletely The unsuccessful.unsuccessful. backbone replacements TheThe backbackbonebone evaluated replacementsreplacements lead to evaluated aevaluated destabilization leadlead toto of aa thedestabilizationdestabilization duplexes formed of of the the by duplexes duplexes all modified formed formed sequences by by al all lmodified modified with their sequences sequences RNA complements. with with their their RNA RNA Only comple- comple- dimers ofments.ments. type 1Only Onlyand dimers 3dimersshowed of of notypetype significant 1 1 and and 3 3 showed improvementshowed no no significant significant of duplex improvement improvement stabilization. of Inof duplex allduplex instances, stabi- stabi- thelization.lization. incorporation In In all all instances, instances, of heterocyclic the the incorporation incorporation backbone of modificationsof heterocyclic heterocyclic leads backbone backbone to reduction modifications modifications of the thermalleads leads to to stabilityreductionreduction of of duplexes.of the the thermal thermal stability stability of of duplexes. duplexes.

FigureFigure 4. 4. Thymidine Thymidine dinucleoside dinucleoside analogues analogues withwith with imidazoleim imidazoleidazole andand and triazoletriazole triazole modiﬁedmodified modified backbones.backbones. backbones.

AnotherAnother type type of of coupling coupling between between two two pyrimidinepyri pyrimidinemidine analoguesanalogues analogues hashas has beenbeen been accomplished accomplished byby Lazrek Lazrek et etet al. al. (Figure (Figure(Figure 5) 5)5 )[[138]. [138].138 The ].The The novel novel novel branched branched branched nucleoside nucleoside nucleoside dimers dimers dimers containing containing containing a a 1,2,3- 1,2,3- a 1,2,3-triazolyltriazolyltriazolyl linkage linkage linkage have have been havebeen synthesized synthesized been synthesized using using 1,3-dipolar 1,3-dipolar using 1,3-dipolar cycloaddition cycloaddition cycloaddition of of N N-3-3 or or of C C-5N-5 -3acet- acet- or 0 0 Cyleneylene-5 acetylene nucleosides nucleosides nucleosides with with 3 3′-azido-3′ with-azido-3 3 -azido-3′-deoxythymidine′-deoxythymidine-deoxythymidine (AZT). (AZT). (AZT).The The coupling coupling The coupling reactions reactions reactions under under Biomedicines 2021, 9, 628 6 of 35 BiomedicinesBiomedicines 2021 2021, ,9 9, ,628 628 66 ofof 3434

underrefluxreflux reﬂux always always always provide provide provide a a mixture mixture a mixture of of the the of two thetwo twopo possiblessible possible 4- 4- and and 4- and 5-substi 5-substi 5-substitutedtutedtuted isomers. isomers. isomers. As As Asthe the theauthorsauthors authors suggest, suggest, suggest, such such such modified modified modiﬁed nucleosides nucleosides nucleosides may may may be be beof of interest ofinterest interest as as aspotential potential potential candidates candidates candidates for for fornucleoside-basednucleoside-based nucleoside-based therapeutics. therapeutics. therapeutics.

Bzl-OBzl-O TT Bzl-OBzl-O TT OO OO

NN NN NN NN NN OO NN OO NN NHNH OO NN NN OO RR-O-O Ac-OAc-O 33 OO OO

RR11 RR22 OAcOAcOAcOAc 5656 FigureFigureFigure 5. 5. 5.N N N-3-3-3 or or orC C -5C-5-5 branched branched branched nucleoside nucleoside nucleoside dimers dimers dimers containing co containingntaining a a 1,2,3-triazolyla 1,2,3-triazolyl 1,2,3-triazolyl linkage. linkage. linkage.

BecauseBecauseBecause of of of thethe the importantimportant important rolerole role ofof of AZTAZT AZT inin in chemotherapychemotherapy chemotherapy scientistsscientists scientists areare are constantlyconstantly constantly look-look- look- inginging for for for its its its valuable valuable valuable analogues. analogues. analogues. AlsoAlso Also inin in this this this case, case, case, an an an inestimable inestimable inestimable support support support is is is given given given by by by click click click chemistrychemistrychemistry [[33,55,79]. 33[33,55,79].,55,79]. UsingUsing Using thisthis this approach,approach, approach, thethe the TorrenceTorrence Torrence groupgroup group hashas has reportedreported reported thethe the ﬁrstfirst first syn-syn- syn- thesisthesisthesis ofof of nucleosidenucleoside nucleoside 1,2,3-triazole 1,2,3-triazole derivatives derivativesderivatives using usingusing CuAAC CuAAC [139] [139] [139 ](Scheme (Scheme (Scheme 1). 11).). For For For this this this re- re- reaction,action,action, Sharpless’s Sharpless’s original original conditions conditions using usingusing NaAsc NaAsc toto reduce reduce Cu(II)Cu(II) Cu(II) toto to Cu(I)Cu(I) Cu(I) inin in waterwater water are are are employed.employed.employed. Dinucleosides DinucleosidesDinucleosides 7 77 and andand 88 ,8, in,in in which whichwhich two twotwo thymidines thymidinesthymidines are are are linked linked linked by byby triazole, triazole,triazole, have havehave 0 0 beenbeenbeen synthesizedsynthesized synthesized byby by reacting reacting AZT AZT or or 5 55′-azidothymidine′-azidothymidine-azidothymidine with with 3 3 3′-′-O-OO-propargylthymidine-propargylthymidine-propargylthymidine in in inexcellentexcellent excellent yields.yields. yields. Generally,Generally, Generally, 1% 1% CuSO CuSO4 4 catalystcatalyst is is enough enoughenough toto carrycarry out outout this thisthis reaction. reaction.reaction.The TheThe reactionreactionreaction proceeds proceeds proceeds to to to completion completion completion at at at ambient ambient ambient temperature temperature temperature within within within 12 12 12 h. h. h. All All All novel novel novel thymidine thymidine thymidine analoguesanaloguesanalogues withwith with a a broadbroad scope scope of ofof modifications modiﬁcationsmodifications have have have been been been examined examined examined for for for their their their antiviral antiviral antiviral ac- ac- activity,tivity,tivity, but but but they they they have have have been been been found found found to to to be be be inactive inactive inactive against against 13 13 viruses. viruses.

SchemeSchemeScheme 1. 1. 1.One of the ﬁrst examples of AZT dimer analogues obtained by CuAAC.

InInIn 2007, 2007, 2007, Nuzzi Nuzzi Nuzziet et al.et al. al.published published published some some some model model model studies studies studies towards towards towards the synthesisthethe synthesis synthesis of thymidine of of thymi- thymi- oligonucleotidesdinedine oligonucleotides oligonucleotides with triazolewith with triazole triazole internucleosidic internucleosidic internucleosidic linkages linkages linkagesvia Cu(I)-promoted via via Cu(I)-promoted Cu(I)-promoted ligation ligation ligation strat- egystrategystrategy [140]. [140]. [140]. These These These artiﬁcial artificial artificial molecular molecular molecular fragments frag fragmentsments are more are are more more stable stable stable than than thethan natural the the natural natural products. prod- prod- Theseucts.ucts. These initialThese initial resultsinitial results results have demonstrated have have demonstrated demonstrated a promising a a promising promising usefulness usefulness usefulness of such of structuresof such such structures structures in search in in towardssearchsearch towards towards antisense antisense antisense exploitation. exploitation. exploitation. Also, anAlso, Also, explorative an an explorative explorative study study study has been has has been takenbeen taken taken to prepare to to pre- pre- aparepare trinucleotide—a a a trinucleotide—a trinucleotide—a triazole triazole triazole linked linked linked codon codon codon in a newin in a a new classnew class class of oligothymidine of of oligothymidine oligothymidine analogues analogues analogues as shownasas shown shown by structuresby by structures structures9, 10 9 9,, ,10 and10, ,and and11 11 (Scheme11 (Scheme (Scheme2). 2). The2). The The authors authors authors the the the of of workof work work assure, assure, assure, that that that this this this studystudystudy is isis a aa part partpart of ofof a aa longstanding longstandinglongstanding research researchresearch program programprogram to toto develop developdevelop antisense antisenseantisense nucleotides nucleotidesnucleotides in inin theirtheirtheir laboratory. laboratory. laboratory. Biomedicines 2021, 9, 628 7 of 35 BiomedicinesBiomedicines 20212021,, 99,, 628628 77 ofof 3434

SchemeSchemeScheme 2. 2.2. Synthesis SynthesisSynthesis of ofof non-natural non-naturalnon-natural dinucleotides dinucleotidesdinucleotides and andand trinucleotide. trinucleotide.trinucleotide.

SubsequentSubsequentSubsequent applications applicationsapplications of ofof such suchsuch interesting interestinginteresting oligomers oligomersoligomers may maymay be bebe found foundfound in inin the thethe literature literatureliterature alreadyalreadyalready dedicated dedicateddedicated to toto modiﬁed modifiedmodified ONs. ONs.ONs. One OneOne of ofof the ththee ﬁrst firstfirst who whowho indicated indicatedindicated that thatthat topic, topic,topic, were werewere Kumar KumarKumar etetet al. al.al.[ 141[141].[141].]. The TheThe click clickclick reaction reactionreaction is is usedis usedused to ligationtoto ligationligation of two ofof twotwo single singlesingle stranded strandedstranded oligdeoxynucleotides oligdeoxynucleo-oligdeoxynucleo- 0 0 (ODNs),tidestides (ODNs),(ODNs), one with oneone a with 5with-alkyne aa 55′′-alkyne-alkyne and the andand other thethe with otherother a 3 withwith-azide aa 33 (Scheme′′-azide-azide (Scheme(Scheme3). A covalent 3).3). AA covalentcovalent catena- tioncatenationcatenation leads to leadsleads intramolecular toto intramolecularintramolecular circularization circularizationcircularization and produces andand producesproduces a DNA strand aa DNADNA with strandstrand an unnatural withwith anan extendedunnaturalunnatural backbone extendedextended at backbonebackbone the ligation atat thethe point. ligationligation The point.point. product TheTheis productproduct obtained isis obtainedobtained at room temperatureatat roomroom tem-tem- withinperatureperature 2 h. withinwithin 22 h.h.

SchemeSchemeScheme 3. 3.3. Schematic SchematicSchematic template-mediated template-mediatedtemplate-mediated click-ligation click-ligationclick-ligation of ofof two twotwo oligonucleotides. oligonucleotides.oligonucleotides.

AnotherAnotherAnother excitingexciting example example ofof of CuAACCuAAC CuAAC temptemp templatedlatedlated nucleicnucleic nucleic acidacid acid reactionsreactions reactions usesuses uses 33′′--OO 3-pro--pro-0-O- propargylpargylpargyl 5-methyldeoxycytidine5-methyldeoxycytidine 5-methyldeoxycytidine atat thethe at the33′′-terminus-terminus 30-terminus ofof anan of ODNODN an ODN strand,strand, strand, andand aa and 55′′-azide-azide a 50-azide atat thethe at55′′-strand-strand the 50-strand terminusterminus terminus [142].[142]. AsAs [142 aa ]. result,result, As a thethe result, triazoletriazole the triazolelinkagelinkage is linkageis similarsimilar is inin similar lengthlength in toto length aa naturalnatural to aphosphate-sugarphosphate-sugar natural phosphate-sugar linkagelinkage [143],[143], linkage whichwhich [143 ],isis whichtoleratedtolerated is tolerated byby manymany by differentdifferent many different enzymes,enzymes, enzymes, suchsuch asas suchDNADNA as andand DNA RNARNA and polymerasespolymerases RNA polymerases [144,145].[144,145]. [144 Thus,Thus,,145]. DNADNA Thus, strandsstrands DNA strands withwith thesethese with terminalterminal these terminal groupsgroups groupscancan bebe used canused be toto used constructconstruct to construct genesgenes thatthat genes areare that susubsequentlybsequently are subsequently translatedtranslated translated andand transcribedtranscribed and transcribed inin E.E. colicoli in[116,142].[116,142].E. coli [116 Moreover,Moreover,,142]. Moreover, usingusing thethe using CuAACCuAAC the CuAACapproaapproach,ch, approach, epigeneticepigenetic epigenetic modificationsmodifications modifications cancan bebe incor-incor- can beporatedporated incorporated intointo thethe into genegene the toto genedeterminedetermine to determine thethe influeinflue thencence influence ofof suchsuch trial oftrial such [116].[116]. trial ThisThis [116 workwork]. This indicatesindicates work 0 0 indicatesthatthat CuAACCuAAC that CuAACofof 55′′-azido-azido of 5 andand-azido 33′′--OO and-propargyl-propargyl 3 -O-propargyl modificationsmodifications modifications mightmight mightemergeemerge emerge asas aa non-enzy-non-enzy- as a non- enzymaticmaticmatic tooltool toolforfor ligationligation for ligation thatthat thatcouldcould could complementcomplement complement oror replacereplace or replace ligaseligase ligase systemssystems systems [142,146,147].[142,146,147]. [142,146,147 ThisThis]. Thisquickquickquick clickclick click methodologymethodologymethodology waswas was alsoalso also usedused used byby byOsmanOsman Osman etet al.al.et al.forforfor thethe the samesame same purpose—ligationpurpose—ligation purpose—ligation ofof 0 0 of33′′-- 3OO--propargyl-propargylO-propargyl andand and 55′′-azide-azide 5 -azide DNA-modifiedDNA-modified DNA-modified strandsstrands strands throthro throughughugh biocompatiblebiocompatible biocompatible triazoletriazole triazole unitunit unit[148].[148]. [148 TheThe]. CuAACTheCuAAC CuAAC ofof oligonucleotidesoligonucleotides of oligonucleotides isis aa promisingpromising is a promising ligationligation ligation systemsystem system becausebecause because itit isis asas it rapidrapid is as rapidbutbut moremore but more selectiveselective selective than,than, than, e.g.e.g.,, T4e.g.T4 DNA,DNA T4 DNA ligase.ligase. ligase. TriazoleTriazole Triazole linklink linkageageage cancan can effectivelyeffectively effectively mimicmimic mimic thethe thephosphate-sugarphosphate-sugar phosphate-sugar backbonebackbone backbone ofof ofDNA,DNA, DNA, leadingleading leading toto to unprecedentedunprecedented unprecedented tolerancetolerance ofof thethethe ligated ligatedligated strandsstrandsstrands by byby polymerases. polymerases.polymerases. TheTheThe click clickclick,,, bis-click bis-clickbis-click,,, and andand stepwise stepwisestepwise clickclickclick reactions reactionsreactions have havehave been beenbeen used usedused for forfor the thethe synthesis synthesissynthesis of ofof a aa covalentlycovalentlycovalently closed closedclosed ssDNA ssDNAssDNA circle circlecircle and andand dsDNA dsDNAdsDNA [ [149–151]149[149–151]–151] (Scheme (Scheme(Scheme4 ).4).4). This ThisThis chemistry chemistrychemistry makes makesmakes ititit possible possiblepossible to toto receive receivereceive cyclic, cyclic,cyclic, branched, branched,branched, and andand even evevenen bicyclic bicyclicbicyclic ONs ONsONs [ 152[152].[152].]. Cyclization CyclizationCyclization has hashas been beenbeen foundfoundfound tototo bebebe moremore efficient efficientefficient in in solutionsolution thanthan than onon on solidsolid solid supportsupport support andand and inin in bothboth both cases,cases, cases, itit itcancan can bebe Biomedicines 2021, 9, 628 8 of 35 Biomedicines 2021, 9, 628 8 of 34

beassisted assisted by by microwave microwave irradiation irradiation (MW, (MW, at 60 at 60°C ◦forC for60 min). 60 min). Cyclic Cyclic DNA DNA and and RNA RNA pre- presentsent unusual unusual chemical chemical and and biological biological properties properties in in comparison comparison with linearlinear counterparts,counterparts, andand have have been been evaluated evaluated for for several several biological biological applications, applications, including including antisense, antisense, triplex, triplex, andand diagnostic diagnostic ones. ones. CyclicCyclic DNADNA miniduplexes miniduplexes areare very very stable, stable, resistant resistant to to intracellular intracellular enzymaticenzymatic degradation,degradation, and are more more readily readily taken taken up up by by cells cells [149]. [149 ].Oligonucleotide Oligonucleotide ap- aptamerstamers are are used used inin vivo vivo asas decoys decoys to to sequester sequester DNA-bind DNA-bindinging proteins.proteins. TheyThey mustmust remain remain doubledouble stranded stranded in in the the cell cell and and be be resistant resistant to to DNases. DNases. The The branched branched cyclic cyclic ONs ONs could could be be consideredconsidered as as mimics mimics of of a a lariat lariat structure structure (lariat (lariat mimics). mimics). These These molecules molecules are are structurally structurally relatedrelated toto natural intermediates intermediates formed formed during during RNA RNA splicing splicing [153]. [153 In]. turn, In turn,triazole-linked triazole- linkeddumbbell dumbbell ODNs ODNs can exist can as exist potential as potential decoy decoy molecules molecules [154]. [ 154Described]. Described triazole-cross- triazole- cross-linkedlinked ODNs ODNs werewere synthesized synthesized with withthe use the of use the of CuAAC the CuAAC and andODNs ODNs possessing possessing N-3- N-3-(azidoethyl)thymidine and N-3-(propargyl)thymidine at the 30- and 50-termini. The (azidoethyl)thymidine and N-3-(propargyl)thymidine at the 3′- and 5′-termini.0 The newly newlysynthesized synthesized dumbbell dumbbell ODNs ODNsshowed showed excellent excellent stability stability against 3 against′-exonuclease 3 -exonuclease and high andthermal high stability, thermal stability,which are which necessary are necessary for decoy for molecules decoy molecules to achieve to biological achieve biological responses responsesleading to leading alteration to alteration of gene expression. of gene expression. This click This methodology click methodology can be valuable can be valuable in nano- in nanotechnological applications involving DNA scaffolds [155,156]. technological applications involving DNA scaffolds [155,156].

SchemeScheme 4. 4.Cyclization Cyclization and and bicyclization bicyclization of of oligonucleotides. oligonucleotides.

AnotherAnother scientificscientific ideaidea waswas presented presented inin order order to to investigate investigate the the efficiency efficiency of of sugar sugar labelinglabeling ofof DNA under under various various conditions conditions [157 [157]. ].For For this this purpose, purpose, synthetic synthetic alkyne-mod- alkyne- modifiedified nucleoside nucleoside triphosphates triphosphates (dU*TP) (dU *wereTP) wereincorporated incorporated into long into DNA long DNAstrands strands by the byPCR the reaction, PCR reaction, as shown as in shown Scheme in Scheme5. Then 5the. Then alkyne-modified the alkyne-modified DNA could DNA be derivatized could be derivatizedusing the click using reaction the click with reactionvarious azide with molecules, various azide and molecules,in this case of and sugars—galactose in this case of sugars—galactoseazide [158–161]. This azide result [158– showed161]. This the result decr showedeased click the reaction decreased yield click for reaction DNA yieldmodified for DNAwith modifiedshort-chain with alkyne short-chain nucleoside alkyne (ethynylur nucleosideidine), (ethynyluridine), rather than for rather DNA than containing for DNA al- containingkyne-modified alkyne-modified uridine with uridine an attached with an long attacheder spacer. longer Authors spacer. also Authors compared also compared the click thereaction click reactionefficiency efficiency of dsDNA of with dsDNA ssDNA with 300 ssDNA-bases 300-bases long DNA long modified DNA modifiedwith 5-ethynyl- with 5-ethynyluridine.uridine. It turned Itout turned that both out thatsingle both strand singles, the strands, minus- the and minus- the plus-strand, and the plus-strand, reacted in reactedsignificantly in significantly higher yield higher compared yield to compared dsDNA. toThe dsDNA. study motivated The study Su motivated et al. to investigateSu et al. toin investigatemore detail in the more click detail reaction the on click DNA, reaction one of on the DNA, most challenging one of the mostbiomolecular challenging sub- biomolecularstrates. This work substrates. is of great This practical work is valu of greate because practical labelled value DNA because are labelledused in biomedical DNA are usedapplications, in biomedical for example applications, in genetic for example analysis, in genome genetic analysis,screening, genome and high-throughput screening, and high-throughputDNA sequencing. DNA Professor sequencing. Carell Professoralso included Carell synthesis also included of labelled synthesis nucleotides of labelled and nucleotidessynthesis of and probes synthesis for the of sequencing probes for theof epigenetic sequencing markers of epigenetic [162]. markers [162].

Biomedicines 2021, 9, 628 9 of 35 Biomedicines 2021, 9, 628 9 of 34

Biomedicines 2021, 9, 628 9 of 34

Scheme 5. Illustrative depiction of DNA functionalization using click chemistry. R–N3 any azide- Scheme 5. Illustrative∗ depiction of DNA functionalization using click chemistry. R–N3 any azide- molecule, dU TP is a uridine derivative modified with a terminal alkyne functionality. 3 molecule,Scheme 5. dU Illustrative∗TP is a uridine depiction derivative of DNA modified functionalization with a terminal usingalkyne click chemistry. functionality. R–N any azide- molecule, dU∗TP is a uridine derivative modified with a terminal alkyne functionality. TheThe CuAACCuAAC reaction reaction allowed allowed for for the the labeling labeling of bothof both small small synthetic synthetic oligonucleotides oligonucleo- andtides largeThe and CuAAClarge gene gene fragments. reaction fragments. allowed Interesting Interesting for methodsthe me labelingthods of DNAofof DNA both modification modificationsmall synthetic can can beoligonucleo- be achieved achieved intidesin postpost and syntheticsynthetic large gene labelinglabeling fragments. [[163].163]. Interesting This strategy me isthods is characterized characterized of DNA modification by by a a high high degree degreecan be ofachieved of modu- mod- ularity,larity,in post because becausesynthetic changing changing labeling the [163]. the label label This does does strategy not not require requireis characterized a novel a novel nucleotide nucleotideby a high synthesis. degree synthesis. of The modu- Thesub- substitutionstitutionlarity, because of 5-position of 5-positionchanging in thepyrimidines in pyrimidineslabel does or not the or require the 7-position 7-position a novel in purinesnucleotide in purines are synthesis. are generally generally The well well sub- ac- acceptedceptedstitution by byof polymerases 5-position polymerases in for pyrimidines for enzymatic enzymatic incorporationor incorporationthe 7-position into intoinPCR purines PCR and primer and are primergenerally extension extension well prod- ac- products.ucts.cepted That by That polymerasesis why is why it is possible it for is possible enzymatic to attach to attach incorporation alkynyl-labels, alkynyl-labels, into which PCR which resultsand resultsprimer in DNA in extension DNA bearing bearing prod- var- variousiousucts. modifications;That modifications; is why it is however, possible however, tothe theattach copper copper alkynyl-labels, ions ions damage damage which DNA DNA results by breakingbreaking in DNA strands.strands. bearing This Thisvar- problemproblemious modifications; cancan bebe overcomeovercome however, bybythe thethe use usecopper ofof thethe ions Cu(I)-stabilizingCu(I)-stabilizing damage DNA ligand, ligand,by breaking forfor exampleexample strands. TBTA.TBTA. This TheTheproblem nextnext limitation canlimitation be overcome isis thatthat the theby direct thedirect use incorporation incorporation of the Cu(I)-stabilizing ofof azides azides into into ligand, synthetic synthetic for example DNA DNA strands strands TBTA. isisThe intrinsicallyintrinsically next limitation difficult,difficult, is that becausebeca theuse direct thethe azideazide incorporation groupgroup reactsreacts of azides withwith the theinto phosphorus(III) phosphorus(III) synthetic DNA atom atomstrands of of theisthe intrinsically phosphoramiditephosphoramidite difficult, group.group. beca Here,useHere, the wewe azide cancan noticenoticegroup thatthatreacts properlyproperly with the preparedprepar phosphorus(III)ed buildingbuilding blocks–atomblocks– of nucleosidethenucleoside phosphoramidite dimersdimers may group. be very very Here, helpful, helpful, we can as as noticenew new nucleic nucleicthat properly acid acid motifs motifs prepar earlier earliered building described described blocks– in the in theliteraturenucleoside literature [41–43,151,154,163–167]. dimers [41–43 may,151 ,be154 very,163 –helpful,167 Some]. Some as examples new examples nucleic of such of acid such structures motifs structures earlier are arepresented described presented in in Fig- the in Figureureliterature 6. 6. [41–43,151,154,163–167]. Some examples of such structures are presented in Fig- ure 6.

