molecules Review Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach Karolina Bartosik , Katarzyna Debiec , Anna Czarnecka , Elzbieta Sochacka and Grazyna Leszczynska * Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; [email protected] (K.B.); [email protected] (K.D.); [email protected] (A.C.); [email protected] (E.S.) * Correspondence: [email protected]; Tel.: +48-42-6313150 Academic Editor: Katherine Seley-Radtke Received: 20 June 2020; Accepted: 20 July 2020; Published: 23 July 2020 Abstract: The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels. Keywords: phosphoramidite chemistry; RNA solid-phase synthesis; post-synthetic RNA modification; convertible nucleoside 1. Introduction Recently, a significant interest has been observed in the use of modified oligoribonucleotides in the fields of molecular biology, biochemistry, and medicine [1–3]. The representative sites of RNA modifications are the 50- and 30-ends of oligoribonucleotides, and the 20-positions of the ribose, phosphodiester residues, and nucleobase moieties. Notably, among 150 modified nucleosides identified in cellular RNA sequences, more than 95% of functional groups are installed in the nucleobase heterocycles (Figure1)[ 4,5]. Improved mass spectrometry approaches and highly sensitive chemical screens in cellular RNA isolation are still extending this list with new base-modifying units, e.g., cyclic N6-threonylcarbamoyladenosines (ct6A, ms2ct6A) [6,7], 2-methylthiomethylenethio-N6-isopentenyl adenosine (msms2i6A) [8], uniquely lipophilic 5-substituted 2-geranylthiouridines (mnm5ges2U, cmnm5ges2U) [9] and 2-methylthiocytidine (ms2C) [10]. RNA modifications affect the structure, stability, and biological activity of RNA biomolecules, particularly the translation process [11]. Experiments of the site-specific incorporation of nucleic base modifications have proved to be a particularly useful method for precisely assessing the role of various functional groups in the structure, function, and biosynthesis of RNA molecules [12–17]. Molecules 2020, 25, 3344; doi:10.3390/molecules25153344 www.mdpi.com/journal/molecules Molecules 2020, 25, 3344 2 of 37 Molecules 2020, 25, x FOR PEER REVIEW 2 of 37 FigureFigure 1. 1.Modified Modified positions positions distributed distributed in naturally existing existing RNA RNA nucleobases; nucleobases; Rb: Rb: ribose. ribose. ValuableValuable data data on on RNA RNA structure structure andand cellularcellular activity can can be be also also extracted extracted from from biochemical, biochemical, biophysical,biophysical, and and structural structural studies studies involving involving unnatural unnatural base-modified base-modified RNARNA constructs.constructs. The The insertion insertion of artificialof artificial purine purine/pyrimidine/pyrimidine in the in place the ofplace naturally of naturally existing existing nucleosides nucleosides may change may change the local the hydrogen local bondinghydrogen system, bonding stacking system, interactions, stacking interactions, or puckering or puckering of ribose of residues, ribose residues, giving answersgiving answers as to which as modificationsto which modifications or interactions or areinteractions essential forare theessential functional for RNAthe functional structure RNA [18–20 structure]. Important [18–20]. details onImportant RNA structure, details conformational on RNA structure, dynamics, conformational and RNA’s dynamics, homo- and and RNA’s heterotropic homo- and interactions heterotropic can be providedinteractions by oligoribonucleotides can be provided by oligoribonucleotides site-specifically labeled site-specifically with fluorescent labeled dyes,with fluorescent nitroxide radicals,dyes, nitroxide radicals, disulfide crosslinks, or photolabile groups [21–28]. disulfide crosslinks, or photolabile groups [21–28]. For years, chemically modified oligonucleotides have been extensively studied for the development For years, chemically modified oligonucleotides have been extensively studied for the development of new antisense and siRNA therapeutics [1,29–31]. Several nucleobase modifications were found to of new antisense and siRNA therapeutics [1,29–31]. Several nucleobase modifications were found improve the drug-like properties of oligonucleotides such as cellular uptake, stability in the cell, target to improvespecificity, the and drug-like binding affinity properties [31–33], of and oligonucleotides reduce undesirable such protein as cellular binding uptake, and immune stability stimulation in the cell, target[34,35]. specificity, Gratifyingly, and bindingthe incorporation affinity [of31 the–33 first-generation], and reduce undesirableinternucleotide protein linkage binding modifications and immune and stimulationsecond-generation [34,35]. sugar Gratifyingly, modifications the resulted incorporation in the current of the approval first-generation of two RNAi-based internucleotide therapeutics, linkage modificationsOnpattro (patisiran) and second-generation and Givlaari (givosiran) sugar [31]. modifications resulted in the current approval of two RNAi-basedGrowing therapeutics, interest in Onpattrothe modified (patisiran) RNA oligomers and Givlaari creates (givosiran) the need [31to ].develop chemistry that permitsGrowing the site-specific interest in theintroduction modified of RNA functionalizable oligomers groups creates into the RNA. need toThe develop most general chemistry way to that permitsaddress the the site-specific issue of site introduction selectivity is of through functionalizable the use of groups solid-phase into RNA.synthesis The phosphoramidite most general way to addresschemistry, the which issue ofinvolves site selectivity two strategies: is through (1) theclassical use of modified solid-phase monomer synthesis approach phosphoramidite via the chemistry,preparation which of a involves modified two phosphoramidite strategies: (1) classicalbuilding modifiedblock and monomer its subsequent approach incorporation via the preparation into the of aRNA modified chain; phosphoramidite and (2) a post-synthetic building RNA block modification and its subsequent approach incorporation based on selective into the chemical RNA chain; andreaction(s) (2) a post-synthetic of precursor RNA unit(s) modification in the full-length approach oligonucleotide based on selective prepared chemical by the reaction(s) classical method. of precursor unit(s) inThis the review full-length for oligonucleotidethe first time summarizes prepared by the the methods classical method.of site-specific incorporation of nucleobase-modified units into RNA oligomers via the post-synthetic strategy. We focused on the This review for the first time summarizes the methods of site-specific incorporation of nucleobase functionalization due to the high abundance of nucleobase modifications in the nature nucleobase-modified units into RNA oligomers via the post-synthetic strategy. We focused on and the high convenience of the post-synthetic strategy to construct the nucleobase-labeled chemical the nucleobase functionalization due to the high abundance of nucleobase modifications in the nature or biophysical probes. The scope of post-synthetically reactive precursor oligonucleotides has been andlimited the high to RNA convenience oligomers of prepared the post-synthetic by the phosphoramidite strategy to construct chemistry. the To nucleobase-labeled facilitate the selection chemical of or biophysicalpost-synthetic probes. methods The best scope suited of to post-synthetically the assumed chemical–biological reactive precursor objectives, oligonucleotides a large number has of been limitedexperimental to RNA details oligomers have prepared been included. by the phosphoramidite chemistry. To facilitate the selection of post-synthetic methods best suited to the assumed chemical–biological objectives, a large number of experimental2. Solid-Phase details Synthesis have been of Modified included. RNA Oligomers via Phosphoramidite Chemistry 2. Solid-PhaseThe most Synthesis current method of Modified for the RNA incorporation Oligomers of via a modified Phosphoramidite nucleoside Chemistry into a site-specific position of oligoribonucleotides is the phosphoramidite chemistry, which was introduced by CaruthersThe most in current 1981 [36,37]. method The for preparation the incorporation of RNA of oligomers a modified by nucleoside