Figure 6. 1,4- and 1,5-bisnucleosides 1,2,3-triazole derivatives of thymidine and adenosine. FigureFigure 6.6.1,4- 1,4- andand 1,5-bisnucleosides1,5-bisnucleosides 1,2,3-triazole1,2,3-triazolederivatives derivatives of of thymidine thymidine and and adenosine. adenosine. As a part of the study of modified DNA, Seela and co-workers got interested in the use ofAsAs CuAAC aa partpart of as the an studyefficient study of of modifiedway modified to labe DNA, DNA,l DNA Seela Seela [168]. and and They co-workers co-workers have examined got got interested interested ODNs in con- the in thetaininguse useof CuAAC ofalkyne-labelled CuAAC as an as efficient an 7-deazapurines efficient way way to labe to and labell DNA pyrimidines, DNA [168]. [168 They]. and They have later have examined 7-alkynyl-7-deaza-2 examined ODNs ODNs con-′- containingtainingdeoxyinosine alkyne-labelled alkyne-labelled [169]. One 7-deazapurines of 7-deazapurines such examples and and ispyrimidines, presented pyrimidines, below and and later and later 7-alkynyl-7-deaza-2 applies 7-alkynyl-7-deaza- in receiving′- 0 2oligonucleotidesdeoxyinosine-deoxyinosine [169]. [ 169containing ].One One of of suchthe such octa examples examplesdiynyl derivativeis is presented presented of belowdU with and AZT appliesapplies via CuAAC inin receivingreceiving reac- oligonucleotidesoligonucleotidestion on solid-phase containing containing support the andthe octadiynyl octain solutiondiynyl derivative (tderivativeo link the of of dUDNA dU with strands)with AZT AZTvia [170] CuAACvia (SchemeCuAAC reaction 6).reac- In ontheirtion solid-phase on research, solid-phase support the supportfluorescence and in and solution inproperties solution (to link (tofo the linkoligonucleotides DNA the DNA strands) strands) [ 170for] the (Scheme[170] visualization (Scheme6). In their 6). Inof research,DNAtheir research,was the under fluorescence the special fluorescence emphasis properties properties [171–173]. of oligonucleotides of In oligonucleotidesparticular, for the visualizationgeneration for the visualization of offluorescence DNA was of underbyDNA the specialwas click under emphasisreaction special [151,171], [171 emphasis–173]. and In [171–173]. particular, studying In the theparticular, generation mechanisms the of generation fluorescenceof its quenching, of fluorescence by the clickwere reactionby the [click151, 171reaction], and studying[151,171], the and mechanisms studying ofthe its mechanisms quenching, were of its presented quenching, [173 ,174were]. Biomedicines 2021, 9, 628 10 of 35 Biomedicines 2021,, 99,, 628628 10 of 34

In turn, the introduction of a dye at the 2-position of the ribose sugar to DNA has been also presented [173,174]. In turn, the introduction of aa dyedye atat thethe 2-position2-position ofof thethe riboseribose sugarsugar done by Berndl et al. [175]. to DNA has been also done by Berndl et al. [175].[175].

Scheme 6. Synthesis example of a derivative containing the octadiynyl analogue of dU with AZT. SchemeScheme 6. 6.Synthesis Synthesis example example of of a a derivative derivative containing containing the the octadiynyl octadiynyl analogue analogue of of dU dU with with AZT. AZT.

TheThe number number of of reports reports related related to to click click chemistry chemistry and and DNA DNA is is still still limited, limited, due due to to the the requiredrequired long long reaction reaction time time (from (from hours hours to to days), days), elevated elevated temperatures temperatures (37–80 (37–80◦ C),°C), and and lacklack ofof anyany easyeasy procedure procedure for for coupling coupling of of water-insoluble water-insoluble organic organic azides. azides. Hence, Hence, we wecan cannotice notice the thenext next step step in developing in developing click click reaction reaction (CuAAC) (CuAAC) by using by using MW [176]. MW [This176]. highly This highlyefficient efﬁcient method method allowed allowed for 1,3-dipolar for 1,3-dipolar cycloaddition cycloaddition reaction reactionss to be carried to be carriedout on fully out ondeprotected fully deprotected ONs, affording ONs, affording 1,4-regioisomeric 1,4-regioisomeric 1,2,3-triazoles 1,2,3-triazoles in a very in ashort very time short (10–15 time (10–15min). The min). universality The universality of the technique of the technique was also was demonstrateddemonstrated also demonstrated inin thethe synthesissynthesis in the synthesis ofof triazole-triazole- of triazole-linkedlinked nucleoside nucleoside dimers. dimers. First examples First examples of such ofreactions such reactions have been have investigated been investigated for 3′′,5,5′′- 0 0 fordithymidine 3 ,5 -dithymidine in order into reduce order to the reduce reaction the time reaction [177,178]. time Zerrouki’s [177,178]. group Zerrouki’s reported group the reportedexamination the examinationof chemical conditions of chemical for conditions CuAAC. forThey CuAAC. tested two They different tested two salts, different namely salts,CuI and namely CuSO CuI4,, andand and clickclick CuSO reactionsreactions4, and werewere click performedperformed reactions were eieither performed on classical either heating on or classical micro- heatingwave activation or microwave (Scheme activation 7). Both (Scheme systems7 led). Both to products, systems but led during to products, reaction but using during CuI reactionthe formation using CuIof a side the formation product was of aobserved side product at room was temperature. observed at Click room reaction temperature. using Click reaction4 using CuSO gave a single product, same as in the case of classical heating CuSO4 gavegave aa singlesingle product,product,4 samesame asas inin thethe cacase of classical heating (80 °C, 5 h) and MW (80(80◦ °C,C, 5 200 h) andW, 3 MW min), (80 giving◦C, 200 identical W, 3 min), result givings (80% identical yields). results This (80%work yields).was continued This work to- (80 °C, 200 W, 3 min), giving identical result0 0 s (80% yields). This work was continued to- waswards continued tri-, tetra-, towards and 3 tri-,′′,5,5′′-pentathymidine tetra-, and 3 ,5 -pentathymidine synthesis using synthesisthis fast and using efficient this fast proce- and efﬁcientdure to proceduregenerate a tonew generate family aof new oligonucleotide family of oligonucleotide analogues [178]. analogues [178].

Scheme 7. SchemeScheme 7. 7.Different conditions for performing the CuAAC reaction.

FurtherFurther scientific scientific reports reports show show the the research research directions directions of triazole-linked of triazole-linked nucleoside nucleoside for longerfor longer chains chains design design and and synthesis, synthesis, and and even even double-strand double-strand formation formation of such of such modified modi- nucleicfied nucleic acids. acids. Isobe Isobeet al. work et al. work onwork a new onon aa triazole-linked newnew triazole-linkedtriazole-linked analogue analogueanalogue of DNA ofof using DNADNA click usingusing chem- clickclick istrychemistry [179]. These[179]. These scientists scientists managed managed to get theto get artificial the artificial 10-mer 10-merTLDNA TL onDNA a solid-phase on a solid- synthesis,phase synthesis, that after that examined after examined could form could a stableform a double stable stranddouble with strand the with complementary the comple- strandmentary of naturalstrand of DNA natural (Scheme DNA8 ).(Sch Theeme melting 8). The temperature melting temperature (T m) of the (T correspondingm) of the corre- ◦ duplexsponding is about duplex 40 isC about higher 40 than °C higher that of thethan isosequential that of the isosequential unmodified duplex. unmodified The stability duplex. mayThe bestability due to may the neutralbe due backboneto the neutral that doesbackbo notne have that any does repulsive not have interactions any repulsive with inter- the anionicactions phosphatewith the anionic backbone phosphate of the backbone natural target. of the Because natural target. similar Because triazole similar linker triazole with a TL longerlinker methylenewith a longer bridge methylene destabilizes bridge the destabil doubleizes strand, the thedouble six-bond strand, periodicity the six-bond of DNAperio- isdicity crucial of TL toDNA form is stable crucial double to form strands. stable Thus,double the strands. monomer Thus, for the the monomer elongation for reactionthe elon- 0 0 shouldgation havereaction an acetyleneshould have group an acetylene at the 5 -position group at and the an5′′-position azide group and atan the azide 3 -position. group at Unfortunately,the 3′′-position. this Unfortunately, stabilization this was stabilizat later shownion was to belater sequence-dependent shown to be sequence-depend- [180]. It is worthent [180]. noticing It is thatworth the noticing elongation that reaction the elongation did not requirereaction an did excess not require of the substrate, an excess in of con- the Biomedicines 2021, 9, 628 11 of 35 Biomedicines 2021, 9, 628 11 of 34

trast to the need in a standard phosphoramidite DNA synthesis. Additionally, microwave substrate, in contrast to the need in a standard phosphoramidite DNA synthesis. Addi- irradiation helps to considerably shorten the reaction time. Since the ﬁrst coupling reaction tionally, microwave irradiation helps to considerably shorten the reaction time. Since the proceeded smoothly and the results were encouraging, the research group improved a first coupling reaction proceeded smoothly and the results were encouraging, the research route for the solution-phase synthesis of 7-mer and 8-mer TLDNA [180], which is aimed at group improved a route for the solution-phase synthesis of 7-mer and 8-mer TLDNA [180], preparation in a larger scale of triazole-oligomers, and even for other deoxyribonucleoside which is aimed at preparation in a larger scale of triazole-oligomers, and even for other analogues bearing adenine, cytosine, and guanine nucleobases [181]. deoxyribonucleoside analogues bearing adenine, cytosine, and guanine nucleobases [181].

Me3Si H H

T T T O O O

N3 N N N N H + N N "click" T 9 T O O T O O H OH N O H O N O O O SchemeScheme 8. 8.Solid-phase Solid-phase synthesis synthesis of of 10-mer 10-merTL TLDNA.DNA.

ChandrasekharChandrasekhar andand Idris Idris rightly rightly noticed noticed that that the the triazole-linkage triazole-linkage throughout throughout the the chainchain is is a a limiting limiting factor factor in in the the whole whole synthesis synthesis [182 [182].]. That That is is why why they they firstly firstly proposed proposed receivingreceiving a a ready ready dimer dimer nucleoside nucleoside phosphoramidite phosphoramidite with with a a triazole triazole linkage linkage inside inside ( 12(12) ) (Figure(Figure7). 7). Some Some related related studies studies were were presented presente byd by the the group group of of Florentiev Florentiev [ 180 [180,183].,183]. The The groupgroup reported reported an an improved improved synthesis synthesis of theof the dinucleoside dinucleoside phosporamidite phosporamidite block block12 and 12 and its utilizationits utilization in ONs in ONs synthesis, synthesis, and additionallyand additionally DNA DNA binding binding affinity affinity verification. verification. However, How- shortever, linkages short linkages with four-bond with four-bond triazolyl triazolyl internucleotide internucleotide distort distort the double the double helix, ashelix, well as aswell the as longer the longer triazole triazole internucleotide internucleotide linkages linkages [134 [134,178].,178]. It confirmsIt confirms that that the the six-bond six-bond backbonebackbone distance distance is is crucial crucial for for the the double-strand double-strand formation. formation. The The search search for for backbonebackbone- modifiedmodified oligonucleotide oligonucleotide analogues analogues is is still still intriguing intriguing since since the the discovery discovery of of the the CuAAC CuAAC reaction.reaction. The The same same researchersresearchers obtainedobtained dithymidinedithymidine phosphoramiditephosphoramidite analogues with with a atriazole triazole linker linker and and an additional methylene group ofof typetype 1313,, whichwhich was was then then incorpo- incorpo- ratedrated in in oligonucleotides oligonucleotides using using solid-phase solid-phase synthesis synthesis with with standard standard phosphoramididte phosphoramididte protocolsprotocols [ 180[180].]. The The hybridization hybridization ability ability and and thermal thermal stability stability of of the the duplexes duplexes verified verified destabilizationdestabilization effect, effect, for for which which the the geometry geometry is is probably probably responsible. responsible. Here, Here, we we can can also also seesee that that the the synthetic synthetic strategy strategy based based on on the the use use of of dinucleoside dinucleoside phosphoramidite phosphoramidite blocks blocks withwith a a triazole triazole linkerlinker isis employed. The The effect effect of of single single and and multiple multiple modifications modifications on onsta- stabilitybility of of mixed-base mixed-base duplexes duplexes was was assessed assessed and and compared compared with with previously previously published published data data[183,184]. [183,184 However,]. However, these these results results did did not not disc discourageourage the the authors authors to continuecontinue thisthis work, work, and a paper on triazole-linked oligonucleotides with high affinity to DNA complements and a paper on triazole-linked oligonucleotides with high affinity to DNA complements was published a year later [185]. They obtained a new dinucleotide with a triazole internu- was published a year later [185]. They obtained a new dinucleotide with a triazole inter- cleotide modification type 14, that demonstrated DNA binding affinities similar to those nucleotide modification type 14, that demonstrated DNA binding affinities similar to of unmodified oligonucleotides, with simultaneous protection from nuclease hydrolysis those of unmodified oligonucleotides, with simultaneous protection from nuclease hy- due to the modification (Figure7). ONs bearing these triazole fragments at the 3 0-terminus, drolysis due to the modification (Figure 7). ONs bearing these triazole fragments at the 3′- 50-terminus or in the middle of the chain hybridized to complementary ssDNAs and did terminus, 5′-terminus or in the middle of the chain hybridized to complementary ssDNAs not cause any significant changes in duplex geometry. Then, the UV-melting experiments and did not cause any significant changes in duplex geometry. Then, the UV-melting ex- proved a slight destabilization effect of duplexes (almost 4 times lower than for other periments proved a slight destabilization effect of duplexes (almost 4 times lower than for triazole modifications). Furthermore, these modified oligonucleotides were tested as PCR other triazole modifications). Furthermore, these modified oligonucleotides were tested primers and were tolerated by polymerases. This finding shows a great potential of these as PCR primers and were tolerated by polymerases. This finding shows a great potential new oligonucleotide analogues as the most promising triazole DNA mimics in terms of hybridizationof these new properties, oligonucleotide compatibility analogues with as biosystems,the most promising and stability triazole in biological DNA mimics liquids. in Theterms new of modifications hybridization have properties, a relatively compatibility insignificant with impact biosystems, on duplex and geometry, stability thereforein biologi- thecal authors liquids. hypothesized The new modifications that it might have be tolerated a relatively by ON-recognizinginsignificant impact enzymes. on duplex It is worth geom- noticing,etry, therefore that at the the authors same time hypothesized Madhuri andthat Kumarit might also be tolerated worked onby theON-recognizing same six-atom en- triazolidezymes. It linker is worth group noticing, of compounds that at the15 sameand 16time[186 Madhuri]. They synthesizedand Kumar also two worked dimer blocks, on the same six-atom triazolide linker group of compounds 15 and 16 [186]. They synthesized Biomedicines 2021, 9, 628 12 of 35 BiomedicinesBiomedicines 2021 2021, ,9 9, ,628 628 1212 ofof 3434

then incorporated them to oligonucleotides, and UV-Tm with complementary DNA and twotwo dimer dimer blocks, blocks, then then incorporated incorporated them them to to oligonucleotides, oligonucleotides, and and UV-T UV-Tmm with with comple- comple- RNA were presented. mentarymentary DNA DNA and and RNA RNA were were presented. presented.

FigureFigureFigure 7.7. 7. PhosphoramiditePhosphoramidite Phosphoramidite triazole-linkedtriazole-linked triazole-linked dimersdimers dimers T–T.T–T. T–T.

AtAtAt thethe the samesame same time,time, time, El-SagheerEl-Sagheer El-Sagheer andand and BrownBrown Brown workedwo workedrked onon on DNADNA DNA strands strands containing containing an an un- un- naturalnaturalnatural triazole triazoletriazole linkage, linkage,linkage, that thatthat had hadhad been beenbeen synthesized synthesizedsynthesized by click byby clickclick ligation ligationligation between betweenbetween ONs ONswithONs AZT withwith andAZTAZT 4 and0 -propargyloamido-5and 4 4′-propargyloamido-5′-propargyloamido-50-derivative′-derivative′-derivative of 50-deoxymethylthymidine of of 5 5′-deoxymethylthymidine′-deoxymethylthymidine(Scheme (Scheme (Scheme9)[187 9) ].9) [187]. This[187]. isThisThis the is firstis the the examplefirst first example example of triazole-modified of of triazole-modified triazole-modified oligonucleotides, oligonucleotides, oligonucleotides, which which which serves serves serves as PCR as as template,PCRPCR tem- tem- whichplate,plate, meanswhichwhich means polymerasemeans polymerase polymerase chain reaction chain chain reaction amplification.reaction amplification. amplification. From the From From beginning the the beginning beginning it was planned, it it was was thatplanned,planned, the biological that that the the biological aspectbiological of theaspect aspect use of ofof the thesethe use use compounds of of these these compounds compounds is the most is is important the the most most important here.important The productionhere.here. The The production production of synthetic of of genessynthetic synthetic is prohibitive genes genes is is pr pr duringohibitiveohibitive solid-phase during during solid-phase solid-phase synthesis andsynthesis synthesis it is caused and and it it byisis caused ancaused upper by by limitan an upper upper of around limit limit of 150of around around bases in150 150 oligonucleotides, ba basesses in in oligonucleotides, oligonucleotides, an imperfect an an couplingimperfect imperfect efficiency, coupling coupling andefficiency,efficiency, chemical and and modifications chemical chemical modifica modifica by side-reactionstionstions by by side-reactions side-reactions during the during during process. the the So, process. process. there isSo, So, a there needthere foris is a a anneedneed inexpensive for for an an inexpensive inexpensive and simple and and method simple simple formethod method oligonucleotide for for oligonucleotide oligonucleotide synthesis synthesis synthesis in a controlled in in a a controlled controlled manner andmannermanner large and and scale. large large Such scale. scale. constructs Such Such constructs constructs could becould could used be be in used used living in in living cells;living enzyme-freecells; cells; enzyme-free enzyme-free synthesis synthe- synthe- of genes,sissis ofof genes, whichgenes, which allowwhich theallowallow incorporation thethe incorporationincorporation of site-specific ofof site-specificsite-specific base analogues basebase analoguesanalogues for studying forfor studyingstudying DNA replication/repair;DNADNA replication/repair; replication/repair; or as or fluorescentor as as fluorescent fluorescent dyes dyes fordyesin for for vivo in in vivo visualization.vivo visualization. visualization. Hence, Hence, Hence, the searchthe the search search for aforfor polymerase-compatible a a polymerase-compatible polymerase-compatible triazole triazole triazole linkage linkage linkage with with with the the the initial initial initial experiments experiments experiments focused focused focused onon on non-non- non- enzymaticenzymaticenzymatic DNADNA DNA strandstrand strand ligationligation ligation combinedcombined combined withwith with reproduciblereproducible reproducible amplification.amplification. amplification. AnAn An additionaladditional additional advantageadvantageadvantage ofof of thethe the triazoletriazole triazole unitsunits units isis is fact,fact, fact, thatthat that thisthis this chemicalchemical chemical linkage linkage is is compatible compatible with with enzy- enzy- maticmaticmatic processing,processing, processing, inin in particularparticular particular PCR,PCR, andand doesdoes not not suffersuffer suffer fromfrom from degradationdegradation degradation duedue toto endo-endo- endo- and exonucleases. It is worth noting here that DNA sequencing of the PCR amplicon andand exonucleases. exonucleases. It It is is worth worth noting noting here here th thatat DNA DNA sequencing sequencing of of the the PCR PCR amplicon amplicon and and and clones revealed the presence of a single thymine at the ligation site instead of the clonesclones revealedrevealed thethe presencepresence ofof aa singlesingle thymthymineine atat thethe ligationligation sitesite insteadinstead ofof thethe twotwo two thymine bases that were present in the original template. This is the first time that thyminethymine bases bases that that were were present present in in the the original original template. template. This This is is the the first first time time that that highly highly highly efficient non-enzymatic DNA strand ligation has been combined with reproducible efficientefficient non-enzymatic non-enzymatic DNA DNA strand strand ligation ligation has has been been combined combined with with reproducible reproducible am- am- amplification. And reverse transcription of RNA analogues containing triazole linkages plification.plification. And And reverse reverse transcription transcription of of RNA RNA analogues analogues containing containing triazole triazole linkages linkages will will will be also envisaged. bebe also also envisaged. envisaged.

SchemeSchemeScheme 9. 9.9. Synthesis SynthesisSynthesis and andand polymerase polymerasepolymerase chain chainchain reactionreaction reaction ampliﬁcation amamplificationplification of ofof DNA DNADNA strandsst strandsrands containing containingcontaining an anan unnaturalunnaturalunnatural triazoletriazole triazole linkage.linkage. linkage.

InInIn recent recentrecent years, years,years, this thisthis research researchresearch group groupgroup has hashas dealt dedealtalt with withwith the thethe subject subjectsubject of click ofof clickclick chemistry chemistrychemistry in the inin nucleicthethe nucleic nucleic acids acids acids ﬁeld field field in a diversein in a a diverse diverse range range range of applications, of of applications, applications, namely: namely: namely: strand strand strand ligation ligation ligation [80,141 [80,141] [80,141]] and cross-linkingandand cross-linking cross-linking using using using CuAAC CuAAC CuAAC [188] [188] or[188] even or or receivingeveneven receiving receiving stable stable cyclicstable cyclic DNAcyclic DNA miniduplexesDNA miniduplexes miniduplexes [149]. However,[149].[149]. However,However, the above-described thethe above-describedabove-described experiment experimeexperime encouragedntnt encouragedencouraged them them tothem carry toto carry outcarry further outout furtherfurther and moreandand more more detailed detailed detailed research research research on the on on applicationthe the application application of artiﬁcial of of artificial artificial DNA DNA DNA and and and RNA RNA RNA linkages linkages linkages in livingin in liv- liv- systemsinging systemssystems [143 ]. [143].[143]. So, within So,So, withinwithin 10 years, 1010 years,years, they they explainedthey explainedexplained the correlation thethe correlationcorrelation between betweenbetween structure structurestructure and Biomedicines 2021, 9, 628 13 of 34

Biomedicines 2021, 9, 628 13 of 35

and dynamics [189–191]; proved the biocompatibility of the artificial linker and the fact it dynamicscould mimic [189 the–191 DNA]; proved phosphodiester the biocompatibility group [144]; of showed the artificial efficient linker RNA and synthesis the fact by it a couldtriazole-modified mimic the DNA DNA phosphodiester template [145]; group synthesized [144]; showed hairpin efficientand hammerhead RNA synthesis ribozymes by a triazole-modifiedwith the triazole linkage DNA template [192]; obtained [145]; synthesized a click-linked hairpin gene andthat hammerheadwas functional ribozymes in human withcells the[193], triazole and thus linkage confirmed [192]; obtained that the aimportance click-linked of gene epigenetically that was functional modified ingenes human and cellsgenomes [193], increases and thus [116]. confirmed Furthermore, that the they importance synthesized of epigenetically some cyclic single-stranded modified genes DNA and genomesconstructs increases containing [116 a]. single Furthermore, triazole, theyand synthesizedused them as some templates cyclic for single-stranded RCA reaction DNA (syn- constructsthesis of the containing cyclic oligonucleotide) a single triazole, [194]. and It is used astonishing them as in templates the case of for triazole-modified RCA reaction (synthesisoligodeoxynucleotides, of the cyclic oligonucleotide)that their influence [194 on]. thermodynamic It is astonishing destabilization in the case of of triazole- the du- modifiedplexes, despite oligodeoxynucleotides, the artificial linkage that (that their is influence produced on by thermodynamic click ligation) destabilizationis read through ofcorrectly the duplexes, and efficiently despite theby artificialDNA polymerases, linkage (that and is producedis functional by in click vivo ligation). Moreover, is read the throughresultant correctly synthetic and ribozyme efficiently with by DNAa triazole polymerases, backbone and mimic is functional at the catalyticin vivo site. Moreover, saves its thecatalytic resultant activity. synthetic This ribozyme is also the with first a triazoledemonstration backbone of mimictranscription at the catalytic through site a heavily saves itsmodified catalytic DNA activity. backbone This is linkage also the and first suggestion demonstration that ofclick-ligated transcription DNA through could abe heavily useful modifiedfor the direct DNA synthesis backbone oflinkage biologically and suggestionactive RNA that and click-ligated proteins. Careful DNA design could behas useful given forrise the to directbiocompatible synthesis triazole of biologically linkage active that is RNA treated and as proteins. a normal Careful one by design DNA hasand given RNA risepolymerases, to biocompatible and the triazolegene containing linkage that triazole is treated linkages as a has normal been oneshown by DNAto function and RNA in E. polymerases,coli. This is how and these the gene scientists containing have pioneered triazole linkages a chemical has been method shown of linking to function DNA in strandsE. coli. Thisthat isis howtolerated these by scientists living systems have pioneered [144]. The a artificial chemical DNA method linkage of linking was developed DNA strands using thatclick is chemistry, tolerated bya highly living efficient systems chemical [144]. The re artificialaction, to DNA join DNA linkage strands was developed together without using clickdisrupting chemistry, the genetic a highly code. efficient It means chemical long reaction, DNA can to be join created DNA strandsquickly togetherand efficiently without by disruptingchemical methods. the genetic The code. genetic It means code longcould DNA still be can correctly be created read—this quickly andopens efficiently up all sorts by chemicalof possibilities. methods. It all The means, genetic that code the could fundam stillental be correctly genetic read—thispolymer of opens all living up allorganisms sorts of possibilities.on Earth, can It be all chemically means, that modified the fundamental to embrace genetic novel polymer functions of allthat living do not organisms exist in onna- Earth,ture. can be chemically modified to embrace novel functions that do not exist in nature. InIn 2012, 2012, Krishna Krishna and and Caruthers Caruthers had had developed developed the the 1,2,3-triazolylphosphonate 1,2,3-triazolylphosphonate inter-inter- nucleotidenucleotide (TP)(TP) linkage,linkage, in which which a a 1,2,3-triazole 1,2,3-triazole moiety moiety is is bonded bonded via via the the C4 C4 atom atom to the to thephosphonate phosphonate system system [195] [195 (Scheme] (Scheme 10). 10 The). Theresu resultinglting TP linkages TP linkages 18 are 18 formed are formed in a intwo- a two-stepstep process. process. In the In the first first step, step, an an intern internucleotideucleotide linkage linkage containing containing an alkyne groupgroup17 17 waswas introduced introduced using using the the classic classic phosphoramidite phosphoramidite method. method. Such Such an an alkynylphosphonate alkynylphosphonate clickableclickablebackbone backbone hashas versatile versatile possibilitiespossibilities ofof biological biological applications.applications. InIn thethe secondsecond step,step, thethe CuAAC CuAAC reactionreaction withwith selected selected azidesazides waswas used used toto form form a a triazole triazole system, system, before before the the synthesizedsynthesized oligonucleotideoligonucleotide was detached detached from from the the solid solid support. support. In this In this way, way, it was it was pos- possiblesible to toobtain obtain oligonucleotide oligonucleotide chimeras chimeras built built from from 16 16 to to 23 23 mers mers with various modifica-modifications. Further studies allowed to state that such modified systems were resistant to 50-and tions.0 Further studies allowed to state that such modified systems were resistant to 5′-and 33-exonucleoases,′-exonucleoases, and and the the stability stability of of duplexes duplexes with with RNA RNA did did notnot changechange significantly.significantly. The The resultsresults of of these these experiments experiments may may encourage encourage to to continue continue the the investigation investigation of of TP TP ODNs ODNs as as potentialpotential antagomirs antagomirs for for targeting targeting microRNA microRNA expression. expression.

SchemeScheme 10. 10.Alkynyl Alkynyl phosphonatephosphonate DNA: DNA: a a versatile versatile clickable clickable backbone backbone for for biological biological applications. applications.

2.2.2.2. (TL)RNA(TL)RNA SkeletonSkeleton TheThe “RNA“RNA World”World” is is various various and and interesting interesting because because of of diversiﬁed diversified structures structures and and functionsfunctions ofof ribonucleicribonucleic acidsacids [196[196].]. InIn nature,nature, RNARNA appearedappeared earlierearlier thanthan DNADNA [[197].197]. Because the DNA molecule has a more difﬁcult accessibility in the cell, it causes many Biomedicines 2021, 9, 628 14 of 35

problems in gene therapy. Therefore, scientists rather use mRNA—a smaller and simpler molecule that is possible to prepare in the laboratory [198,199]. This is the medium of genetic code and acts as a pattern in protein biosynthesis. The short lifetime of mRNA, also produced for therapeutic purposes, limits its use due to enzymatic degradation in the cell. The scientists would like mRNA used in medicinal preparations to “live” longer than its natural counterpart. Of course, the biochemical and therapeutic contribution of compounds classified into the RNA family is enormous. Next to the mRNA group, a huge group of ncR- NAs is present [200–205]. In particular, they are involved in drug development [206–210] and cancer therapy [211–213]. They can also act as valuable therapeutic tools in gene regulation, e.g., siRNA [214], circRNA [215], miRNA [216], where triazole-modified nucleic acid elements are present in almost all described cases. In turn, the internal RNA modifications can also be achieved by the functionality transfer reaction (FTR), followed by click chemistry with a variety of clickable molecules [217]. The benefits of this method have been demonstrated by its specificity, rapidity, broad applicability, and procedure simplicity. Additionally, oligonucleotides with propargylated nucleoside as clickable moieties and a bifunctional azide have been synthesized by the CuAAC reaction [218]. This strategy has been used to obtain cross-linking in DNA–DNA and DNA–RNA duplexes for study of their stability and hybridization properties. This procedure shows how to receive 20-O-propargyl-2-(amino)adenosine phosphoramidite to the formation of homodimers and heterodimers while pointing to significant stability for duplexes with terminal ligation. Using click chemistry, small molecules are applied for creating new RNA conjugates [219–221], for example, alkyne-modified ATP [222] or SAM-adenosine conjugates [223]. Other, less frequent uses of triazolo-modified RNAs are applied to searching for chemical probes in nucleic acid chemistry [224,225] and in physicochemical methods like EPR spectroscopy [226] and cat-ELCCA assays [227] in biomedical sciences. Catalytic RNAs, named ribozymes, can catalyze cleavage or ligation of phosphodiester bonds, and many other reactions in all life forms [15]. Since nucleic acid catalysts (and aptamers) can perform additional reactions, including solutions to problems not encountered in nature, scientists have started to fathom their options. New ribozymes are being identified through a method called SELEX and can be used as therapeutics to block functions of their targets in vivo, or as diagnostic sensors to detect them in vitro. Here, the click chemistry approach comes to aid in exploring the RNA chemistry and biochemistry. An avenue to the development of new catalysts have been presented by El-Sagheer and Brown [228]. They described a strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes and DNA–RNA chimeras, too (Scheme 11). The CuAAC reaction was used to produce catalytically active hairpin and hammerhead ribozymes containing a novel nucleic acid backbone mimic at the catalytic site, which exhibit activity against target molecules. In the same study, the constructed DNA–RNA hybrid duplexes had the same conformation and stability as their natural counterparts. These properties gave evidence for the potential compatibility of triazole- modified oligoribonucleotides with natural nucleic acids. It proved the ability to synthesize long RNA strands by a combination of solid-phase support and click ligation, resulting in the synthesis of many biologically important RNA molecules. A year later, the same authors presented an efficient RNA synthesis by in vitro transcription of a triazole-modified DNA template [145]. As it turned out, click chemistry has enabled the large DNA strands to produce biologically active RNA and proteins. The modified nucleic acid containing triazole linkage is correctly read-through by DNA polymerases and is functional in bacteria [144]. This work shows that T7-RNAP enzyme (T7 RNA polymerase) can transcribe through modified DNA strands to synthesize RNA, which is evidence for the surprising biocompatibility of TLDNA. And this is a first example of transcription through a fully synthetic analogue of a DNA backbone. Therefore, click ligation is a strategy for overcoming the size limit of DNA and RNA obtained by solid-phase phosphoramidite chemistry (without the use of enzymes), and DNA constructs can be used directly to synthesize biologically active RNA constructs and proteins. Biomedicines 2021, 9, 628 15 of 35 Biomedicines 2021, 9, 628 15 of 34

SchemeScheme 11. 11.DNA–RNA DNA–RNA hybrids hybrids obtained obtained in in CuAAC CuAAC reaction. reaction.

ChemicalA year later, synthesis the same of RNAauthors is less presented efficient an than efficient DNA RNA owing synthesis to problems by in caused vitro tran- by thescription presence of ofa triazole-modified the 20-hydroxyl group DNA of template ribose, which[145]. As requires it turned selective out, click protection chemistry during has oligonucleotideenabled the large assembly. DNA strands This reduces to produce the coupling biologically efficiency active of RNA phosphoramiditeand proteins. The monomersmodified nucleic due to acid steric containing hindrance triazole and side-reactions linkage is correctly that occur read-through during the by removal DNA poly- (or 0 0 0 prematuremerases and loss) is offunctional the 2 -protecting in bacteria groups, [144]. and This 3 towork 2 phosphate shows that migration. T7-RNAP Additionally, enzyme (T7 theRNA solid-phase polymerase) RNA can synthesis transcribe dictates through the sizemodified of constructs—about DNA strands to 50 synthesize nucleotides RNA, in length—andwhich is evidence longer for ones the remainsurprising difficult biocompa to prepare.tibility of Here, TLDNA. it is And worth this noting is a first that example most biologicallyof transcription important through RNA a fully molecules synthetic like an ribozymes,alogue of a aptamers,DNA backbone. and riboswitches Therefore, click are significantlyligation is a longer.strategy Hence, for overcoming a huge need the for size new limit approaches of DNA toand the RNA synthesis obtained of RNA. by solid- The researchphase phosphoramidite group of Micura chemistry has reported (without a versatile the use tool of enzymes), for oligoribonucleotide and DNA constructs synthesis, can 0 whichbe used consists directly in to the synthesize introduction biologic of clickableally active 2 -azidoRNA constructs derivatives and of proteins. all four canonical nucleosidesChemical as buildingsynthesis blocksof RNA into is less RNA efficient [229,230 than] (Scheme DNA owing 12). Into problems contrast to caused nucleo- by 0 0 0 sidesthe presence modified of atthe the 2′-hydroxyl 3 and 5 positions,group of ribose, compounds which requires modified selective at the 2 protectionposition can during be incorporatedoligonucleotide at anyassembly. position This of thereduces synthetic the co oligonucleotides.upling efficiency of The RNA only phosphoramidite problem is the incompatibilitymonomers due ofto azidessteric hindrance in the phosphoramidite and side-reactions technique that occur (Staudinger during reaction)the removal [231 (or]. Therefore,premature in loss) most of cases the 2 of′-protecting oligonucleotide groups, modification and 3′ to 2′ withphosphate CuAAC, migrat nucleosidesion. Addition- con- tainingally, the an solid-phase alkyne substituent RNA synthesis are introduced dictates into the the size chain. of constructs—about However, the diester 50 nucleotides19 can be incorporatedin length—and into longer the formed ones remain oligonucleotide difficult to strand prepare. by Here, the triester it is worth P(V) noting technique that with most thebiologically help of the important MSNT activator RNA molecules [232]. The like continuation ribozymes, of theaptamers, automated and synthesis riboswitches did not are influence the azido group and the resulting siRNA can be labeled with fluorine. As the significantly longer. Hence, a huge need for new approaches to the synthesis of RNA. The authors claim, this work is addressed to siRNA applications and bioconjugation, where research group of Micura has reported a versatile tool for oligoribonucleotide synthesis, the 20-N group is well accepted in the guide strand (antisense) and causes an increased which consists3 in the introduction of clickable 2′-azido derivatives of all four canonical resistance to nucleases. The rare and exceptional 20-azido modifications are compatible nucleosides as building blocks into RNA [229,230] (Scheme 12). In contrast to nucleosides with 20-fluoro and/or 20-O-methyl modifications to achieve siRNAs of rich modification Biomedicines 2021, 9, 628 modified at the 3′ and 5′ positions, compounds modified at the 2′ position can be incorpo-16 of 34 patterns and tunable properties. rated at any position of the synthetic oligonucleotides. The only problem is the incompat- ibility of azides in the phosphoramidite technique (Staudinger reaction) [231]. Therefore, in most cases of oligonucleotide modification with CuAAC, nucleosides containing an alkyne substituent are introduced into the chain. However, the diester 19 can be incorporated into the formed oligonucleotide strand by the triester P(V) technique with the help of the MSNT activator [232]. The continuation of the automated synthesis did not influence the azido group and the resulting siRNA can be labeled with fluorine. As the authors claim, this work is addressed to siRNA applications and bioconjugation, where the 2′-N3 group is well accepted in the guide strand (antisense) and causes an increased resistance to nucleases. The rare and exceptional 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and SchemeScheme 12.12.RNA RNA synthesis synthesis using using phosphoramidite phosphoramidite chemistry chemistr iny in the the presence presence of freeof free azide azide moiety moiety at tunable properties. C2’at C2’ position position and and structure structure ofMSNT. of MSNT.

TheThe synthesissynthesis and properties of of triazoletriazole-linkedlinked RNA RNA have have been been the the subject subject of ofexplo- ex- plorationsrations undertaken undertaken by research by research groups groups of Ro ofzners Rozners [233] and [233 ]Isobe and [234,235]. Isobe [234 The,235 first]. The one ﬁrstmade one efforts made towards efforts towardsthe preparation the preparation TLRNA byTL couplingRNA by coupling3′-O-propargyl 30-O-propargyl and 5′-azidori- and 5bonucleosides0-azidoribonucleosides followedfollowed by their by study their using study usingspectroscopic spectroscopic techniques techniques (Scheme(Scheme 13). 13 To). evaluate the effect of triazole linkage on thermal stability of RNA, they synthesized several self-complementary sequences. The UV thermal melting analysis, CD, and NMR spec- tra showed that the triazole linkage strongly destabilized RNA double helix by about 7– 14 °C depending on the sequence and location of the modifications. So, the triazole moiety was not a good mimic of the phosphate linkage in RNA. In contrast, Isobe showed how long triazole linkage stabilized DNA [179,184]. On the other hand, RNA tolerates better the triazole linkage than DNA, despite being two atoms longer than the phosphate [228]. The second group of scientists, after their successful work on duplex-forming TLDNA, received triazole-linked trinucleotide analogues of RNA [234]. As it turned out, the synthesis of ribonucleoside analogues was more robust than in the case of deoxyribonucleoside analogues, which then underwent the elongation reaction via solid-phase synthesis using CuAAC reaction. The conditions to receive 3-mer TLRNA were inappropriate for longer oligonucleotides, therefore the authors re-optimized them for 11-mer TLRNA. The triazole- modified RNA was designed to explore the duplex formation, which was found to be inferior to the natural RNA oligonucleotide (in forming duplexes) [235].

Scheme 13. Synthesis of triazole-linked r(UTRA) dimer.

TLDNA has been demonstrated to be a competent enzymatic substrate, but the biologically non degradable linkage of TLRNA may provide an intriguing opportunity to explore the sophisticated function of RNA, for instance, RNA interference, that requires the enzymatic recognition of the oligonucleotides [234]. The design and synthesis of such constructs will certainly be investigated in the near future. In bioconjugation chemistry the CuAAC allows for rapid labeling and ligation of RNA [236,237]. Once clickable groups are installed on RNA with free 2′-hydroxyl groups, they can be rapidly click labeled or conjugated together for biological studies. For example, triazole-linked lipid-oligonucleotide conjugates have been shown to be nontoxic, and this displays a broad applicability of click chemistry to any synthetic or natural RNA [64]. This strategy enables the capture dynamic aspects of RNA metabolism in living systems [237] and to study of the epitranscriptome marks such as new detection methods for m6A or mechanism-based trapping of an inosine-generating ADAR enzyme [238]. Biomedicines 2021, 9, 628 16 of 34

Scheme 12. RNA synthesis using phosphoramidite chemistry in the presence of free azide moiety at C2’ position and structure of MSNT.

Biomedicines 2021, 9, 628 The synthesis and properties of triazole-linked RNA have been the subject of 16explo- of 35 rations undertaken by research groups of Rozners [233] and Isobe [234,235]. The first one made efforts towards the preparation TLRNA by coupling 3′-O-propargyl and 5′-azidoribonucleosides followed by their study using spectroscopic techniques (Scheme 13). To Toevaluate evaluate the the effect effect of triazole of triazole linkage linkage on thermal on thermal stability stability of RNA, of RNA, they they synthesized synthesized sev- severaleral self-complementary self-complementary sequences. sequences. The TheUV thermal UV thermal melting melting analysis, analysis, CD, and CD, NMR and NMR spec- spectratra showed showed that that the thetriazole triazole linkage linkage strongly strongly destabilized destabilized RNA RNA double double helix helix by byabout about 7– ◦ 7–1414 °C Cdepending depending on on the the sequence sequence and and location location of ofthe the modifications. modiﬁcations. So, So, the the triazole triazole moiety moi- etywas was not not a good a good mimic mimic of ofthe the phosphate phosphate linkage linkage in in RNA. RNA. In In contrast, contrast, Isobe showed howhow longlong triazoletriazole linkagelinkage stabilizedstabilized DNADNA [[179,184].179,184]. OnOn thethe otherother hand,hand, RNARNA toleratestolerates betterbetter thethe triazoletriazole linkagelinkage thanthan DNA,DNA, despitedespite beingbeing twotwo atomsatoms longerlonger thanthan thethe phosphatephosphate [[228].228]. TL TheThe secondsecond group of scientists, scientists, after after their their successful successful work work on on duplex-forming duplex-forming TLDNA,DNA, re- receivedceived triazole-linked triazole-linked trinucleotide trinucleotide analogues analogues of of RNA RNA [234]. [234]. As As it it turned turned out, the synthe- sissis ofof ribonucleosideribonucleoside analoguesanalogues waswas moremore robustrobust thanthan inin thethe casecase ofof deoxyribonucleosidedeoxyribonucleoside analogues,analogues, whichwhich thenthen underwentunderwent thethe elongationelongation reactionreaction viavia solid-phasesolid-phase synthesissynthesis usingusing TL CuAAC reaction. The conditions to receive 3-mer TLRNA were inappropriate for longer CuAAC reaction. The conditions to receive 3-mer RNA were inappropriateTL for longer oligonucleotides,oligonucleotides, thereforetherefore the the authorsauthors re-optimized re-optimized them them for for 11-mer 11-mer TLRNA. The triazole- modiﬁed RNA was designed to explore the duplex formation, which was found to be modified RNA was designed to explore the duplex formation, which was found to be inferior to the natural RNA oligonucleotide (in forming duplexes) [235]. inferior to the natural RNA oligonucleotide (in forming duplexes) [235].

Scheme 13. Synthesis of triazole-linked r(UTRA) dimer. Scheme 13. Synthesis of triazole-linked r(UTRA) dimer.

TLTLDNA has been demonstrated demonstrated to to be be a a comp competentetent enzymatic enzymatic substrate, substrate, but but the the bio- bi- ologicallylogically non non degradable degradable linkage linkage of of TLTLRNARNA may may provide provide an anintriguing intriguing opportunity opportunity to ex- to exploreplore the the sophisticated sophisticated function function of ofRNA, RNA, for for instance, instance, RNA RNA interference, interference, that that requires requires the theenzymatic enzymatic recognition recognition of the of theoligonucleotides oligonucleotides [234]. [234 The]. Thedesign design and andsynthesis synthesis of such of such con- constructsstructs will will certainly certainly be inve be investigatedstigated in the inthe near near future. future. InIn bioconjugationbioconjugation chemistrychemistry thethe CuAACCuAAC allowsallows forfor rapidrapid labelinglabeling andand ligationligation ofof RNARNA [[236,237].236,237]. OnceOnce clickableclickable groupsgroups areare installedinstalled onon RNARNA withwith freefree 220′-hydroxyl-hydroxyl groups,groups, theythey cancan be be rapidly rapidly click click labeled labeled or or conjugated conjugated together together for for biological biological studies. studies. For For example, exam- triazole-linkedple, triazole-linked lipid-oligonucleotide lipid-oligonucleotide conjugates conjugates have have been been shown shown to be to nontoxic, be nontoxic, and thisand displaysthis displays a broad a broad applicability applicability of click of click chemistry chemistry to any to synthetic any synthetic or natural or natural RNA RNA [64]. This[64]. strategyThis strategy enables enables the capture the capture dynamic dynamic aspects aspects of RNA of metabolismRNA metabolism in living in living systems systems [237] and[237] to and study to study of the of epitranscriptome the epitranscriptome marks marks such such as new as new detection detection methods methods for mfor6A m or6A mechanism-basedor mechanism-based trapping trapping of anof inosine-generatingan inosine-generating ADAR ADAR enzyme enzyme [238 [238].]. There are two major tools of biological significance in the current RNA research, namely RNA interference (RNAi) and RNA bioconjugation. RNAi is a post-transcriptional gene silencing mechanism induced by siRNA and miRNA. Here, the structural and functional repertoire of RNAs can be significantly manipulated by chemical modifications. There are high expectations for the development of siRNA and miRNA as therapeutics agents to treat diseases [229,239]. However, for applications of siRNA as therapeutic agents, chemical modification is obligatory to enhance nuclease resistance, to prevent immune activation, to decrease off-target effects, and to improve pharmacokinetic and pharmacody- namic properties. Additionally, the modifications should help these agents to penetrate cell membranes and improve siRNA delivery, and this remains one of the major challenges. Short-interfering RNAs containing various and functional spacers at the central region of the sense strand are biocompatible and suitable gene silencing substrates [240]. For example, placing cholesterol may offer a new method to be used in delivery system. The use of new functional groups and small molecules in this place allows for the creation of a new diverse class of biofunctionally active modified siRNAs, e.g., cell-targeting molecules. This has enormous potential for modern medicine, and some candidate siRNAs are in Biomedicines 2021, 9, 628 17 of 34

There are two major tools of biological significance in the current RNA research, namely RNA interference (RNAi) and RNA bioconjugation. RNAi is a post-transcriptional gene silencing mechanism induced by siRNA and miRNA. Here, the structural and functional repertoire of RNAs can be significantly manipulated by chemical modifications. There are high expectations for the development of siRNA and miRNA as therapeutics agents to treat diseases [229,239]. However, for applications of siRNA as therapeutic agents, chemical modification is obligatory to enhance nuclease resistance, to prevent immune activation, to decrease off-target effects, and to improve pharmacokinetic and phar- macodynamic properties. Additionally, the modifications should help these agents to penetrate cell membranes and improve siRNA delivery, and this remains one of the major challenges. Short-interfering RNAs containing various and functional spacers at the central region of the sense strand are biocompatible and suitable gene silencing substrates [240]. Biomedicines 2021, 9, 628 For example, placing cholesterol may offer a new method to be used in delivery system.17 of 35 The use of new functional groups and small molecules in this place allows for the creation of a new diverse class of biofunctionally active modified siRNAs, e.g., cell-targeting molecules. This has enormous potential for modern medicine, and some candidate siRNAs clinicalare in clinical trials. Astrials. stated As stated above, above, chemical chemic modificational modification of siRNAs of siRNAs is necessary is necessary to improve to im- theirprove pharmacokinetic their pharmacokinetic properties. properties. One of One such of methodssuch methods is to generateis to generate modified modified siRNAs siR- thatNAs contain that contain triazole triazole backbone backbone linkages. linkages. This study This is study undertaken is undertaken by the research by the groupresearch of Desaulniers,group of Desaulniers, namely new namely siRNA new design siRNA and desi thengn the and evaluation then the of evaluation their gene-silencing of their gene- po- tentialsilencing [241 potential,242]. Utilizing [241,242]. the Utilizing well-known the we CuAACll-known reaction, CuAAC the reaction, triazole backbone the triazole dimers back- (UbonetU anddimers CtU) (U basedtU and onCtU) a PNA-likebased on a structure PNA-like were structure synthesized were synthesized and incorporated and incorpo- into variousrated into siRNAs various targeting siRNAs representative targeting representa gene transcriptstive gene of transcripts an exogenous of an and exogenous endogenous and geneendogenous (Scheme gene 14). The(Scheme triazole-modified 14). The triazole-mod siRNAs wereified found siRNAs to bewere capable found of to gene be capable silencing of ofgene reporter silencing genes of and reporter thus proved genes theand compatibility thus proved asthe substrates compatibility within as the substrates RNAi pathway. within Thethe modificationsRNAi pathway. were The designed modifications to impart were potentially designed favorable to impart properties potentially to siRNAs favorable in orderproperties to target to siRNAs endogenous in order genes, to target for instance endogenous the oncogene genes, forBCL2 instance. This the is the oncogene first example BCL2. whereThis is a the triazole first inexample place of where a phosphodiester a triazole in backbone place of showsa phosphodiester compatibility backbone withinsiRNA shows duplexes and mimics the silencing of gene expression when positioned within the internal compatibility within siRNA duplexes and mimics the silencing of gene expression when Watson–Crick double-stranded region of siRNAs. positioned within the internal Watson–Crick double-stranded region of siRNAs.

Scheme 14. Synthesis of triazole-linked CTRU dimer based on a PNA-type scaffold. Scheme 14. Synthesis of triazole-linked CTRU dimer based on a PNA-type scaffold.

Short-interferingShort-interfering RNAsRNAs (siRNAs)(siRNAs) areare naturallynaturally occurringoccurring biomoleculesbiomolecules usedused forfor post-post- transcriptionaltranscriptional genegene regulation,regulation, soso theythey cancan bebe promisingpromising therapeutictherapeutic agentsagents byby silencingsilencing genegene expressionexpression ofof overexpressedoverexpressed deleteriousdeleterious genes. genes. siRNAssiRNAs oligonucleotidesoligonucleotides containingcontaining commerciallycommercially availableavailable 220-modifications′-modifications (2(20′-OMe-OMe oror 220′-F)-F) cancan bebe combinedcombined withwith aa triazole-triazole- linkedlinked backbone backbone modification modification in thein the siRNA siRN designA design and theseand siRNAsthese siRNAs are amendable are amendable within thewithin RNAi the pathway. RNAi pathway. The chemically The chemically modified modified siRNAs enhancesiRNAs gene-silencingenhance gene-silencing activity and ac- biologicaltivity and stabilitybiological with stability high nuclease with high resistance, nuclease and resistance, keep a similarand keep immune a similar response immune as unmodifiedresponse as siRNAs.unmodified RNA siRNAs. interference RNA mightinterference be a promising might be alternative a promising to conventionalalternative to chemotherapyconventional chemotherapy of genetic disorders, of genetic cancer, disord viralers, infections, cancer, viral and infections, other diseases and forother which dis- theeases target for which mRNA the can target be identified. mRNA can However, be identified. the short However, interfering the short RNAs interfering (siRNAs) RNAs need to(siRNAs) be chemically need to modified be chemically to be efficient modified therapeutic to be efficient agents, therapeutic in order to agents, improve in nuclease order to resistance,improve nuclease crossing resistance, of cell membranes, crossing of biodistribution, cell membranes, and biodistribution, pharmacokinetics. and pharmacokinetics.Chemically modified mRNA cap structures show very promising anti-cancer properties and therefore, are being extensively studied as a target for drug discovery. A dinucleotide mRNA cap analogue obtained via click chemistry is a focus of attention for different research groups around the world. All these efforts are directed towards the improvement of intracellular delivery m7G caps mRNA through a cell membrane by the conjugation with cell-penetrating peptides [243], the introduction of a propargylated moiety to form a stable complex with initiation factor eIF4E [244] or for labeling strategy [245] (Figure8). Large RNAs, such as long non-coding RNAs or mRNAs, have exciting potential as gene therapy drugs or vaccine antigens, and for such molecules post-transcriptional chemical modification will be crucial. Moreover, breakthrough technology to isolate exclu- sive capped mRNA opens new possibilities in the area of personalized medicine. For years, advanced research on therapeutic mRNA with a modified cap has also been conducted by Darzynkiewicz and Jemielity [246]. The modified 50-ends of all mRNAs have been used for visualizing mRNAs in cells by fluorescent labeling, as small-molecule inhibitors of translation and/or in cancer immunotherapy and gene therapy [247,248]. The novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain are more durable and act with efficiency similar to their natural counterparts. Biomedicines 2021, 9, 628 18 of 34

Chemically modified mRNA cap structures show very promising anti-cancer properties and therefore, are being extensively studied as a target for drug discovery. A dinucleotide mRNA cap analogue obtained via click chemistry is a focus of attention for different research groups around the world. All these efforts are directed towards the improvement of intracellular delivery m7G caps mRNA through a cell membrane by the conjugation with cell-penetrating peptides [243], the introduction of a propargylated moiety to form a stable complex with initiation factor eIF4E [244] or for labeling strategy [245] (Figure 8). Large RNAs, such as long non-coding RNAs or mRNAs, have exciting potential as gene therapy drugs or vaccine antigens, and for such molecules post-transcriptional chemical modification will be crucial. Moreover, breakthrough technology to isolate ex- clusive capped mRNA opens new possibilities in the area of personalized medicine. For years, advanced research on therapeutic mRNA with a modified cap has also been conducted by Darzynkiewicz and Jemielity [246]. The modified 5′-ends of all mRNAs have been used for visualizing mRNAs in cells by fluorescent labeling, as small-molecule in- Biomedicines 2021, 9, 628 hibitors of translation and/or in cancer immunotherapy and gene therapy [247,248].18 of The 35 novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain are more durable and act with efficiency similar to their natural counterparts.

FigureFigure 8. 8.mRNA mRNA m m77GG 55’-cap0-cap clickableclickable analogues.analogues.

TheThe Nobel Nobel Prize Prize in in Chemistry Chemistry 2020 2020 has has been been awarded awarded for for genetic genetic scissors—a scissors—a tool tool for for rewritingrewriting the the code code of of life. life. It It is is talking talking about about the the gene gene technology’s technology’s sharpest sharpest tools, tools, namely namely thethe CRISPR/Cas9 CRISPR/Cas9 genomegenome editingediting method,method, thatthatcan can change change the the DNA DNA with with extremely extremely high high precisionprecision [[249].249]. This This technology technology has has revolutionized revolutionized the the molecular molecular biology—it biology—it creates creates new newpossibilities possibilities for plant for plant breeding breeding or is orcontributing is contributing to new to innovative new innovative therapies therapies for cancer for cancerand inherited and inherited diseases diseases [250]. Additionally, [250]. Additionally, in this case, in this scientists case, scientists are looking are looking for new for so- newlutions solutions using usingclick chemistry click chemistry approach approach [84] (Scheme [84] (Scheme 15). CRISPR/Cas915). CRISPR/Cas9 system system is using is usingguide guide RNAs RNAs (sgRNAs) (sgRNAs) to target to target catalytically catalytically inactive inactive Cas9 Cas9 (dCas9) (dCas9) [81,83]. [81,83 It]. gives It gives the theopportunities opportunities to study to study pervasive pervasive antisense antisense transcription, transcription, namely namely the use the of use CuAAC of CuAAC reac- reactiontion to construct to construct DNA DNA templates templates for forsgRNA sgRNA expression, expression, thus thus generating generating novel novel sense sense and andantisense antisense transcripts. transcripts. All this All evidence this evidence confirms confirms that stable that stable artificial artificial triazole triazole DNA DNAlinker linkeris biocompatible is biocompatible for PCR, for PCR, read read and and accura accuratelytely copied copied by by DNA DNA and and RNA polymerases.polymerases. Furthermore,Furthermore, click click chemistry chemistry is anis efficientan efficien methodt method for sgRNA for sgRNA template template construction construction which combinedwhich combined with dCas9 with allowsdCas9 forallows advanced for advanc geneed expression gene expression analysis. analysis. Another Another strategy strat- is so-calledegy is so-calledsplit-and-click split-and-clickapproach, approach, where clicked where sgRNAs clicked provide sgRNAs easier provide access easier to individual access to orindividual pools of modifiedor pools of sgRNAs modified (that sgRNAs are functional (that are functional in cells) [82 in]. cells) Click [82]. ligation Click of ligation the two of componentsthe two components generates generates an artificial an artificial triazole linkagetriazole that linkage is tolerated that is tolerated in functionally in functionally critical regionscritical ofregions the sgRNA of the and sgRNA allows and for efficientallows for DNA efficient cleavage DNAin vitrocleavageas well in asvitro gene-editing as well as in cells without no unexpected off-target effects. Importantly, the site-specific incorporation gene-editing in cells 0without no unexpected off-target effects. Importantly, the site-spe- Biomedicines 2021, 9, 628 ofcific nucleotides incorporation with of 2 nucleotides-sugar modification with 2′-sugar or bridged modification nucleic or acids bridged can nucleic enhance acids19 target of can 34 specificity and improve sgRNAs stability. enhance target specificity and improve sgRNAs stability.

SchemeScheme 15.15.The The mechanismmechanism ofof preparingpreparing sgRNAsgRNA by by click click chemistry. chemistry.

2.3.2.3. (TL)LNA(TL)LNA andand (TL)BNA(TL)BNA SkeletonSkeleton LockedLocked nucleicnucleic acidsacids (LNAs)(LNAs) areare widelywidely usedused inin RNARNA therapeuticstherapeutics as as LNA-modiﬁedLNA-modified antisenseantisense oligonucleotidesoligonucleotides [251]. [251]. From From the the structural structural point point of ofview, view, LNA LNA is an is anRNA RNA de- derivativerivative in inwhich which the the ribose ribose ring ring is isconstrained constrained by by a methylene a methylene linkage linkage between between the the 2′- 0 0 2oxygen-oxygen and and the the 4 4′-carbon-carbon [252,253]. [252,253]. This This conformation conformationalal restriction causes anan increasedincreased bindingbinding afﬁnityaffinity toto complementarycomplementary DNADNA andand RNARNA sequences,sequences, butbut thethe susceptibilitysusceptibility toto RNaseRNase HH shouldshould bebe optimizedoptimized forfor individual individual applications. applications. ThisThis providesprovides aa new new chemical chemical approachapproach forfor the control of of gene gene expression, expression, an an important important consideration consideration for for antisense antisense ap- applications.plications. RNase RNase H H is is an an enzyme enzyme that that normally normally cleaves cleaves the RNA strandstrand ofof RNA–DNARNA–DNA hybrids formed during lagging strand synthesis. Campbell and Wengel have found that oligonucleotides containing only LNA units form hybrids with RNA that activate RNase H to a lower degree than corresponding DNA oligonucleotides [253]. LNA units are linked by the same phosphate linkage as in DNA and RNA, allowing for synthesis of LNA oligomers using standard reagents and automated synthesizers. To be effective inside the cells or in cell extracts, antisense oligomers must be resistant to digestion by nucleases. Their therapeutic potential lies in hybridization with sequestering miRNAs—an important class of regulatory ncRNAs that can bind to partially complementary sites located in the 3′ untranslated regions (UTRs) of target mRNAs. That is why one of the most promising modifications of antisense and/or antigen oligonucleotides is the introduction of triazole or triazole-bridged nucleic acids into LNAs [254,255]. Chemical synthesis of triazole(bridged) structures also requires the intramolecular Huisgen 1,3-dipolar cycloaddition involving the ethynyl group and the azide group. It is worth emphasizing that bridged nucleic acids (BNAs) are attractive construction placed beyond LNAs and peptide nucleic acids (PNAs), with high binding affinity to ssRNA and/or dsDAN [256,257]. Novel attributes of LNAs and other classes of bridged nucleic acid analogues are extensively explored in different synthetic oligonucleotide-based therapeutics, so straightforward synthesis of their monomers could be very useful [254,255,258–261] (Figure 9).

Figure 9. Examples of clickable monomers for LNAs and BNAs synthesis.

First attempts towards design and synthesis of LNA-based nucleoside dimers using CuAAC reaction were done in the laboratory of Prasad [262,263] (Scheme 16). In total, they received three triazole-linked LNA dimers of type TL–t– TL, TL–t– ABzL, and TL–t– CBzL. In the next step, they obtained three triazole-linked xylonucleoside dimers of type TL–t– TxL, TL–t– ABzxL, and TL–t– CBzxL. It is noteworthy to point out the effort they made in order to stich two unique nucleoside scaffolds together through a triazole ring. The optimized method can easily be adopted for automated synthesis of triazole-linked LNA-based Biomedicines 2021, 9, 628 19 of 34

Scheme 15. The mechanism of preparing sgRNA by click chemistry.

2.3. (TL)LNA and (TL)BNA Skeleton Locked nucleic acids (LNAs) are widely used in RNA therapeutics as LNA-modified antisense oligonucleotides [251]. From the structural point of view, LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2′- oxygen and the 4′-carbon [252,253]. This conformational restriction causes an increased Biomedicines 2021, 9, 628 binding affinity to complementary DNA and RNA sequences, but the susceptibility19 of 35to RNase H should be optimized for individual applications. This provides a new chemical approach for the control of gene expression, an important consideration for antisense applications. RNase H is an enzyme that normally cleaves the RNA strand of RNA–DNA hybridshybrids formedformed duringduring lagginglagging strandstrand synthesis.synthesis. CampbellCampbell andand WengelWengel havehave foundfound thatthat oligonucleotidesoligonucleotides containingcontaining onlyonly LNALNA unitsunits formform hybridshybrids withwith RNARNA thatthat activateactivate RNaseRNase HH toto aa lowerlower degreedegree thanthan correspondingcorresponding DNADNA oligonucleotides oligonucleotides [253[253].]. LNALNA unitsunits areare linkedlinked byby thethe samesame phosphatephosphate linkage linkage as as inin DNADNA andand RNA,RNA, allowingallowing forfor synthesissynthesis of of LNALNA oligomersoligomers usingusing standardstandard reagentsreagents andand automatedautomated synthesizers.synthesizers. ToTo be be effectiveeffective inside inside the the cellscells oror inin cellcell extracts,extracts, antisenseantisense oligomersoligomers mustmust bebe resistantresistant toto digestiondigestion byby nucleases.nucleases. TheirTheir therapeutictherapeutic potential potential lies lies in hybridizationin hybridization with with sequestering sequestering miRNAs—an miRNAs—an important im- class of regulatory ncRNAs that can bind to partially complementary sites located in the portant0 class of regulatory ncRNAs that can bind to partially complementary sites located 3inuntranslated the 3′ untranslated regions regions(UTRs) (UTRs) of target of target mRNAs. mRNAs. That isThat why is onewhy of one the of most the most promising prom- modiﬁcationsising modifications of antisense of antisense and/or and/or antigen antigen oligonucleotides oligonucleotides is the introduction is the introduction of triazole of tri- or triazole-bridgedazole or triazole-bridged nucleic acids nucleic into LNAsacids [into254 ,255LNAs]. Chemical [254,255]. synthesis Chemical of triazole(bridged)synthesis of tria- structureszole(bridged) also structures requires the also intramolecular requires the intramolecular Huisgen 1,3-dipolar Huisgen cycloaddition 1,3-dipolar involvingcycloaddi- the ethynyl group and the azide group. It is worth emphasizing that bridged nucleic tion involving the ethynyl group and the azide group. It is worth emphasizing that acids (BNAs) are attractive construction placed beyond LNAs and peptide nucleic acids bridged nucleic acids (BNAs) are attractive construction placed beyond LNAs and pep- (PNAs), with high binding afﬁnity to ssRNA and/or dsDAN [256,257]. Novel attributes tide nucleic acids (PNAs), with high binding affinity to ssRNA and/or dsDAN [256,257]. of LNAs and other classes of bridged nucleic acid analogues are extensively explored in Novel attributes of LNAs and other classes of bridged nucleic acid analogues are exten- different synthetic oligonucleotide-based therapeutics, so straightforward synthesis of their sively explored in different synthetic oligonucleotide-based therapeutics, so straightfor- monomers could be very useful [254,255,258–261] (Figure9). ward synthesis of their monomers could be very useful [254,255,258–261] (Figure 9).

FigureFigure 9.9. ExamplesExamples ofof clickableclickable monomersmonomers forfor LNAsLNAs andand BNAsBNAs synthesis.synthesis.

FirstFirst attemptsattempts towards design and and synthesis synthesis of of LNA-based LNA-based nucleoside nucleoside dimers dimers using us- ingCuAAC CuAAC reaction reaction were were done done in the in thelaboratory laboratory of Prasad of Prasad [262,263] [262, 263(Scheme] (Scheme 16). In16 ).total, In total,they received they received three triazole-linked three triazole-linked LNA dimers LNA dimers of type ofTL type–t– TL T, LT–t–L–t– T AL,TBzLL, and–t– ATBzLL–t–, andCBzL. TInL–t– the C nextBzL. Instep, the they next obtained step, they three obtained triazole-linked three triazole-linked xylonucleoside xylonucleoside dimers of type dimers TL–t– ofTxL type, TL–t–TL A–t–BzxL T,xL and, TL T–t–L–t– A CBzxLBzxL,. andIt is TnoteworthyL–t– CBzxL .to It point is noteworthy out the effort to point they made out the in effort order Biomedicines 2021, 9, 628 20 of 34 theyto stich made two in unique order tonucleoside stich two scaffolds unique nucleoside together through scaffolds a triazole together ring. through The optimized a triazole ring.method The can optimized easily be method adopted can for easily automated be adopted synthesis for automated of triazole-linked synthesis LNA-based of triazole- linked LNA-based oligonucleotides. Such structures were tested by Sharma et al. for their biologicaloligonucleotides. and biophysical Such structures properties were tested through by Sharma incorporation et al. for their into biological gene-silencing and bio- oligonu- cleotides,physical properties namely antisensethrough incorporation oligonucleotides into gene-silencing (ASOs) and double-strandedoligonucleotides, namely siRNAs [260]. antisense oligonucleotides (ASOs) and double-stranded siRNAs [260]. They presented ac- They presented accurate analyzes of good hybridization afﬁnity (when incorporated at the curate analyzes of good hybridization affinity (when incorporated at the 3′ or 5′ termini 30 or 50 termini of siRNAs, but with extremely low binding afﬁnity at internal positions), of siRNAs, but with extremely low binding affinity at internal positions), outstanding nu- outstandingclease stability, nuclease and high stability, gene silencing and high activity gene by silencing both RNase activity H and by Ago2 both based RNase mech- H and Ago2 basedanisms. mechanisms.

SchemeScheme 16. SynthesisSynthesis of of triazole-linked triazole-linked LNA-dimers LNA-dimers using using Cu(I)-catalyzed Cu(I)-catalyzed click reaction. click reaction. Independently, Kumar et al. have studied the combination of triazole-linked LNAs and six-atom triazole linkage [264,265] (Figure 10). It is known that oligonucleotides containing triazole linkage form less stable duplexes with complementary RNA/DNA targets compared to unmodified DNA strands; however (TL)LNAs with internal triazole-3′-LNA linkages bind to complementary RNA with similar affinity and specificity to unmodified oligonucleotides, and the introduction of triazole moiety significantly improves the thermal stability of the modified duplex. The combined triazole-LNA linkages have demonstrated an extreme resistance to nuclease degradation (3′-exonuclease digestion). The experiment has also shown that the (TL)LNAs with LNA on either side of the triazole linkage have the highest stability against degradation by DNase, in addition to the strongest affinity for RNA targets. Thus, the combination of LNA and the triazole linkage provides a new class of potentially significant antisense oligonucleotide candidates.

Figure 10. Triazole-linked DNA and LNA backbones.

2.4. (TL)PNA Skeleton Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the phosphodiester backbone is replaced with a polyamide structure. From a chemical point of view, PNA molecules resemble more peptides than nucleic acids. Despite the differences in relation to nucleic acids, PNA molecules with a strictly defined sequence show the ability to bind to complementary DNA or RNA molecules using Watson–Crick hydrogen bonds, blocking or reducing in this way the expression of relevant genes [266,267]. PNA is one of the most promising new molecules for recognition of nucleic acids because of Biomedicines 2021, 9, 628 20 of 34

oligonucleotides. Such structures were tested by Sharma et al. for their biological and biophysical properties through incorporation into gene-silencing oligonucleotides, namely antisense oligonucleotides (ASOs) and double-stranded siRNAs [260]. They presented accurate analyzes of good hybridization affinity (when incorporated at the 3′ or 5′ termini of siRNAs, but with extremely low binding affinity at internal positions), outstanding nuclease stability, and high gene silencing activity by both RNase H and Ago2 based mechanisms.

Biomedicines 2021, 9, 628 20 of 35

Scheme 16. Synthesis of triazole-linked LNA-dimers using Cu(I)-catalyzed click reaction.

Independently,Independently, Kumar Kumaret et al. al.have have studied studied the the combination combination of of triazole-linked triazole-linked LNAs LNAs andand six-atomsix-atom triazoletriazole linkage [264,265] [264,265] (Figure (Figure 10). 10). It is It known is known that that oligonucleotides oligonucleotides con- containingtaining triazole triazole linkage linkage form form less lessstable stable duplexes duplexes with withcomplementary complementary RNA/DNA RNA/DNA targets targetscompared compared to unmodified to unmodified DNA strands; DNA strands; however however (TL)LNAs (TL)LNAs with internal with internal triazole-3 triazole-′-LNA 0 3linkages-LNA linkages bind to complementary bind to complementary RNA with RNA similar with affinity similar and affinity specificity and to specificity unmodified to unmodifiedoligonucleotides, oligonucleotides, and the introduction and the introduction of triazole of moiety triazole significantly moiety significantly improves improves the ther- themal thermal stability stability of the modified of the modified duplex. duplex. The combined The combined triazole-LNA triazole-LNA linkages linkages have demon- have 0 demonstratedstrated an extreme an extreme resistance resistance to nuclease to nuclease degradation degradation (3′-exonuclease (3 -exonuclease digestion). digestion). The ex- Theperiment experiment has also has shown also shown that the that (TL)LNAs the (TL)LNAs with withLNA LNA on either on either side sideof the of triazole the triazole link- linkageage have have the the highest highest stability stability against against degradation degradation by by DNase, DNase, in addition toto thethe strongest strongest affinityaffinity for for RNA RNA targets. targets. Thus, Thus, the the combination combinatio ofn of LNA LNA and and the the triazole triazole linkage linkage provides provides a newa new class class of potentiallyof potentially significant significant antisense antisense oligonucleotide oligonucleotide candidates. candidates.

FigureFigure 10. 10.Triazole-linked Triazole-linked DNA DNA and and LNA LNA backbones. backbones.

2.4.2.4. (TL)PNA (TL)PNA Skeleton Skeleton PeptidePeptide nucleic nucleic acids acids (PNAs) (PNAs) are are oligonucleotide oligonucleotide analogues analogues in which in which the phosphodi- the phos- esterphodiester backbone backbone is replaced is replac with aed polyamide with a polyamide structure. Fromstructure. a chemical From pointa chemical of view, point PNA of moleculesview, PNA resemble molecules more resemble peptides more than peptides nucleic acids. than nucleic Despite acids. the differences Despite the in relationdifferences to nucleicin relation acids, to PNAnucleic molecules acids, PNA with molecules a strictly wi definedth a strictly sequence defined show sequence the ability show to bindthe abil- to complementaryity to bind to complementary DNA or RNA molecules DNA or using RNA Watson–Crick molecules using hydrogen Watson–Crick bonds, blocking hydrogen or reducingbonds, blocking in this way or reducing the expression in this ofway relevant the expression genes [266 of, 267relevant]. PNA genes is one [266,267]. of the most PNA promisingis one of the new most molecules promising for recognition new molecules of nucleic for recognition acids because of ofnucleic their highacids affinity because for of RNA as well as to single and double-stranded DNA targets. Moreover, PNAs have received great attention due to their resistance to nuclease and protease digestion, stability in serum, and thermal stability. These compounds, due to the formation of stronger complementary bonds with DNA or RNA, were considered as excellent candidates for antisense drugs in gene therapy since their discovery [268,269]. Their disadvantage is that they are too large of molecules and it is difficult for them to penetrate inside the cell. Moreover, due to their hydrophilic nature, they are quickly excreted with urine. Therefore, prior to the introduction of such drugs, chemical modifications of PNAs or appropriate vectors should be developed to increase the bioavailability of these new antisense drugs [270,271]. The research group of Wissinger has synthesized PNAs analogues bearing a triazole in lieu of the amide bond using a click cycloaddition [272] (Figure 11). PNA oligomers possessing an azide terminal group were obtained by standard Fmoc procedure [273], and then coupled with various alkyne monomers in the CuAAC reaction with various alkyne monomers. In the main product, dimer 20, the modification in the form of a triazole linker shows a little effect on the hybridization ability of the modified PNA strand and the sequence accuracy of the synthesized strand. These results show that the triazole group is a good substitute for an amide linker in PNA, and such clickable peptide nucleic acids (cPNA) are an important addition in nucleic acid applications. Biomedicines 2021, 9, 628 21 of 34

their high affinity for RNA as well as to single and double-stranded DNA targets. More- over, PNAs have received great attention due to their resistance to nuclease and protease digestion, stability in serum, and thermal stability. These compounds, due to the formation of stronger complementary bonds with DNA or RNA, were considered as excellent candidates for antisense drugs in gene therapy since their discovery [268,269]. Their disadvantage is that they are too large of molecules and it is difficult for them to penetrate inside the cell. Moreover, due to their hydrophilic nature, they are quickly excreted with urine. Therefore, prior to the introduction of such drugs, chemical modifications of PNAs or appropriate vectors should be developed to increase the bioavailability of these new antisense drugs [270,271]. The research group of Wissinger has synthesized PNAs analogues bearing a triazole in lieu of the amide bond using a click cycloaddition [272] (Figure 11). PNA oligomers possessing an azide terminal group were obtained by standard Fmoc procedure [273], and then coupled with various alkyne monomers in the CuAAC reaction with various alkyne monomers. In the main product, dimer 20, the modification in the form of a triazole linker shows a little effect on the hybridization ability of the modified PNA strand and the se- Biomedicines 2021, 9, 628 quence accuracy of the synthesized strand. These results show that the triazole group21 of is 35 a good substitute for an amide linker in PNA, and such clickable peptide nucleic acids (cPNA) are an important addition in nucleic acid applications.

FigureFigure 11. 11.Structure Structure of of PNA PNA linked linked with with triazole triazole moiety. moiety.

AmongAmong others,others, two two research research groups groups have have published published synthetic synthetic protocols protocols for new for newPNA PNAoligonucleotides oligonucleotides that contain that contain a triazole a triazole linkage linkage [274,275]. [274 The,275 ].first The example first example refers to refers a tri- toazole-linked a triazole-linked nucleoside nucleoside dimer, dimer, and the and seco thend second one, one,to a totrimer. a trimer. Efthymiou Efthymiou and andDe- Desaulnierssaulniers showed showed a amodified modified uracil–uracil uracil–uracil dimer dimer and and its compatibility withwith hybridizationhybridization toto a a complementary complementary sequence sequence when when appended appended at at the the 5 50 ′or or3 30′ endend ofof DNADNA oligonucleotide.oligonucleotide. TheThe second second mentioned mentioned work, work, which which has has been been performed performed in Zerrouki’s in Zerrouki’s laboratory, laboratory, displays dis- theplays synthesis the synthesis of a very of a interesting very interesting azido-heterotrimer, azido-heterotrimer, which which may undergo may undergo further further click reactionclick reaction providing providing polytriazole polytriazole PNA analogues. PNA analogues. All these All efforts these efforts have been have made been inmade order in toorder synthesize to synthesize chemically chemically modified modified oligonucleotides oligonucleotides with improvedwith improved properties, properties, and theand resultsthe results confirm confirm that 1,4-disubstitutedthat 1,4-disubstitute 1,2,3-triazoled 1,2,3-triazole is a good is a mimicgood mimic of a trans of -amidea trans-amide bond. bond. 2.5. (TL)G-Quadruplexes 2.5. (TL)G-QuadruplexesG-quadruplexes (G4s) are a family of nucleic acid structures based on the formation of G-quartersG-quadruplexes of four guanines (G4s) thatare area family arranged of nucleic in a plane acid to structures form several based G-tetrad, on the stabilizedformation byof hydrogenG-quarters bonds of four and guanines a metal that cation. are G-quadruplexesarranged in a plane represent to form unusual several four-stranded G-tetrad, sta- structures found in the human DNA and RNA molecules which show unique properties bilized by hydrogen bonds and a metal cation. G-quadruplexes represent unusual four- including resistance to nucleases and fast folding. Interestingly, RNA and PNA also formed stranded structures found in the human DNA and RNA molecules which show unique RNA–PNA hybrids of G-quadruplex structures [276]. Their main place of occurrence are properties including resistance to nucleases and fast folding. Interestingly, RNA and PNA genome regions with high transcriptional activity, the promoter regions and telomeres, so also formed RNA–PNA hybrids of G-quadruplex structures [276]. Their main place of oc- they undoubtedly take part in the regulation of gene expression. currence are genome regions with high transcriptional activity, the promoter regions and G-quadruplexes are components of biologically important structures, they are com- telomeres, so they undoubtedly take part in the regulation of gene expression. monly found in telomeres and oncogenes, which translates into their functions in ageing G-quadruplexes are components of biologically important structures, they are com- and disease development, including malignant tumors [277]. Such DNA–RNA hybrid monly found in telomeres and oncogenes, which translates into their functions in ageing G-quadruplex structure may also be a valuable target for anti-cancer agents directed against and disease development, including malignant tumors [277]. Such DNA–RNA hybrid G- telomeres [278]. Telomeres are present at the ends of all eukaryotic chromosomes and play quadruplex structure may also be a valuable target for anti-cancer agents directed against an important role in critical processes underlying genome stability, ageing, and cancer [279]. Understanding the chemistry of human telomere DNA and RNA biology will allow for the developing of chemical approaches to discover anticancer agents [280]. The related enzyme, telomerase, was first recognized as a unique and exciting anticancer target about five years after its discovery. Telomerase is activated in 80–90% of human tumors of all cancer types [281]. No other tumor-associated gene is as widely expressed as telomerase in cancers. In turn, the pioneer of DNA research on G quadruplexes, Shankar Balasubramanian, has managed to prove that formation of a G-quadruplex structure in the promoter region of the c-myc and c-kit protooncogenes inhibits their transcription and prevents the development of tumors. He has stated that in the future it will be possible to create synthetic molecules that block the proliferation of cells during the growth of cancerous tissue. So, G quadruplexes began to attract the attention of oncologists in the early 1990s, in the hope that they could help to fight cancers. The clinical experiments’ evidence for use of telomerase or telomeres as targets in cancer therapy are encouraging and are exciting prospects in future medical strategies. DNA–RNA hybrid G-quadruplex structure is technically difficult to study by tradi- tional methods, such as NMR and crystallography, since various G-quadruplexes may coexist as a mixture. Click chemistry has helped overcome this difficulty. This reaction can trap a particular species or produce a snapshot of various structures that are present in a complex solution [282] (Scheme 17). The detected “azido–alkyne cycloaddition” product of Biomedicines 2021, 9, 628 22 of 34

telomeres [278]. Telomeres are present at the ends of all eukaryotic chromosomes and play an important role in critical processes underlying genome stability, ageing, and cancer [279]. Understanding the chemistry of human telomere DNA and RNA biology will allow for the developing of chemical approaches to discover anticancer agents [280]. The related enzyme, telomerase, was first recognized as a unique and exciting anti- cancer target about five years after its discovery. Telomerase is activated in 80–90% of human tumors of all cancer types [281]. No other tumor-associated gene is as widely expressed as telomerase in cancers. In turn, the pioneer of DNA research on G quadruplexes, Shankar Balasubramanian, has managed to prove that formation of a G-quadruplex structure in the promoter region of the c-myc and c-kit protooncogenes inhibits their transcription and prevents the development of tumors. He has stated that in the future it will be possible to create synthetic molecules that block the proliferation of cells during the growth of cancerous tissue. So, G quadruplexes began to attract the attention of oncologists in the early 1990s, in the hope that they could help to fight cancers. The clinical experiments’ evidence for use of telomerase or telomeres as targets in cancer therapy are encouraging and are exciting prospects in future medical strategies. DNA–RNA hybrid G-quadruplex structure is technically difficult to study by tradi- tional methods, such as NMR and crystallography, since various G-quadruplexes may Biomedicines 2021, 9, 628 coexist as a mixture. Click chemistry has helped overcome this difficulty. This reaction22 of 35 can trap a particular species or produce a snapshot of various structures that are present in a complex solution [282] (Scheme 17). The detected “azido–alkyne cycloaddition” product of the alkyne- and azido-labeled RNA and DNA coupling identifies that DNA–RNA thehybrid alkyne- G-quadruplex and azido-labeled structure RNA can and be formed DNA coupling from human identiﬁes telomeric that DNA–RNA DNA and RNA hybrid se- G-quadruplex structure can be formed from human telomeric DNA and RNA sequences. quences.

SchemeScheme 17. 17.Illustrative Illustrative depiction depiction of of the the use use of of click click chemistry chemistry in in G-quadruplexes. G-quadruplexes.

ClickClick chemistry chemistry can can be be a verya very useful useful method method for for a search, a search, identification, identification, and and designing design- therapeutics,ing therapeutics, and above and above all, studying all, studying the functions the functions of these of biomolecules these biomolecules [282,283 [282,283].]. DNA aptamersDNA aptamers bearing bearing triazole triazole internucleotide internucleotide linkages linkages can be alsocan foundbe also in found such applicationsin such appli- ascations novel thrombin-bindingas novel thrombin-binding aptamers aptamers for anticoagulant for anticoagulant effects [284 effects], human [284], telomere human telo- bi- ologymere [280biology,285, 286[280,285,286],], and furthermore, and furthermore, to design to and design synthesize and synthesize a click-light-up a click-light-upprobe to investigateprobe to investigate whether DNA-RNA whether G-quadruplexesDNA-RNA G-quadruplexes exist in living exist cells in [287 living]. Human cells [287]. telomere Hu- structuresman telomere are difficult structures to probe are difficult in living to cells probe because in living the concentrationcells because the of G-quadruplexes concentration of isG-quadruplexes too low to be detected is too low in to the be presence detected ofin onlythe presence a few dozen of only chromosome a few dozen ends, chromosome so em- ployingends, so a light-switchingemploying a light-switching probe is possible probe to find is possible the process to find of DNA–RNA the process G-quadruplex of DNA–RNA formationG-quadruplex [285]. formation Owing to the[285]. fact Owing that DNA to the aptamers fact that areDNA increasingly aptamers recognizedare increasingly as drug rec- candidates,ognized as hereindrug candidates, click chemistry herein also click enables chemistry the synthesis also enables and the development synthesis and of smalldevel- moleculesopment of that small target molecules G-quadruplex that target nucleic G-quad acidsruplex to elucidate nucleic drugacids specificityto elucidate [283 drug]. specificity [283]. 3. Conclusions 3. ConclusionsIn conclusion, oligonucleotides modified at the phosphodiester linkage are important class of compounds, especially in the field of antisense and antigen strategies. We are In conclusion, oligonucleotides modified at the phosphodiester linkage are important interested in the replacement of the phosphodiester linkage of the natural oligonucleotides class of compounds, especially in the field of antisense and antigen strategies. We are in- with novel, neutral, and heterocyclic linkages based on their stability and rigidity, and terested in the replacement of the phosphodiester linkage of the natural oligonucleotides therefore, report here an efficient synthesis of the 1,2,3-triazole units to prepare triazolo- modified oligonucleotides. In this review we have demonstrated the usefulness of the CuAAC or RuAAC ligation strategy in nucleic acid chemistry. The viability of the method has been evidenced in the preparation of clickable nucleoside and nucleotide monomers, triazole-linked dimers and trimers units, and triazolo-modified oligonucleotides. The chemical modifications used in the oligonucleotide design can bypass many pharmacological shortcomings and limitations. This shows the importance of non-natural oligonucleotides acting as inhibitors of target deleterious genes responsible for various diseases. Oligonucleotides containing triazole in place of the phosphodiester linkage have been known before the area of click chemistry—the first works about the synthesis and thermal stability of triazole-linked DNA were reported by von Matt et al. But a catalyzed version of the Huisgen [3 + 2] cycloaddition, so-called click chemistry, has just enabled an intensive development of this chemistry area. The main goal of this work was to present efficient routes to design nucleic acids and their components dedicated for gene expression control. The use of gene silencing agents is a topic of intense research because these molecules can act as inhibitors of any target genes, expression of which causes diseases, among others viral infections, cancer growth, and inflammation. As naturally occurring ONs suffer from degradation due to endo- and exonucleases, modifications have become necessary to enhance the stability and improve pharmacokinetic properties from a therapeutic point of view. Modified nucleosides and nucleotides continue to attract significant focus in medical applications as building blocks for gene silencing, such as antisense, antigen, and RNA interference. The third generation Biomedicines 2021, 9, 628 23 of 35

of nucleic acids therapeutics—newest and most promising with enhanced binding affinity and biostability—are PNAs, LNAs, tcDNA, and CeNA. Fundamental knowledge from many fields, including chemistry, structural biology, cell biology, and medicine, has opened up the possibility of development of novel strategies against cancer and other diseases. This review highlights the contribution of click chemistry to drug development, but also to labelling and imaging for better visualization. The click chemistry covers chemical synthesis of small molecules as well as oligonucleotides by the bio-orthogonal ligation process and their application as drugs and tools in molecular medicine. The intention of this article is to provide both inside and inspiration in the field and stimulate further research to develop new chemical approaches. Thus, click reaction is one of the most popular interdisciplinary reaction that bridges the gap between chemistry and biology.

Author Contributions: Conceptualization, D.B. and J.B.; writing—original draft preparation, D.B.; writing—review and editing, J.B.; visualization, D.B.; supervision, J.B.; funding acquisition, D.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Centre, Poland, grant number 2019/35/D/ NZ7/03637. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

A adenosine Ac acetyl protecting group ADAR adenosine deaminases Ago2 argonaute RISC catalytic component 2 ASO antisense oligonucleotide asRNA antisense ribonucleic acid ATP adenosine triphosphate AZT 30-azido-30-deoxythymidine B heterocyclic base BCL2 B-cell lymphoma 2 proteins BNA bridged nucleic acids Bzl benzoyl protecting group C cytidine cat-ELCCA catalytic enzyme-linked click chemistry assay CD circular dichroism spectroscopy CeNA cyclohexene nucleic acids ceRNA competing endogenous ribonucleic acid circRNA circular ribonucleic acid cPNA clickable peptide nucleic acids CRISP-Cas9 clustered regularly interspaced short palindromic repeats associated protein 9 CuAAC copper(I)-catalyzed alkyne-azide cycloaddition CuI copper(I) iodide CuSO4 copper(II) sulfate DIPEA N,N-diisopropylethylamine DMF dimethylformamide DMTr 4,40 -dimethoxytrityl protecting group Biomedicines 2021, 9, 628 24 of 35

DNA 20- deoxyribonucleic acid dsDNA double-stranded DNA dU 20-deoxyuridine dU*TP alkyne-modified 20-deoxyuridine triphosphate eIF4E eukaryotic translation initiation factor 4E endo-siRNA endogenous siRNA EPR electron paramagnetic resonance spectroscopy eRNA enhancer ribonucleic acid EtOH ethanol FTR functionality transfer reaction G4 G-quadruplex gsRNA germline small ribonucleic acid HCV hepatitis C virus HIV human immunodeficiency virus iPr isopropyl protecting group lincRNA intervening/intergenic ribonucleic acid LNA locked nucleic acids lncRNA long noncoding ribonucleic acid Me methyl min minute(s) miRNA micro ribonucleic acid mRNA messenger ribonucleic acid 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole or MSNT (2,4,6-trimethylphenyl)-(3-nitro-1,2,4-triazol-1-yl) sulfone MW microwave irradiation NaAsc sodium ascorbate NAT natural antisense transcript ncRNA non-coding ribonucleic acid NMR nuclear magnetic resonance spectroscopy ODN oligodeoxynucleotide ON oligonucleotide PCR polymerase chain reaction piRNA piwi-interacting ribonucleic acid PNA peptide nucleic acids pRNA promoter ribonucleic acid rasiRNA repeat associated siRNA RCA rolling circle amplification reaction RNA ribonucleic acid RNAi RNA interference RNAi RNA interference RNaseP ribonuclease P rRNA ribosomal ribonucleic acid rt room temperature RuAAC ruthenium(II)-catalyzed alkyne-azide cycloaddition SAM S-adenosyl-L-methionine scaRNA small Cajal body-specific ribonucleic acid sdRNA snoRNA-derived ribonucleic acid systematic evolution of ligands by exponential enrichment or in vitro selection, SELEX or in vitro evolution sgRNA single guide RNA siRNA small interfering ribonucleic acid snoRNA small nucleolar ribonucleic acid snRNA small nuclear ribonucleic acid ssDNA single-stranded DNA stRNA small temporal ribonucleic acid T thymidine TBDMS tert-butyldiphenylsilyl protecting group TBS tert-butyl(dimethyl)silyl protecting group Biomedicines 2021, 9, 628 25 of 35

TBTA tris(benzyltriazolylmethyl)amine tcDNA tricyclo-DNA tiRNA tRNA-derived stress-induced ribonucleic acid (TL)BNA triazole-modified BNA (TL)DNA triazole-modified DNA (TL)LNA triazole-modified LNA (TL)PNA triazole-modified PNA (TL)quadruplexes triazole-modified quadruplexes (TL)RNA triazole-modified RNA Tm melting temperature TP 1,2,3-triazolylphosphonate internucleotide linkage tRNA transfer ribonucleic acid U uridine UTR untranslated region of mRNA UV ultraviolet

References 1. Becker, S.; Schneider, C.; Crisp, A.; Carell, T. Non-canonical nucleosides and chemistry of the emergence of life. Nat. Commun. 2018, 9, 5174. [CrossRef][PubMed] 2. Lönnberg, H. Chemistry of Nucleic Acids; De Gruyter: Berlin, Germany, 2020. 3. Duffy, K.; Arangundy-Franklin, S.; Holliger, P. Modiﬁed nucleic acids: Replication, evolution, and next-generation therapeutics. BMC Biol. 2020, 18, 112. [CrossRef][PubMed] 4. Mattick, J.S. The Genetic Signatures of Noncoding RNAs. PLoS Genet. 2009, 5, 1–12. [CrossRef] 5. Nie, P.; Bai, Y.; Mei, H. Synthetic Life with Alternative Nucleic Acids as Genetic Materials. Molecules 2020, 25, 3483. [CrossRef] 6. Barciszewski, J.; Marquez, V.E.; Vasseur, J.-J.; Markiewicz, W.T. Chemical Biology of Nucleic Acids. ACS Chem. Biol. 2015, 10, 1358–1361. [CrossRef] 7. Benner, S.A.; Karalkar, N.B.; Hoshika, S.; Laos, R.; Shaw, R.W.; Matsuura, M.; Fajardo, D.; Moussatche, P. Alternative Watson-Crick Synthetic Genetic Systems. Cold Spring Harb. Perspect. Biol. 2016, 8, a023770. [CrossRef][PubMed] 8. Grosjean, H.; Westhof, E. An integrated, structure- and energy-based view of the genetic code. Nucleic Acids Res. 2016, 44, 8020–8040. [CrossRef] 9. Biondi, E.; Benner, S.A. Artiﬁcially Expanded Genetic Information Systems for New Aptamer Technologies. Biomedicines 2018, 6, 53. [CrossRef] 10. International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 2001, 409, 860–921. [CrossRef] 11. Venter, J.C.; Adams, M.D.; Myers, E.W.; Li, P.W.; Mural, R.J.; Sutton, G.G.; Smith, H.O.; Yandell, M.; Evans, C.A.; Holt, R.A.; et al. The Sequence of the Human Genome. Science 2001, 291, 1304–1351. [CrossRef][PubMed] 12. Wright, M.W.; Bruford, E.A. Naming ‘junk’: Human non-protein coding RNA (ncRNA) gene nomenclature. Hum. Genom. 2011, 5, 90–98. [CrossRef] 13. Cech, T.R.; Steitz, J.A. The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones. Cell 2014, 157, 77–94. [CrossRef] 14. Mattick, J.S.; Makunin, I.V. The Role of MicroRNAs in Cancer. Hum. Mol. Genet. 2006, 15, R17–R29. [CrossRef] 15. Walter, N.G.; Engelke, D.R. Ribozymes: Catalytic RNAs that cut things, make things, and do odd and useful jobs. Biologist 2002, 49, 199–203. 16. Bachellerie, J.P.; Cavaillé, J.; Hüttenhofer, A. The expanding snoRNA world. Biochimie 2002, 84, 775–790. [CrossRef] 17. Kiss, T. Biogenesis of small nuclear RNPs. J. Cell Sci. 2004, 117, 5949–5951. [CrossRef] 18. Turner, J.D.; Williamson, R.; Almefty, K.K.; Nakaji, P.; Porter, R.; Tse, V.; Kalani, M.Y. The many roles of microRNAs in brain tumor biology. Neurosurg. Focus 2010, 28, E3. [CrossRef] 19. Szymanski, M.; Barciszewska, M.Z.; Erdmann, V.A.; Barciszewski, J. A new frontier for molecular medicine: Noncoding RNAs. Biochem. Biophys. Acta 2005, 1756, 65–75. [CrossRef] 20. Espinosa, C.E.S.; Slack, F.J. The Role of MicroRNAs in Cancer. Yale J. Biol. Med. 2006, 79, 131–140. 21. Staedel, C.; Tran, T.P.A.; Giraud, J.; Darfeuille, F.; Di Giorgio, A.; Tourasse, N.J.; Salin, F.; Uriac, P.; Duca, M. Modulation of oncogenic miRNA biogenesis using functionalized polyamines. Sci. Rep. 2018, 8, 1667. [CrossRef] 22. Costales, M.G.; Aikawa, H.; Li, Y.; Childs-Disney, J.L.; Abegg, D.; Hoch, D.G.; Velagapudi, S.P.; Nakai, Y.; Khan, T.; Wang, K.W.; et al. Small-molecule targeted recruitment of a nuclease to cleave an oncogenic RNA in a mouse model of metastatic cancer. Proc. Natl. Acad. Sci. USA 2020, 117, 2406–2411. [CrossRef] 23. Boriack-Sjodin, P.A.; Ribich, S.; Copeland, R.A. RNA-modifying proteins as anticancer drug targets. Nat. Rev. Drug. Discov. 2018, 17, 435–453. [CrossRef][PubMed] 24. Pang, J.C.; Kwok, W.K.; Chen, Z.; Ng, H.-K. Oncogenic role of microRNAs in brain tumors. Acta Neuropathol. 2009, 117, 599–611. [CrossRef] 25. Souckova, K.; Ivkovic, T.C.; Slaby, O. Non-coding RNA therapy in cancer. Precis. Med. Investig. Pract. Provid. 2020, 20, 211–220. Biomedicines 2021, 9, 628 26 of 35

26. Zhang, J.; Lau, M.W.; Ferré-D’Amaré, A.R. Ribozymes and Riboswitches: Modulation of RNA Function by Small Molecules. Biochemistry 2010, 49, 9123–9131. [CrossRef][PubMed] 27. Garst, A.D.; Edwards, A.L.; Batey, R.T. Riboswitches: Structures and mechanisms. Cold Spring Harb. Perspect. Biol. 2011, 3, a003533. [CrossRef][PubMed] 28. Conde, J.; Edelman, E.R.; Artzi, N. Target-responsive DNA/RNA nanomaterials for microRNA sensing and inhibition: The jack-of-all-trades in cancer nanotheranostics? Adv. Drug Deliv. Rev. 2015, 81, 169–183. [CrossRef] 29. Storz, G. An Expanding Universe of Noncoding RNAs. Science 2002, 296, 1260–1263. [CrossRef][PubMed] 30. Kuhlmann, J.D.; Baraniskin, A.; Hahn, S.A.; Mosel, F.; Bredemeier, M.; Wimberger, P.; Kimmig, R.; Kasimir-Bauer. S. Circulating U2 small nuclear RNA fragments as a novel diagnostic tool for patients with epithelial ovarian cancer. Clin. Chem. 2014, 60, 206–213. [CrossRef] 31. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599. [CrossRef] 32. Tornøe, C.W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1–3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67, 3057–3064. [CrossRef][PubMed] 33. Amblard, F.; Cho, J.H.; Schinazi, R.F. Cu(I)-Catalyzed Huisgen Azide−Alkyne 1,3-Dipolar Cycloaddition Reaction in Nucleoside, Nucleotide, and Oligonucleotide Chemistry. Chem. Rev. 2009, 109, 4207–4220. [CrossRef] 34. Efthymiou, T.; Gong, W.; Desaulniers, J.-P. Chemical Architecture and Applications of Nucleic Acid Derivatives Containing 1,2,3-Triazole Functionalities Synthesized via Click Chemistry. Molecules 2012, 17, 12665–12703. [CrossRef] 35. Tiwari, V.K.; Mishra, B.B.; Mishra, K.B.; Mishra, N.; Singh, A.S.; Chen, X. Cu-Catalyzed Click Reaction in Carbohydrate Chemistry. Chem. Rev. 2016, 116, 3086–3240. [CrossRef] 36. Zavgorodny, S.G.; Pechenov, A.E.; Shvets, V.I.; Miroshnikov, A.I. S,X-Acetals in Nucleoside Chemistry. III1. Synthesis of 20-and 30-O-Azidomethyl Derivatives of Ribonucleosides. Nucleos. Nucleot. Nucl. 2000, 19, 1977–1991. [CrossRef][PubMed] 37. Baraniak, D.; Baranowski, D.; Ruszkowski, P.; Boryski, J. 3’-O- and 5’-O-Propargyl derivatives of 5-fluoro-2’-deoxyuridine: Synthesis, cytotoxic evaluation and conformational analysis. Nucleos. Nucleot. Nucl. 2016, 35, 178–194. [CrossRef][PubMed] 38. Castro, V.; Rodríguez, H.; Albericio, F. CuAAC: An Efficient Click Chemistry Reaction on Solid Phase. ACS Comb. Sci. 2016, 18, 1–14. [CrossRef] 39. Kropp, H.M.; Dürr, S.L.; Peter, C.; Diederichs, K.; Marx, A. Snapshots of a modified nucleotide moving through the confines of a DNA polymerase. Proc. Natl. Acad. Sci. USA 2018, 115, 9992–9997. [CrossRef] 40. Lingala, S.; Nordstrøm, L.U.; Mallikaratchy, P.R. Synthesis of stable azide and alkyne functionalized phosphoramidite nucleosides. Tetrahedron Lett. 2019, 60, 211–213. [CrossRef] 41. Michalska, L.; Wawrzyniak, D.; Szymańska-Michalak, A.; Barciszewski, J.; Boryski, J.; Baraniak, D. Synthesis and biological assay of new 20-deoxyuridine dimers containing a 1,2,3-triazole linker. Part, I. Nucleos. Nucleot. Nucl. 2019, 38, 218–235. [CrossRef] 42. Baraniak, D.; Baranowski, D.; Ruszkowski, P.; Boryski, J. Nucleoside dimers analogues with 1,2,3-triazole linkage: Conjugation of floxuridine and thymidine provides novel tools for cancer treatment. Part II. Nucleos. Nucleot. Nucl. 2019, 38, 807–835. [CrossRef] [PubMed] 43. Baraniak, D.; Ruszkowski, P.; Baranowski, D.; Framski, G.; Boryski, J. Nucleoside dimers analogs containing floxuridine and thymidine with unnatural linker groups: Synthesis and cancer line studies. Part III. Nucleos. Nucleot. Nucl. 2019, 38, 980–1005. [CrossRef][PubMed] 44. Jawalekar, A.M.; Malik, S.; Verkade, J.M.M.; Gibson, B.; Barta, N.S.; Hodges, J.C.; Rowan, A.; van Delft, F.L. Oligonucleotide Tagging for Copper-Free Click Conjugation. Molecules 2013, 18, 7346–7363. [CrossRef] 45. Sharma, V.K.; Sharma, R.K.; Singh, S.K. Antisense oligonucleotides: Modifications and clinical trials. Med. Chem. Commun. 2014, 5, 1454–1471. [CrossRef] 46. Guo, F.; Li, Q.; Zhou, C. Synthesis and biological applications of fluoro-modified nucleic acids. Org. Biomol. Chem. 2017, 15, 9552–9565. [CrossRef] 47. Chittepu, P.; Sirivolu, V.R.; Seela, F. Nucleosides and oligonucleotides containing 1,2,3-triazole residues with nucleobase tethers: Synthesis via the azide-alkyne ‘click’ reaction. Bioorg. Med. Chem. 2008, 16, 8427–8439. [CrossRef][PubMed] 48. Guo, J.; Xu, N.; Li, Z.; Zhang, S.; Wu, J.; Kim, D.H.; Marma, M.S.; Meng, Q.; Cao, H.; Li, X.; et al. Four-color DNA sequencing with 30-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc. Natl. Acad. Sci. USA 2008, 105, 9145–9150. [CrossRef] 49. Okholm, A.; Kjems, J.; Astakhova, K. Fluorescence detection of natural RNA using rationally designed “clickable” oligonucleotide probes. RSC Adv. 2014, 4, 45653–45656. [CrossRef] 50. Horisawa, K. Specific and quantitative labeling of biomolecules using click chemistry. Front. Physiol. 2014, 5, 457. [CrossRef] 51. Saito, Y.; Hudson, R.H.E. Base-modified fluorescent purine nucleosides and nucleotides for use in oligonucleotide probes. J. Photochem. Photobiol. C 2018, 36, 48–73. [CrossRef] 52. Venkatesham, A.; Pillalamarri, S.R.; Wit, F.D.; Lescrinier, E.; Debyser, Z.; Van Aerschot, A. Propargylated Purine Deoxynucleosides: New Tools for Fluorescence Imaging Strategies. Molecules 2019, 24, 468. [CrossRef] 53. Bag, S.S.; Das, S.K. Triazolyl C-nucleosides via the intermediacy of β-10-ethynyl-20-deoxyribose derived from a Nicholas reaction: Synthesis, photophysical properties and interaction with BSA. Tetrahedron 2019, 75, 3024–3037. [CrossRef] Biomedicines 2021, 9, 628 27 of 35

54. Klimkowski, P.; De Ornellas, S.; Singleton, D.; El-Sagheer, A.H.; Brown, T. Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting. Org. Biomol. Chem. 2019, 17, 5943–5950. [CrossRef] 55. Prasher, P.; Sharma, M. Tailored therapeutics based on 1,2,3-1H-triazoles: A mini review. Med. Chem. Commun. 2019, 10, 1302–1328. [CrossRef][PubMed] 56. Pujari, S.S.; Seela, F. Parallel Stranded DNA Stabilized with Internal Sugar Cross-Links: Synthesis and Click Ligation of Oligonucleotides Containing 20-Propargylated Isoguanosine. J. Org. Chem. 2013, 78, 8545–8561. [CrossRef][PubMed] 57. Xiong, H.; Seela, F. Cross-Linked DNA: Site-Selective “Click” Ligation in Duplexes with Bis-Azides and Stability Changes Caused by Internal Cross-Links. Bioconjug. Chem. 2012, 23, 1230–1243. [CrossRef][PubMed] 58. Tera, M.; Taji, Z.H.; Luedtke, N.W. Intercalation-enhanced “Click” Crosslinking of DNA. Angew. Chem. Int. Ed. 2018, 130, 15631–15635. [CrossRef] 59. Best, M.D. Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules. Biochemistry 2009, 48, 6571–6584. [CrossRef] 60. Grammel, M.; Hang, H. Chemical reporters for biological discovery. Nat. Chem. Biol. 2013, 9, 475–484. [CrossRef] 61. Ren, X.; Gerowska, M.; El-Sagheerac, A.H.; Brown, T. Enzymatic incorporation and fluorescent labelling of cyclooctyne-modified deoxyuridine triphosphates in DNA. Bioorg. Med. Chem. 2014, 22, 4384–4390. [CrossRef] 62. Seo, S.; Onizuka, K.; Nishioka, C.; Takahashi, E.; Tsuneda, S.; Abe, H.; Ito, Y. Phosphorylated 5-ethynyl-20-deoxyuridine for advanced DNA labeling. Org. Biomol. Chem. 2015, 13, 4589–4595. [CrossRef] 63. Sallustrau, A.; Bregant, S.; Chollet, C.; Audisio, D.; Taran, F. Scalable and practical synthesis of clickable Cu-chelating azides. Chem. Commun. 2017, 53, 7890–7893. [CrossRef][PubMed] 64. Godeau, G.; Staedel, C.; Barthélémy, P. Lipid-Conjugated Oligonucleotides via “Click Chemistry” Efficiently Inhibit Hepatitis C Virus Translation. J. Med. Chem. 2008, 51, 4374–4376. [CrossRef] 65. Singh, Y.; Murat, P.; Defrancq, E. Recent developments in oligonucleotide conjugation. Chem. Soc. Rev. 2010, 39, 2054–2070. [CrossRef][PubMed] 66. Zheng, M.; Zheng, L.; Zhang, P.; Li, J.; Zhang, Y. Development of Bioorthogonal Reactions and Their Applications in Bioconjuga- tion. Molecules 2015, 20, 3190–3205. [CrossRef] 67. Neumann, S.; Biewend, M.; Rana, S.; Binder, W.H. The CuAAC: Principles, Homogeneous and Heterogeneous Catalysts, and Novel Developments and Applications. Macromol. Rapid Commun. 2020, 41, 1900359. [CrossRef] 68. Erbas-Cakmak, S.; Leigh, D.A.; McTernan, C.T.; Nussbaumer, A.L. Artificial Molecular Machines. Chem. Rev. 2015, 115, 10081–10206. [CrossRef] 69. Chandrasekaran, A.R.; Rusling, D.A. Triplex-forming oligonucleotides: A third strand for DNA nanotechnology. Nucleic Acids Res. 2018, 46, 1021–1037. [CrossRef][PubMed] 70. Valsangkar, V.; Chandrasekaran, A.R.; Wang, R.; Haruehanroengra, P.; Levchenko, O.; Halvorsen, K.; Sheng, J. Click-based functionalization of a 20-O-propargyl-modified branched DNA nanostructure. J. Mater. Chem. B 2017, 5, 2074–2077. [CrossRef] 71. Takezawa, Y.; Shionoya, M. Supramolecular DNA Three-Way Junction Motifs with a Bridging Metal Center. Front. Chem. 2020, 7, 925. [CrossRef] 72. Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Click chemistry for drug development and diverse chemical-biology applications. Chem. Rev. 2013, 113, 4905–4979. [CrossRef] 73. Garner, A.L. at-ELCCA: Catalyzing Drug Discovery Through Click Chemistry. Chem. Commun. 2018, 54, 6531–6539. [CrossRef] [PubMed] 74. Corso, A.D.; Pignataro, L.; Belvisi, L.; Gennari, C. Innovative Linker Strategies for Tumor-Targeted Drug Conjugates. Chem. Eur. J. 2019, 25, 14740–14757. [CrossRef][PubMed] 75. Rodrigues, T.; Bernardes, G.J.L. Machine learning for target discovery in drug development. Curr. Opin. Chem. Biol. 2020, 56, 16–22. [CrossRef] 76. Isobe, H.; Fujino, T. Triazole-linked analogues of DNA and RNA (TLDNA and TLRNA): Synthesis and functions. Chem. Rec. 2014, 14, 41–51. [CrossRef] 77. Astakhova, K.; Ray, R.; Taskova, M.; Uhd, J.; Carstens, A.; Morris, K. “Clicking” Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions. Mol. Pharm. 2018, 15, 2892–2899. [CrossRef][PubMed] 78. Fairbanks, B.D.; Culver, H.R.; Mavila, S.; Bowman, C.N. Towards High-Efficiency Synthesis of Xenonucleic Acids. Trends Chem. 2019, 2, 43–56. [CrossRef] 79. Fantoni, N.Z.; El-Sagheer, A.H.; Brown, T. A Hitchhiker’s Guide to Click-Chemistry with Nucleic Acids. Chem. Rev. 2021. [CrossRef] 80. Brown, T.; El-Sagheer, A.H. Oligonucleotide ligation. U.S. Patent 8,846,883 B2, 30 September 2014. 81. Howe, F.S.; Russell, A.; Lamstaes, A.R.; El-Sagheer, A.; Nair, A.; Brown, T.; Mellor, J. CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape. eLife 2017, 6, e29878. [CrossRef] 82. Taemaitree, L.; Shivalingam, A.; El-Sagheer, A.H.; Brown, T. An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA. Nat. Commun. 2019, 10, 1610. [CrossRef] 83. Filippova, J.; Matveeva, A.; Zhuravlev, E.; Stepanov, G. Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems. Biochimie 2019, 167, 49–60. [CrossRef] Biomedicines 2021, 9, 628 28 of 35

84. Chen, Q.; Zhang, Y.; Yin, H. Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR- mediated genome editing. Adv. Drug Deliv. Rev. 2021, 168, 246–258. [CrossRef] 85. Tolle, F.; Brändle, G.M.; Matzner, D.; Mayer, G. A Versatile Approach Towards Nucleobase-Modified Aptamers. Angew. Chem. Int. Ed. 2015, 54, 10971–10974. [CrossRef] 86. Zhou, J.; Rossi, J. Aptamers as targeted therapeutics: Current potential and challenges. Nat. Rev. Drug Discov. 2017, 16, 181–202. [CrossRef] 87. Cai, S.; Yan, J.; Xiong, H.; Liu, Y.; Peng, D.; Liu, Z. Investigations on the interface of nucleic acid aptamers and binding targets. Analyst 2018, 143, 5317–5338. [CrossRef] 88. Pfeiffer, F.; Tolle, F.; Rosenthal, M.; Brändle, G.M.; Ewers, J.; Mayer, G. Identification and characterization of nucleobase-modified aptamers by click-SELEX. Nat. Protoc. 2018, 13, 1153–1180. [CrossRef] 89. Odeh, F.; Nsairat, H.; Alshaer, W.; Ismail, M.A.; Esawi, E.; Qaqish, B.; Bawab, A.A.; Ismail, S.I. Aptamers Chemistry: Chemical Modifications and Conjugation Strategies. Molecules 2020, 25, 3. [CrossRef][PubMed] 90. Shao, Q.; Chen, T.; Sheng, K.; Liu, Z.; Zhang, Z.; Romesberg, F.E. Selection of Aptamers with Large Hydrophobic 20-Substituents. J. Am. Chem. Soc. 2020, 142, 2125–2128. [CrossRef] 91. Deiters, A.; Schultz, P.G. In vivo incorporation of an alkyne into proteins in Escherichia coli. Bioorganic Med. Chem. Lett. 2005, 15, 1521–1524. [CrossRef][PubMed] 92. Wang, Q.; Parrish, A.R.; Wang, L. Expanding the Genetic Code for Biological Studies. Chem. Biol. 2009, 16, 323–336. [CrossRef] 93. Lu, K.; Duan, Q.-P.; Ma, L.; Zhao, D.-X. Chemical strategies for the synthesis of peptide-oligonucleotide conjugates. Bioconjug. Chem. 2010, 21, 187–202. [CrossRef] 94. Ballikaya, S.; Lee, J.; Warnken, U.; Schnölzer, M.; Gebert, J.; Kopitz, J. De Novo proteome analysis of genetically modified tumor cells by a metabolic labeling/azide-alkyne cycloaddition approach. Mol. Cell. Proteom. 2014, 13, 3446–3456. [CrossRef][PubMed] 95. Shang, S.; Monfregola, L.; Caruthers, M. Peptide-substituted oligonucleotide synthesis and non-toxic, passive cell delivery. Signal Transduct. Target. Ther. 2016, 1, e16019. [CrossRef][PubMed] 96. MacCulloch, T.; Buchberger, A.; Stephanopoulos, N. Emerging applications of peptide–oligonucleotide conjugates: Bioactive scaffolds, self-assembling systems, and hybrid nanomaterials. Org. Biomol. Chem. 2019, 17, 1668–1682. [CrossRef] 97. Stephanopoulos, N. Peptide–Oligonucleotide Hybrid Molecules for Bioactive Nanomaterials. Bioconjug. Chem. 2019, 30, 1915–1922. [CrossRef] 98. Liu, C.; Zou, Y.; Hu, H.; Jiang, Y.; Qin, L. pDobz/pDobb protected diaminodiacid as a novel building block for peptide disulfide-bond mimic synthesis. RSC Adv. 2019, 9, 5438–5444. [CrossRef] 99. Hocek, M. Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing, Cross-linking, and Modulation of Protein–DNA Binding and Transcription. Acc. Chem. Res. 2019, 52, 1730–1737. [CrossRef] 100. Taskova, M.; Madsen, C.S.; Jensen, K.J.; Hansen, L.H.; Vester, B.; Astakhova, K. Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation. Bioconjug. Chem. 2017, 28, 768–774. [CrossRef] 101. Jain, N.; Smith, S.W.; Ghone, S.; Tomczuk, B. Current ADC Linker Chemistry. Pharm. Res. 2015, 32, 3526–3540. [CrossRef] 102. Dovgan, I.; Koniev, O.; Kolodych, S.; Wagner, A. Antibody–Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents. Bioconjug. Chem. 2019, 30, 2483–2501. [CrossRef][PubMed] 103. Bernard, S.; Audisio, D.; Riomet, M.; Bregant, S.; Sallustrau, A.; Plougastel, L.; Decuypere, E.; Gabillet, S.; Kumar, R.A.; Elyian, J.; et al. Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes. Angew. Chem. Int. Ed. 2017, 56, 15612–15616. [CrossRef] 104. Hsu, N.-S.; Lee, C.-C.; Kuo, W.-C.; Chang, Y.-W.; Lo, S.-Y.; Wang, A.H.-J. Development of a Versatile and Modular Linker for Antibody–Drug Conjugates Based on Oligonucleotide Strand Pairing. Bioconjug. Chem. 2020, 31, 1804–1811. [CrossRef] 105. Wan, J.; Li, Y.; Jin, K.; Guo, J.; Xu, J.; Wang, C. Robust Strategy for Antibody–Polymer–Drug Conjugation: Significance of Conjugating Orientation and Linker Charge on Targeting Ability. ACS Appl. Mater. Interfaces 2020, 12, 23717–23725. [CrossRef] 106. Dai, Z.; Zhang, X.-N.; Nasertorabi, F.; Cheng, Q.; Li, J.; Katz, B.B.; Smbatyan, G.; Pei, H.; Louie, S.G.; Lenz, H.-J.; et al. Synthesis of site-specific antibody-drug conjugates by ADP-ribosyl cyclases. Sci. Adv. 2020, 6, eaba6752. [CrossRef] 107. Brune, K.D.; Howarth, M. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front. Immunol. 2018, 9, 1432. [CrossRef] 108. Bhuket, P.R.N.; Luckanagul, J.A.; Ratnatilaka, P.; Wang, Q. Chemical modification of enveloped viruses for biomedical applications. Integr. Biol. 2018, 10, 666–679. [CrossRef][PubMed] 109. Micklefield, J. Backbone modification of nucleic acids: Synthesis, structure and therapeutic applications. Curr. Med. Chem. 2001, 8, 1157–1179. [CrossRef] 110. Alvarez-Salas, L.M. Nucleic Acids as Therapeutic Agents. Curr. Top. Med. Chem. 2008, 8, 1379–1404. [CrossRef][PubMed] 111. Ochoa, S.; Milam, V.T. Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides. Molecules 2020, 25, 4659. [CrossRef] 112. Opalinska, J.B.; Gewirtz, A.M. Nucleic-acid therapeutics: Basic principles and recent applications. Nat. Rev. Drug Discov. 2002, 1, 503–514. [CrossRef] 113. Sharma, V.K.; Rungta, P.; Prasad, A.K. Nucleic acid therapeutics: Basic concepts and recent developments. RSC Adv. 2014, 4, 16618–16631. [CrossRef] Biomedicines 2021, 9, 628 29 of 35

114. Sharma, V.K.; Watts, J.K. Oligonucleotide therapeutics: Chemistry, delivery and clinical progres. Future Med. Chem. 2015, 7, 2221–2242. [CrossRef][PubMed] 115. Hughes, R.A.; Ellington, A.D. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb. Perspect. Biol. 2017, 9, a023812. [CrossRef][PubMed] 116. Kukwikila, M.; Gale, N.; El-Sagheer, A.H.; Brown, T.; Tavassoli, A. Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation. Nat. Chem. 2017, 9, 1089–1098. [CrossRef] 117. Heravi, M.M.; Tamimi, M.; Yahyavi, H.; Hosseinnejad, T. Huisgen’s Cycloaddition Reactions: A Full Perspective. Curr. Org. Chem. 2016, 20, 1591–1647. [CrossRef] 118. Johansson, J.R.; Beke-Somfai, T.; Stålsmeden, A.S.; Kann, N. Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reaction: Scope, Mechanism, and Applications. Chem. Rev. 2016, 116, 14726–14768. [CrossRef] 119. Zhu, L.; Brassard, C.J.; Zhang, X.; Guha, P.M.; Clark, R.J. On the Mechanism of Copper(I)-Catalyzed Azide-Alkyne Cycloaddition. Chem. Rec. 2016, 16, 1501–1517. [CrossRef] 120. Bonandi, L.; Christodoulou, M.S.; Fumagalli, G.; Perdicchia, D.; Rastelli, G.; Passarella, D. The 1,2,3-triazole ring as a bioisostere in medicinal chemistry. Drug Discov. Today 2017, 22, 1572–1581. [CrossRef] 121. Eremeeva, E.; Herdewijn, P. Non canonical genetic material. Curr. Opin. Biotechnol. 2019, 57, 25–33. [CrossRef] 122. Oliveira, B.L.; Guo, Z.; Bernardes, G.J.L. Inverse electron demand Diels–Alder reactions in chemical biology. Chem. Soc. Rev. 2017, 46, 4895–4950. [CrossRef] 123. Quadrelli, P. Modern Applications of Cycloaddition Chemistry; Elsevier: Amsterdam, The Netherlands, 2019. 124. Uhlmann, E.; Peyman, A. Antisense Oligonucleotides: A New Therapeutic Principle. Chem. Rev. 1990, 90, 543–584. [CrossRef] 125. Kurreck, J. Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. 2003, 270, 1628–1644. [CrossRef] 126. Dean, N.M.; Bennett, C.F. Antisense oligonucleotide-based therapeutics for cancer. Oncogene 2003, 22, 9087–9096. [CrossRef] 127. Tamm, I.; Wagner, M. Antisense therapy in clinical oncology. Mol. Biotechnol. 2006, 33, 221–238. [CrossRef] 128. Corey, D.R. Chemical modification: The key to clinical application of RNA interference? J. Clin. Investig. 2007, 117, 3615–3622. [CrossRef] 129. Bennett, C.F.; Swayze, E.E. RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259–293. [CrossRef] 130. Kole, R.; Krainer, A.R.; Altman, S. RNA therapeutics: Beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discov. 2012, 11, 125–140. [CrossRef] 131. Gallas, A.; Alexander, C.; Davies, M.C.; Puri, S.; Allen, S. Chemistry and formulations for siRNA therapeutics. Chem. Soc. Rev. 2013, 42, 7983–7997. [CrossRef] 132. Hu, B.; Zhong, L.; Weng, Y.; Peng, L.; Huang, Y.; Zhao, Y.; Liang, X.-J. Therapeutic siRNA: State of the art. Signal Transduct. Target. Ther 2020, 5, 101. [CrossRef] 133. Wan, W.B.; Seth, P.P. The Medicinal Chemistry of Therapeutic Oligonucleotides. J. Med. Chem. 2016, 59, 9645–9667. [CrossRef] 134. Freier, S.M.; Altmann, K.-H. The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1997, 25, 4429–4443. [CrossRef] 135. Kasahara, Y.; Kuwahara, M. Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries. J. Nucleic Acids 2012, 2012, 156482. [CrossRef][PubMed] 136. von Matt, P.; Lochmann, T.; Altmann, K.-H. Replacement of the phosphodiester linkage in oligonucleotides by heterocycles: Synthesis of thymidine dinucleotide analogs with triazole-modified backbones. Bioorganic Med. Chem. Lett. 1997, 7, 1549–1552. [CrossRef] 137. von Matt, T.; Altmann, K.-H. Replacement of the phosphodiester linkage in oligonucleotides by heterocycles: The effect of triazole-and imidazole-modified backbones on DNA/RNA duplex stability. Bioorganic Med. Chem. Lett. 1997, 7, 1553–1556. [CrossRef] 138. Lazrek, H.B.; Engels, J.W.; Pfleiderer, W. Synthesis of Novel Branched Nucleoside Dimers Containing a 1,2,3-Triazolyl Linkage. Nucleos. Nucleot. 1998, 17, 1851–1856. [CrossRef] 139. Zhou, L.; Amer, A.; Korn, M.; Burda, R.; Balzarini, J.; De Clercq, E.; Kern, E.R.; Torrence, P.F. Synthesis and antiviral activities of 1,2,3-triazole functionalized thymidines: 1,3-dipolar cycloadditionfor efficient regioselective diversity generation. Antivir. Chem. Chemother. 2005, 16, 375–383. [CrossRef] 140. Nuzzi, A.; Massi, A.; Dondoni, A. Model Studies Toward the Synthesis of Thymidine Oligonucleotides with Triazole Inter- nucleosidic Linkages Via Iterative Cu(I)-Promoted Azide–Alkyne Ligation Chemistry. QSAR Comb. Sci. 2007, 26, 1191–1199. [CrossRef] 141. Kumar, R.; El-Sagheer, A.; Tumpane, J.; Lincoln, P.; Wilhelmsson, L.M.; Brown, T. Template-Directed Oligonucleotide Strand Ligation, Covalent Intramolecular DNA Circularization and Catenation Using Click Chemistry. J. Am. Chem. Soc. 2007, 129, 6859–6864. [CrossRef][PubMed] 142. Sanzone, A.P.; El-Sagheer, A.H.; Brown, T.; Tavassoli, A. Assessing the biocompatibility of click-linked DNA in Escherichia coli. Nucleic Acids Res. 2012, 40, 10567–10575. [CrossRef] 143. El-Sagheer, A.H.; Brown, T. A triazole linkage that mimics the DNA phosphodiester group in living systems. Q. Rev. Biophys. 2015, 48, 429–436. [CrossRef] Biomedicines 2021, 9, 628 30 of 35

144. El-Sagheer, A.H.; Sanzone, A.P.; Gao, R.; Tavassoli, A.; Brown, T. Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli. Proc. Natl. Acad. Sci. USA 2011, 108, 11338–11343. [CrossRef][PubMed] 145. El-Sagheer, A.H.; Brown, T. Efficient RNA synthesis by in vitro transcription of a triazole-modified DNA template. Chem. Commun. 2011, 47, 12057–12058. [CrossRef][PubMed] 146. Routh, A.; Head, S.R.; Ordoukhanian, P.; Johnson, J.E. ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3’-Azido cDNAs. J. Mol. Biol. 2015, 427, 2610–2616. [CrossRef][PubMed] 147. Miura, F.; Fujino, T.; Kogashi, K.; Shibata, Y.; Miura, M.; Isobe, H.; Ito, T. Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA. Nucleic Acids Res. 2018, 46, e95. [CrossRef] 148. Osman, E.A.; Gadzikwa, T.; Gibbs, J.M. Quick Click: The DNA-Templated Ligation of 3’-O-Propargyl- and 5’-Azide-Modified Strands Is as Rapid as and More Selective than Ligase. ChemBioChem 2018, 19, 2081–2087. [CrossRef] 149. El-Sagheer, A.H.; Kumar, R.; Findlow, S.; Werner, J.M.; Lane, A.N.; Brown, T. A Very Stable Cyclic DNA Miniduplex with Just Two Base Pairs. ChemBioChem 2008, 9, 50–52. [CrossRef] 150. Yang, H.; Seela, F. “Bis-Click” Ligation of DNA: Template-Controlled Assembly, Circularisation and Functionalisation with Bifunctional and Trifunctional Azides. Chem. Eur. J. 2017, 23, 3375–3385. [CrossRef] 151. Xiong, H.; Seela, F. Stepwise “Click” Chemistry for the Template Independent Construction of a Broad Variety of Cross-Linked Oligonucleotides: Influence of Linker Length, Position, and Linking Number on DNA Duplex Stability. J. Org. Chem. 2011, 76, 5584–5597. [CrossRef] 152. Lietard, J.; Meyer, A.; Vasseur, J.-J.; Morvan, F. New Strategies for Cyclization and Bicyclization of Oligonucleotides by Click Chemistry Assisted by Microwaves. J. Org. Chem. 2008, 73, 191–200. [CrossRef] 153. Padgett, R.A.; Konarska, M.M.; Grabowski, P.J.; Hardy, S.F.; Sharp, P.A. Lariat RNA’s as intermediates and products in the splicing of messenger RNA precursors. Science 1984, 225, 898–903. [CrossRef] 154. Nakane, M.; Ichikawa, S.; Matsuda, A. Triazole-Linked Dumbbell Oligodeoxynucleotides with NF-κB Binding Ability as Potential Decoy Molecules. J. Org. Chem. 2008, 73, 1842–1851. [CrossRef] 155. Vasilyeva, S.V.; Levina, A.S.; Li-Zhulanov, N.S.; Shatskaya, N.V.; Baiborodin, S.I.; Repkova, M.N.; Zarytova, V.F.; Mazurkova, 0 N.A.; Silnikov, V.N. SiO2 nanoparticles as platform for delivery of 3 -triazole analogues of AZT-triphosphate into cells. Bioorg. Med. Chem. 2015, 23, 2168–2175. [CrossRef][PubMed] 156. Cassinelli, V.; Oberleitner, B.; Sobotta, J.; Nickels, P.; Grossi, G.; Kempter, S.; Frischmuth, T.; Liedl, T.; Manetto, A. One-Step Formation of “Chain-Armor”-Stabilized DNA Nanostructures. Angew. Chem. Int. Ed. 2015, 54, 7795–7798. [CrossRef] 157. Wirges, C.T.; Gramlich, P.M.E.; Gutsmiedl, K.; Gierlich, J.; Burley, G.A.; Carell, T. Pronounced Effect of DNA Hybridization on Click Reaction Efficiency. QSAR Comb. Sci. 2007, 26, 1159–1164. [CrossRef] 158. Gierlich, J.; Burley, G.A.; Gramlich, P.M.E.; Hammond, D.M.; Carell, T. Click Chemistry as a Reliable Method for the High-Density Postsynthetic Functionalization of Alkyne-Modified DNA. Org. Lett. 2006, 8, 3639–3642. [CrossRef] 159. Burley, G.A.; Gierlich, J.; Mofid, M.R.; Nir, H.; Tal, S.; Eichen, Y.; Carell, T. Directed DNA Metallization. J. Am. Chem. Soc. 2006, 128, 1398–1399. [CrossRef] 160. Gierlich, J.; Gutsmiedl, K.; Gramlich, P.M.; Schmidt, A.; Burley, G.A.; Carell, T. Synthesis of Highly Modified DNA by a Combination of PCR with Alkyne-Bearing Triphosphates and Click Chemistry. Chem. Eur. J. 2007, 13, 9486–9494. [CrossRef] 161. Gramlich, P.M.E.; Wirges, C.T.; Gierlich, J.; Carell, T. Synthesis of Modified DNA by PCR with Alkyne-Bearing Purines Followed by a Click Reaction. Org. Lett. 2008, 10, 249–251. [CrossRef] 162. Su, M.; Kirchner, A.; Stazzoni, S.; Müller, M.; Wagner, M.; Schröder, A.; Carell, T. 5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites. Angew. Chem. Int. Ed. 2016, 55, 11797–11800. [CrossRef][PubMed] 163. Gramlich, P.M.E.; Wirges, C.T.; Manetto, A.; Carell, T. Postsynthetic DNA modification through the copper-catalyzed azide-alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 2008, 47, 8350–8358. [CrossRef] 164. O’Mahony, G.; Ehrman, E.; Grøtli, M. Synthesis of adenosine-based fluorosides containing a novel heterocyclic ring system. Tetrahedron Lett. 2005, 46, 6745–6748. [CrossRef] 165. Mathew, S.C.; By, Y.; Berthault, A.; Virolleaud, M.-A.; Carrega, L.; Chouraqui, G.; Commeiras, L.; Condo, J.; Attolini, M.; Gaudel-Siri, A.; et al. Expeditious synthesis and biological evaluation of new C-6 1,2,3-triazole adenosine derivatives A1 receptorantagonists or agonists. Org. Biomol. Chem. 2010, 8, 3874–3881. [CrossRef][PubMed] 166. Baker, Y.R.; Traoré, D.; Wanat, P.; Tyburn, A.; El-Sagheer, A.H.; Brown, T. Searching for the ideal triazole: Investigating the 1,5-triazole as a charge neutral DNA backbone mimic. Tetrahedron 2020, 76, 130914. [CrossRef] 167. Kumar, A.S. Design and synthesis of double-headed nucleosides by using click chemistry approach. Chem. Data Collect. 2020, 28, 100468. 168. Seela, F.; Sirivolu, V.R. DNA Containing Side Chains with Terminal Triple Bonds: Base-Pair Stability and Functionalization of Alkynylated Pyrimidines and 7-Deazapurines. Chem. Biodivers. 2006, 3, 509–514. [CrossRef] 169. Seela, F.; Ming, X. Oligonucleotides Containing 7-Deaza-20-deoxyinosine as Universal Nucleoside: Synthesis of 7-Halogenated and 7-Alkynylated Derivatives, Ambiguous Base Pairing, and Dye Functionalization by the Alkyne–Azide ‘Click’ Reaction. Helv. Chim. Acta 2008, 91, 1181–1200. [CrossRef] 170. Seela, F.; Sirivolu, V.R. Nucleosides and Oligonucleotides with Diynyl Side Chains: Base Pairing and Functionalization of 20-Deoxyuridine Derivatives by the Copper(I)-Catalyzed Alkyne–Azide ‘Click’ Cycloaddition. Helv. Chim. Acta 2007, 90, 535–552. [CrossRef] Biomedicines 2021, 9, 628 31 of 35

171. Sirivou, V.R.; Chittepu, P.; Seela, F. DNA with Branched Internal Side Chains: Synthesis of 5-Tripropargylamine-dU and Conjugation by an Azide-Alkyne Double Click Reaction. ChemBioChem 2008, 9, 2305–2316. [CrossRef] 172. Seela, F.; Sirivolu, V.R. Pyrrolo-dColigonucleotides bearing alkynyl side chains with terminal triple bonds: Synthesis, base pairing and fluorescent dye conjugates prepared by the azide–alkyne “click” reaction. Org. Biomol. Chem. 2008, 6, 1674–1687. [CrossRef] 173. Seela, F.; Sirivolu, V.R.; Chittepu, P. Modification of DNA with Octadiynyl Side Chains: Synthesis, Base Pairing, and Formation of Fluorescent Coumarin Dye Conjugates of Four Nucleobases by the Alkyne−Azide “Click” Reaction. Bioconjugate Chem. 2008, 19, 211–224. [CrossRef] 174. El-Sagheer, A.H.; Brown, T. Click chemistry with DNA. Chem. Soc. Rev. 2010, 39, 1388–1405. [CrossRef] 175. Berndl, S.; Herzig, N.; Kele, P.; Lachmann, D.; Li, X.; Wolfbeis, O.S.; Wagenknecht, H.-A. Comparison of a Nucleosidic vs. Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes. Bioconjug. Chem. 2009, 20, 558–564. [CrossRef] 176. Géci, I.; Filichev, V.V.; Pedersen, E.B. Stabilization of Parallel Triplexes by Twisted Intercalating Nucleic Acids (TINAs) Incorporat- ing 1,2,3-Triazole Units and Prepared by Microwave-Accelerated Click Chemistry. Chem. Eur. J. 2007, 13, 6379–6386. [CrossRef] [PubMed] 177. Lucas, R.; Neto, V.; Bouazza, A.H.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y. Microwave-assisted synthesis of a triazole-linked 30–50 dithymidine using click chemistry. Tetrahedron Lett. 2008, 49, 1004–1007. [CrossRef] 178. Lucas, R.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y. A rapid efficient microwave-assisted synthesis of a 30,50- pentathymidine by copper(I)-catalyzed [3 + 2] cycloaddition. Tetrahedron 2008, 64, 5467–5471. [CrossRef] 179. Isobe, H.; Fujino, T.; Yamazaki, N.; Guillot-Nieckowski, M.; Nakamura, E. Triazole-Linked Analogue of Deoxyribonucleic Acid (TLDNA): Design, Synthesis, and Double-Strand Formation with Natural DNA. Org. Lett. 2008, 10, 3729–3732. [CrossRef] [PubMed] 180. Varizhuk, A.; Chizhov, A.; Smirnov, I.; Kaluzhny, D.; Florentiev, V. Triazole-Linked Oligonucleotides with Mixed-Base Sequences: Synthesis and Hybridization Properties. Eur. J. Org. Chem. 2012, 11, 2173–2179. [CrossRef] 181. Fujino, T.; Tsunaka, N.; Guillot-Nieckowski, M.; Nakanishi, W.; Iwamoto, T.; Nakamura, E.; Isobe, H. Synthesis and structures of deoxyribonucleoside analogues for triazole-linked DNA (TLDNA). Tetrahedron Lett. 2010, 51, 2036–2038. [CrossRef] 182. Chandrasekhar, S.; Srihari, P.; Nagesh, C.; Kiranmai, N.; Nagesh, N.; Idris, M.M. Synthesis of Readily Accessible Triazole-Linked Dimer Deoxynucleoside Phosphoramidite for Solid-Phase Oligonucleotide Synthesis. Synthesis 2010, 21, 3710–3714. [CrossRef] 183. Varizhuk, A.; Chizhov, A.; Florentiev, V. Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents. Bioorg. Chem. 2011, 39, 127–131. [CrossRef] 184. Fujino, T.; Yamazaki, N.; Isobe, H. Convergent synthesis of oligomers of triazole-linked DNA analogue (TLDNA) in solution phase. Tetrahedron Lett. 2009, 50, 4101–4103. [CrossRef] 185. Varizhuk, A.M.; Kaluzhny, D.N.; Novikov, R.A.; Chizhov, A.O.; Smirnov, I.P.; Chuvilin, A.N.; Tatarinova, O.N.; Fisunov, G.Y.; Pozmogova, G.E.; Florentiev, V.L. Synthesis of Triazole-Linked Oligonucleotides with High Affinity to DNA Complements and an Analysis of Their Compatibility with Biosystems. J. Org. Chem. 2013, 78, 5964–5969. [CrossRef] 186. Madhuri, V.; Kumar, V.A. Design and Synthesis of Dephosphono DNA Analogues Containing 1,2,3-Triazole Linker and Their UV-Melting Studies with DNA/RNA. Nucleos. Nucleot. Nucl. 2012, 31, 97–111. [CrossRef][PubMed] 187. El-Sagheer, A.; Brown, T. Synthesis and Polymerase Chain Reaction Amplification of DNA Strands Containing an Unnatural Triazole Linkage. J. Am. Chem. Soc. 2009, 131, 3958–3964. [CrossRef] 188. Koˇcalka,P.; El-Sagheer, A.H.; Brown, T. Rapid and Efficient DNA Strand Cross-Linking by Click Chemistry. ChemBioChem 2008, 9, 1280–1285. [CrossRef] 189. Dallmann, A.; El-Sagheer, A.H.; Dehmel, L.; Mügge, C.; Griesinger, C.; Ernsting, N.P.; Brown, T. Structure and Dynamics of Triazole-Linked DNA: Biocompatibility Explained. Chem. Eur. J. 2011, 17, 14714–14717. [CrossRef] 190. El-Sagheer, A.H.; Brown, T. Combined nucleobase and backbone modifications enhance DNA duplex stability and preserve biocompatibility. Chem. Sci. 2014, 5, 253–259. [CrossRef] 191. Shivalingam, A.; Tyburn, A.E.S.; El-Sagheer, A.H.; Brown, T. Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation. J. Am. Chem. Soc. 2017, 139, 1575–1583. [CrossRef] 192. El-Sagheer, A.H.; Brown, T. Click Nucleic Acid Ligation: Applications in Biology and Nanotechnology. Acc. Chem. Res. 2012, 45, 1258–1267. [CrossRef] 193. Birts, C.N.; Sanzone, A.P.; El-Sagheer, A.H.; Blaydes, J.P.; Brown, T.; Tavassoli, A. Transcription of click-linked DNA in human cells. Angew. Chem. Int. Ed. 2014, 53, 2362–2365. [CrossRef] 194. Chen, J.; Baker, Y.R.; Brown, A.; El-Sagheer, A.H.; Brown, T. Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation. Chem. Sci. 2018, 9, 8110–8120. [CrossRef][PubMed] 195. Krishna, H.; Caruthers, M.H. Alkynyl Phosphonate DNA: A Versatile “Click” able Backbone for DNA-Based Biological Applica- tions. J. Am. Chem. Soc. 2012, 134, 11618–11631. [CrossRef] 196. Guo, P.; Haque, F.; Hallahan, B.; Reif, R.; Li, H. Uniqueness, advantages, challenges, solutions, and perspectives in therapeutics applying RNA nanotechnology. Nucleic Acid Ther. 2012, 22, 226–245. [CrossRef] Biomedicines 2021, 9, 628 32 of 35

197. Habibian, M.; Harikrishna, S.; Fakhoury, J.; Barton, M.; Ageely, E.A.; Cencic, R.; Fakih, H.H.; Katolik, A.; Takahashi, M.; Rossi, J.; et al. Effect of 20-50/30-50 phosphodiester linkage heterogeneity on RNA interference. Nucleic Acids Res. 2020, 48, 4643–4657. [CrossRef] 198. Kumar, V.A.; Ganesh, K.N. Structure-Editing of Nucleic Acids for Selective Targeting of RNA. Curr. Top. Med. Chem. 2007, 7, 715–726. [CrossRef][PubMed] 199. Sosson, M.; Pfeffer, D.; Richert, C. Enzyme-free ligation of dimers and trimers to RNA primers. Nucleic Acids Res. 2019, 47, 3836–3845. [CrossRef][PubMed] 200. Wang, X.; He, C. Dynamic RNA Modifications in Posttranscriptional Regulation. Mol. Cell 2014, 56, 5–12. [CrossRef] 201. Chen, K.; Zhao, B.S.; He, C. Nucleic Acid Modifications in Regulation of Gene Expression. Cell Chem. Biol. 2016, 23, 74–85. [CrossRef] 202. Roundtree, I.A.; Evans, M.E.; Pan, T.; He, C. Dynamic RNA Modifications in Gene Expression Regulation. Cell 2017, 169, 1187–1200. [CrossRef] 203. Schaefer, M.; Kapoor, U.; Jantsch, M.F. Understanding RNA modifications: The promises and technological bottlenecks of the ‘epitranscriptome’. Open Biol. 2017, 7, 170077. [CrossRef] 204. Lee, Y.J.; Moon, T.S. Design rules of synthetic non-coding RNAs in bacteria. Methods 2018, 143, 58–69. [CrossRef] 205. Khawar, M.B.; Mehmood, R.; Abbasi, M.H.; Sheikh, N. Multifactorial role of long non-coding RNAs (LncRNAs) in hematopoiesis. Front. Biosci. 2018, 10, 119–126. 206. Yu, A.-M.; Jian, C.; Yu, A.H.; Tu, M.-J. RNA therapy: Are we using the right molecules? Pharmacol. Ther. 2019, 196, 91–104. [CrossRef] 207. Weng, Y.; Xiao, H.; Zhang, J.; Liang, X.-J.; Huang, Y. RNAi therapeutic and its innovative biotechnological evolution. Biotechnol. Adv. 2019, 37, 801–825. [CrossRef][PubMed] 208. Qadir, M.I.; Bukhat, S.; Rasul, S.; Manzoor, H.; Manzoor, M. RNA therapeutics: Identification of novel targets leading to drug discovery. J. Cell. Biochem. 2020, 121, 898–929. [CrossRef][PubMed] 209. Yu, A.-M.; Choi, Y.H.; Tu, M.-J. RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges. Pharmacol. Rev. 2020, 72, 862–898. [CrossRef][PubMed] 210. Costales, M.G.; Childs-Disney, J.L.; Haniff, H.S.; Disney, M.D. How We Think about Targeting RNA with Small Molecules. J. Med. Chem. 2020, 63, 8880–8900. [CrossRef][PubMed] 211. Akman, H.B.; Bensan, A.E.E. Noncoding RNAs and cancer. Turk. J. Biol. 2014, 38, 817–828. [CrossRef] 212. Yu, X.; Zheng, H.; Chan, M.T.V.; Wu, W.K.K. HULC: An oncogenic long non-coding RNA in human cancer. J. Cell. Mol. Med. 2017, 21, 410–417. [CrossRef][PubMed] 213. Wang, W.-T.; Han, C.; Sun, Y.-M.; Chen, T.-Q.; Chen, Y.-Q. Noncoding RNAs in cancer therapy resistance and targeted drug development. J. Hematol. Oncol. 2019, 12, 55. [CrossRef] 214. Selvam, C.; Mutisya, D.; Prakash, S.; Ranganna, K.; Thilagavathi, R. Therapeutic potential of chemically modified siRNA: Recent trends. Chem. Biol. Drug Des. 2017, 90, 665–678. [CrossRef][PubMed] 215. Holdt, L.M.; Kohlmaier, A.; Teupser, D. Circular RNAs as Therapeutic Agents and Targets. Front. Physiol. 2018, 9, 1262. [CrossRef] 216. Seok, H.; Lee, H.; Jang, E.-S.; Chi, S.W. Evaluation and control of miRNA-like off-target repression for RNA interference. Cell. Mol. Life Sci. 2018, 75, 797–814. [CrossRef] 217. Onizuka, K.; Shibata, A.; Taniguchi, Y.; Sasaki, S. Pin-point chemical modification of RNA with diverse molecules through the functionality transfer reaction and the copper-catalyzed azide–alkyne cycloaddition reaction. Chem. Commun. 2011, 47, 5004–5006. [CrossRef] 218. Pujari, S.S.; Leonard, P.; Seela, F. Oligonucleotides with “Clickable” Sugar Residues: Synthesis, Duplex Stability, and Terminal versus Central Interstrand Cross-Linking of 20-O-Propargylated 2-Aminoadenosine with a Bifunctional Azide. J. Org. Chem. 2014, 79, 4423–4437. [CrossRef][PubMed] 219. Wang, W.; Chen, K.; Qu, D.; Chi, W.; Xiong, W.; Huang, Y.; Wen, J.; Feng, S.; Zhang, B. One pot conjugation of small molecules to RNA using click chemistry. Tetrahedron Lett. 2012, 53, 6747–6750. [CrossRef] 220. Peel, B.J.; Hagen, G.; Krishnamurthy, K.; Desaulniers, J.-P. Conjugation and Evaluation of Small Hydrophobic Molecules to Triazole-Linked siRNAs. ACS Med. Chem. Lett. 2015, 6, 117–122. [CrossRef][PubMed] 221. Costales, M.G.; Rzuczek, S.G.; Disney, M.D. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA. Bioorganic Med. Chem. Lett. 2016, 26, 2605–2609. [CrossRef][PubMed] 222. Zheng, Y.; Beal, P.A. Synthesis and evaluation of an alkyne-modified ATP analog for enzymatic incorporation into RNA. Bioorganic Med. Chem. Lett. 2016, 26, 1799–1802. [CrossRef] 223. Atdjian, C.; Coelho, D.; Iannazzo, L.; Ethève-Quelquejeu, M.; Braud, E. Synthesis of Triazole-Linked SAM-Adenosine Conjugates: Functionalization of Adenosine at N-1 or N-6 Position without Protecting Groups. Molecules 2020, 25, 3241. [CrossRef] 224. Sau, S.P.; Hrdlicka, P.J. C20-Pyrene-Functionalized Triazole-Linked DNA: Universal DNA/RNA Hybridization Probes. J. Org. Chem. 2012, 77, 5–16. [CrossRef][PubMed] 225. Velema, W.A.; Kool, E.T. The chemistry and applications of RNA 20-OH acylation. Nat. Rev. Chem. 2020, 4, 22–37. [CrossRef] 226. Haugland, M.M.; El-Sagheer, A.; Porter, R.J.; Peña, J.; Brown, T.; Anderson, E.A.; Lovett, J.E. 20-Alkynylnucleotides: A Sequence- and Spin Label-Flexible Strategy for EPR Spectroscopy in DNA. J. Am. Chem. Soc. 2016, 138, 9069–9072. [CrossRef] Biomedicines 2021, 9, 628 33 of 35

227. Lorenz, D.A.; Garner, A.L. A click chemistry-based microRNA maturation assay optimized for high-throughput screening. Chem. Commun. 2016, 52, 8267–8270. [CrossRef] 228. El-Sagheer, A.H.; Brown, T. New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes. Proc. Natl. Acad. Sci. USA 2010, 107, 15329–15334. [CrossRef] 229. Aigner, A.; Hartl, M.; Fauster, K.; Steger, J.; Bister, K.; Micura, R. Chemical Synthesis of Site-Specifically 20-Azido-Modified RNA and Potential Applications for Bioconjugation and RNA Interference. ChemBioChem 2011, 12, 47–51. [CrossRef][PubMed] 230. Fauster, K.; Hartl, M.; Santner, T.; Aigner, M.; Kreutz, C.; Bister, K.; Ennifar, E.; Micura, R. 20-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling. ACS Chem. Biol. 2012, 7, 581–589. [CrossRef] 231. Staudinger, H.; Meyer, J. Über neue organische Phosphorverbindungen III. Phosphinmethylenderivate und Phosphinimine. Helv. Chim. Acta 1919, 2, 635–646. [CrossRef] 232. Micura, R. Cyclic Oligoribonucleotides (RNA) by Solid-Phase Synthesis. Chem. Eur. J. 1999, 5, 2077–2082. [CrossRef] 233. Mutisya, D.; Selvam, C.; Kennedy, S.D.; Rozners, E. Synthesis and properties of triazole-linked RNA. Bioorganic Med. Chem. Lett. 2011, 21, 3420–3422. [CrossRef][PubMed] 234. Fujino, T.; Endo, K.; Yamazaki, N.; Isobe, H. Synthesis of Triazole-linked Analogues of RNA (TLRNA). Chem. Lett. 2012, 41, 403–405. [CrossRef] 235. Fujino, T.; Suzuki, T.; Ooi, T.; Ikemoto, K.; Isobe, H. Duplex-forming Oligonucleotide of Triazole-linked RNA. Chem. Asian J. 2019, 14, 3380–3385. [CrossRef] 236. Paredes, E.; Das, S.R. Click chemistry for rapid labeling and ligation of RNA. ChemBioChem 2011, 12, 125–131. [CrossRef] 237. Muthmann, N.; Hartstock, K.; Rentmeister, A. Chemo-enzymatic treatment of RNA to facilitate analyses. WIREs RNA 2020, 11, e1561. [CrossRef][PubMed] 238. Palumbo, C.M.; Beal, P.A. Nucleoside analogs in the study of the epitranscriptome. Methods 2019, 156, 46–52. [CrossRef][PubMed] 239. Meter, E.N.V.; Onyango, J.A.; Teske, K.A. A review of currently identified small molecule modulators of microRNA function. Eur. J. Med. Chem. 2020, 188, 112008. [CrossRef] 240. Desaulniers, J.-P.; Hagen, G.; Anderson, J.; McKim, C.; Roberts, B. Effective gene-silencing of siRNAs that contain functionalized spacer linkages within the central region. RSC Adv. 2017, 7, 3450–3454. [CrossRef] 241. Efthymiou, T.C.; Huynh, V.; Oentoro, J.; Peel, B.; Desaulniers, J.-P. Efficient synthesis and cell-based silencing activity of siRNAS that contain triazole backbone linkages. Bioorganic Med. Chem. Lett. 2012, 22, 1722–1726. [CrossRef] 242. Hagen, G.; Peel, B.J.; Samis, J.; Desaulniers, J.-P. Synthesis and in vitro assessment of chemically modified siRNAs targeting BCL2 that contain 20-ribose and triazole-linked backbone modifications. Med. Chem. Commun. 2015, 6, 1210–1215. [CrossRef] 243. Piecyk, M.; Jankowska-Anyszka, M. Chemical conjugation of an mRNA cap analogue with a cell-penetrating peptide as a potential membrane permeable translation inhibitor. Tetrahedron Lett. 2014, 55, 606–609. [CrossRef] 244. Shanmugasundaram, M.; Charles, I.; Kore, A.R. Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety. Bioorg. Med. Chem. 2016, 24, 1204–1208. [CrossRef] 245. Gillingham, D.; Shahid, R. Catalysts for RNA and DNA modification. Curr. Opin. Chem. Biol. 2015, 25, 110–114. [CrossRef] 246. Warminski, M.; Sikorski, P.J.; Kowalska, J.; Jemielity, J. Applications of Phosphate Modification and Labeling to Study (m)RNA Caps. Top. Curr. Chem. 2017, 375, 16. [CrossRef] 247. Mamot, A.; Sikorski, P.J.; Warminski, M.; Kowalska, J.; Jemielity, J. Azido-Functionalized 5’ Cap Analogues for the Preparation of Translationally Active mRNAs Suitable for Fluorescent Labeling in Living Cells. Angew. Chem. Int. Ed. 2017, 56, 15628–15632. [CrossRef] 248. Walczak, S.; Nowicka, A.; Kubacka, D.; Fac, K.; Wanat, P.; Mroczek, S.; Kowalska, J.; Jemielity, J. A novel route for preparing 50 cap mimics and capped RNAs: Phosphate-modified cap analogues obtained via click chemistry. Chem. Sci. 2017, 8, 260–267. [CrossRef] 249. A Nobel Prize for genetic scissors. Nat. Mater. 2021, 20, 1. [CrossRef] 250. Fernholm, A. Genetic Scissors: A Tool for Rewriting the Code of Life; The Royal Swedish Academy of Sciences: Stockholm, Sweden, 2020. 251. Hagedorn, P.H.; Persson, R.; Funder, E.D.; Albæk, N.; Diemer, S.L.; Hansen, D.J.; Møller, M.R.; Papargyri, N.; Christiansen, H.; Hansen, B.R.; et al. Locked nucleic acid: Modality, diversity, and drug discovery. Drug Discov. Today 2018, 23, 101–114. [CrossRef] [PubMed] 252. Braasch, D.A.; Corey, D.R. Locked nucleic acid (LNA): Fine-tuning the recognition of DNA and RNA. Chem. Biol. 2001, 8, 1–7. [CrossRef] 253. Campbell, M.A.; Wengel, J. Locked vs. unlocked nucleic acids (LNA vs.UNA): Contrasting structures work towards common therapeutic goals. Chem. Soc. Rev. 2011, 40, 5680–5689. [CrossRef] 254. Imanishi, T.; Obika, S. BNAs: Novel nucleic acid analogs with a bridged sugar moiety. Chem. Commun. 2002, 1653–1659. [CrossRef] 255. Mitsuoka, Y.; Yamamoto, T.; Kugimiya, A.; Waki, R.; Wada, F.; Tahara, S.; Sawamura, M.; Noda, M.; Fujimura, Y.; Kato, Y.; et al. Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency. J. Org. Chem. 2017, 82, 12–24. [CrossRef] 256. Obika, S.; Rahman, S.M.A.; Fujisaka, A.; Kaawada, Y.; Baba, T.; Imanishi, T. Bridged Nucleic Acids: Development, Synthesis and Properties. Heterocycles 2010, 81, 1347–1392. [CrossRef] Biomedicines 2021, 9, 628 34 of 35

257. Hari, Y. Bridged Nucleosides as Building Blocks of Oligonucleotides: Synthesis and Properties. Heterocycles 2020, 100, 681–717. [CrossRef] 258. Bryld, T.; Sørensen, M.H.; Nielsen, P.; Koch, T.; Nielsen, C.; Wengel, J. Synthesis and antiviral evaluation of novel conformationally locked nucleosides and masked 50-phosphate derivatives thereof. J. Chem. Soc. Perkin Trans. 1 2002, 14, 1655–1662. [CrossRef] 259. Sharma, V.K.; Rungta, P.; Maikhuri, V.K.; Prasad, A.K. An astute synthesis of locked nucleic acid monomers. Sustain. Chem. Process. 2015, 3, 2. [CrossRef] 260. Sharma, V.K.; Singh, S.K.; Krishnamurthy, P.M.; Alterman, J.F.; Haraszti, R.A.; Khvorova, A.; Prasad, A.K.; Watts, J.K. Synthesis and biological properties of triazole-linked locked nucleic acid. Chem. Commun. 2017, 53, 8906–8909. [CrossRef] 261. Yamashita, S.; Nishida, K.; Osawa, T.; Nakanishi, A.; Ito, J.; Hari, Y. Synthesis of Oligonucleotides Containing 20-N- alkylaminocarbonyl-20-amino-LNA (20-urea-LNA) Moieties Using Post-Synthetic Modification Strategy. Molecules 2020, 25, 346. [CrossRef] 262. Singh, S.K.; Sharma, V.K.; Bohra, K.; Olsen, C.E.; Prasad, A.K. Synthesis of Triazole-linked LNA-based Non-ionic Nucleoside Dimers Using Cu(I)- Catalyzed ‘Click’ Reaction. Curr. Org. Synth. 2014, 11, 757–766. [CrossRef] 263. Srivastava, S.; Singh, S.K.; Sharma, V.K.; Mangla, P.; Olsen, C.E.; Prasad, A.K. Design and Synthesis of Triazole-Linked xylo- Nucleoside Dimers. Nucleos. Nucleot. Nucl. 2015, 34, 388–399. [CrossRef][PubMed] 264. Kumar, P.; El-Sagheer, A.H.; Truong, L.; Brown, T. Locked nucleic acid (LNA) enhances binding affinity of triazole-linked DNA towards RNA. Chem. Commun. 2017, 53, 8910–8913. [CrossRef] 265. Kumar, P.; Truong, L.; Baker, Y.R.; El-Sagheer, A.H.; Brown, T. ; Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs). ACS Omega 2018, 3, 6976–6987. [CrossRef] 266. Larsen, H.J.; Bentin, T.; Nielsen, P.E. Antisense properties of peptide nucleic acid. Biochim. Biophys. Acta 1999, 1489, 159–166. [CrossRef] 267. Soomets, U.; Hällbrink, M.; Langel, Ü. Antisense properties of peptide nucleic acid. Front. Biosci. 1999, 4, 782–786. [CrossRef] 268. Cetojević-Simin,D.;ˇ Jakimov, D.; Mrdanović,J.;¯ Bogdanović,V.; Kojić,V.; Andrijević,L.; Bogdanović,G. Peptide nucleic acid: Sequence specific recognition in cancer diagnostics and gene therapy. Arch. Oncol. 2001, 9, 33–37. 269. Piacenti, V.; Langella, E.; Autiero, I.; Nolan, J.C.; Piskareva, O.; Adamo, M.F.A.; Saviano, M.; Moccia, M. A combined experimental and computational study on peptide nucleic acid (PNA) analogues of tumor suppressive miRNA-34a. Bioorg. Chem. 2019, 91, 103165. [CrossRef] 270. Holub, J.M.; Kirshenbaum, K. Tricks with clicks: Modification of peptidomimetic oligomers via copper-catalyzed azide-alkyne [3 + 2] cycloaddition. Chem. Soc. Rev. 2010, 39, 1325–1337. [CrossRef] 271. Amant, A.H.S.; Engbers, C.; Hudson, R.H.E. A solid-phase CuAAC strategy for the synthesis of PNA containing nucleobase surrogates. Artif. Dna Pna Xna 2013, 4, 4–10. [CrossRef] 272. Chouikhi, D.; Barluenga, S.; Winssinger, N. Clickable peptide nucleic acids (cPNA) with tunable affinity. Chem. Commun. 2010, 46, 5476–5478. [CrossRef] 273. Thomson, S.A.; Josey, J.A.; Cadilla, R.; Gaul, M.D.; Hassman, C.F.; Luzzio, M.J.; Pipe, A.J.; Reed, K.L.; Ricca, D.J.; Wiethe, R.W.; et al. Fmoc mediated synthesis of Peptide Nucleic Acids. Tetrahedron 1995, 51, 6179–6194. [CrossRef] 274. Efthymiou, T.C.; Desaulniers, J.-P. Synthesis and properties of oligonucleotides that contain a triazole-linked nucleic acid dimer. J. Heterocyclic Chem. 2011, 48, 533–539. [CrossRef] 275. Vergnaud, J.; Faugerasa, P.-A.; Chaleix, V.; Champavier, Y.; Zerrouki, R. Design of a new oligotriazole peptide nucleic acid analogue (oT-PNA). Tetrahedron Lett. 2011, 52, 6185–6189. [CrossRef] 276. Marin, V.L.; Armitage, B.A. RNA Guanine Quadruplex Invasion by Complementary and Homologous PNA Probes. J. Am. Chem. Soc. 2005, 127, 8032–8033. [CrossRef] 277. Gudanis, D.; Kaniowski, D.; Kulik, K.; Baranowski, D.; Gdaniec, Z.; Nawrot, B. Formation of an RNA Quadruplex-Duplex Hybrid in Living Cells between mRNA of the Epidermal Growth Factor Receptor (EGFR) and a G-Rich Antisense Oligoribonucleotide. Cells 2020, 9, 2375. [CrossRef] 278. Solier, S.; Müller, S.; Rodriguez, R. Whole-genome mapping of small-molecule targets for cancer medicine. Curr. Opin. Chem. Biol. 2020, 56, 42–50. [CrossRef][PubMed] 279. Schärer, O.D. Chemistry and biology of DNA repair. Angew. Chem. Int. Ed. 2003, 42, 2946–2974. [CrossRef][PubMed] 280. Xu, Y. Chemistry in human telomere biology: Structure, function and targeting of telomere DNA/RNA. Chem. Soc. Rev. 2011, 40, 2719–2740. [CrossRef] 281. Kim, N.W.; Piatyszek, M.A.; Prowse, K.R.; Harley, C.B.; West, M.D.; Ho, P.L.; Coviello, G.M.; Wright, W.E.; Weinrich, S.L.; Shay, J.W. Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266, 2011–2015. [CrossRef] 282. Xu, Y.; Suzuki, Y.; Komiyama, M. Click chemistry for the identification of G-quadruplex structures: Discovery of a DNA-RNA G-quadruplex. Angew. Chem. Int. Ed. 2009, 48, 3281–3284. [CrossRef] 283. Saha, P.; Panda, D.; Dash, J. The application of click chemistry for targeting quadruplex nucleic acids. Chem. Commun. 2019, 55, 731–750. [CrossRef] 284. Varizhuk, A.M.; Tsvetkov, V.B.; Tatarinova, O.N.; Kaluzhny, D.N.; Florentiev, V.L.; Timofeev, E.N.; Shchyolkina, A.K.; Borisova, O.F.; Smirnov, I.P.; Grokhovsky, S.L.; et al. Synthesis, characterization and in vitro activity of thrombin-binding DNA aptamers with triazole internucleotide linkages. Eur. J. Med. Chem. 2013, 67, 90–97. [CrossRef] Biomedicines 2021, 9, 628 35 of 35

285. Xu, Y.; Ishizuka, T.; Yang, J.; Ito, K.; Katada, H.; Komiyama, M.; Hayashi, T. Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres. J. Biol. Chem. 2012, 287, 41787–41796. [CrossRef] 286. Xu, Y.; Suzuki, Y.; Ishizuka, T.; Xiao, C.-D.; Liu, X.; Hayashi, T. Finding a human telomere DNA–RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: A protective structure for telomere end. Bioorg. Med. Chem. 2014, 22, 4419–4421. [CrossRef] 287. Ishizuka, T.; Xu, Y. Click Chemistry Takes a Snapshot of DNA-RNA Hybrid G-Quadruplex in Living Cells. J. Nat. Sci. 2016, 2, e237.