Investigation of the Stereochemical-Dependent DNA and RNA Binding of Arginine-Based Nucleopeptides

S S symmetry

Article Investigation of the Stereochemical-Dependent DNA and RNA Binding of Arginine-Based Nucleopeptides

Stefano Tomassi 1 , Francisco Franco Montalban 2 , Rosita Russo 1, Ettore Novellino 3, Anna Messere 1,* and Salvatore Di Maro 1,*

1 DISTABiF, Università degli Studi della Campania “Luigi Vanvitelli”, via A. Vivaldi 43, 81100 Caserta, Italy; [email protected] (S.T.); [email protected] (R.R.) 2 Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, “Campus de Cartuja”, Granada University, 18071 Granada, Spain; ﬀ[email protected] 3 Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy; [email protected] * Correspondence: [email protected] (A.M.); [email protected] (S.D.M.)

Received: 28 March 2019; Accepted: 17 April 2019; Published: 19 April 2019

Abstract: Nucleopeptides represent an intriguing class of nucleic acid analogues, in which nucleobases are placed in a peptide structure. The incorporation of D- and/or L-amino acids in nucleopeptide molecules allows the investigation of the role of backbone stereochemistry in determining the formation of DNA and RNA hybrids. Circular Dichroism (CD) spectroscopic studies indicated the nucleopeptide as having fully l-backbone conﬁguration-formed stable hybrid complexes with RNA molecules. Molecular Dynamics (MD) simulations suggested a potential structure of the complex resulting from the interaction between the l-nucleopeptide and RNA strand. From this study, both the backbone (ionics and H-bonds) and nucleobases (pairing and π-stacking) of the chiral nucleopeptide appeared to be involved in the hybrid complex formation, highlighting the key role of the backbone stereochemistry in the formation of the nucleopeptide/RNA complexes.

Keywords: nucleic acid; nucleopeptides; backbone stereochemistry; solid-phase synthesis; molecular dynamic simulation

1. Introduction Nucleic acid–protein interactions are crucial in numerous cell functions, including transcription, translation and RNA maturation, which in turn modulate numerous physio-pathological processes [1,2]. They regulate genetic information transfer by proper interaction between protein binding domains and specific regions of nucleic acids [3–6]. The formation of highly stable and specific complexes depends on the strict recognition rules regulating the nucleobase association for nucleic acids and on a wide array of aminoacid–nucleobase interactions, including hydrophobic, ionic and hydrogen bonds for the nucleic acid–protein complexes. In the present day, the use of nucleobases constituting DNA and RNA aptamers is a well-established approach to interfere with both nucleic acid and protein targets for the diagnosis and the therapeutic treatment of numerous diseases, including cancer, inflammation and infections [7]. In spite of their great potential, the full capitalization of these chemotypes as therapeutics is still limited by several drawbacks, such as low chemical variability, binding affinity and metabolic stability. In this regard, DNA aptamers containing pyrimidine bases endowed with amino acid residues (SOMAmer) [8,9] represent an enhancement in chemical diversity providing wide application as diagnostic and therapeutic tools. Alternatively, nucleobases have been added to peptides as recognition units by employing nucleobase amino acids (NBA) [10–15]. To date, no study has been reported describing the use of NBA as elements to improve the selective protein recognition of target

Symmetry 2019, 11, 567; doi:10.3390/sym11040567 www.mdpi.com/journal/symmetry Symmetry 2019, 11 FOR PEER REVIEW 2

Alternatively, nucleobases have been added to peptides as recognition units by employing Symmetry 2019, 11, 567 2 of 16 nucleobase amino acids (NBA) [10–15]. To date, no study has been reported describing the use of NBA as elements to improve the selective protein recognition of target nucleic acids. Nevertheless, this basic concept hasnucleic been acids.proposed Nevertheless, in several thisstudies basic [1 concept6] involving has beenpeptide proposed-based molecules, in several studiesin [16] involving order to make novelpeptide-based compounds that molecules, bind DNA in orderand RNA to make specifically, novel compounds such as Peptide that bindNucleic DNA Acid and RNA specifically, (PNA) [17–21]. PNAssuch are as nucleic Peptide acid Nucleic analogues Acid first (PNA) described [17–21]. by PNAs Nielsen are and nucleic coworkers, acid analogues which first described by have been largely appliedNielsen for and chemical coworkers, biology which investigations. have been largely They are applied able to for efficient chemically mimic biology DNA investigations. They are molecules through ablea pseudopeptide to efficiently backbonemimic DNA formed molecules by achiral through N-(2 a-aminoethyl) pseudopeptide glycine backbone (aeg) formed by achiral moieties bearing nucleobasesN-(2-aminoethyl) linked glycinevia a methylene (aeg) moieties carbonyl bearing bridge. nucleobases Although linked PNAs via selectively a methylene carbonyl bridge. recognize DNA andAlthough RNA sequences PNAs selectivelyaffording very recognize stable hybrids, DNA and the RNA absence sequences of chiral aff centersording in very its stable hybrids, the backbone hampers absencethe folding of chiralin specific centers conformations, in its backbone reducing hampers its ability the folding to discriminate in specific highly conformations, reducing structured DNA andits ability RNA to mole discriminatecules. Additionally, highly structured PNAs DNA exhibit and numerous RNA molecules. disadvantages, Additionally, PNAs exhibit including low solubilitynumerous in an disadvantages, aqueous medium including, ambiguous low solubility directional in an selectivity aqueous of medium, DNA/RNA ambiguous directional binding, and low cellselectivity permeability of DNA. /RNA binding, and low cell permeability. To address some of Tothe address above-mentioned some of the issues, above-mentioned several modifications issues, several have modificationsbeen performed have on been performed on the original PNA structurethe original [22– PNA24]. For structure instance, [22 –the24]. incorporation For instance, of the N incorporation-(2-aminoethyl) of-D N-(2-aminoethyl)-D-Lysine-Lysine residues led to the synthesisresidues ledof cationic to the synthesis PNAs, which of cationic combined PNAs, the which nucleic combined acid recognition the nucleic ability acid of recognition ability of PNA with the cell permeabilityPNA with the of cellcationic permeability cell penetrating of cationic peptides cell penetrating (CPP). These peptides new PNA (CPP). variants These new PNA variants displayed advantagesdisplayed in terms advantages of solubility in termsand bindin of solubilityg to DNA and [23 binding,24]. Crystallographic to DNA [23,24 ].studies Crystallographic studies demonstrated that demonstrated the backbone that chirality the backbone could chirality conformationally could conformationally modulate the modulate PNA helical the PNA helical direction, direction, thereby enhancingthereby enhancing the recognition the recognition specificity specificity of the target of the [25 target–27]. [In25 –li27ne]. with In line the with resul thets results achieved by achieved by studyingstudying CPPs, CPPs,the presence the presence of the ofpositive the positive charge charge of Lys of side Lys-chain side-chainss enhanced enhanced their their solubility and solubility and especiallyespecially their theircell permeability cell permeability,, most mostlikely likely through through an endosomal an endosomal pathway pathway or direct or direct cell membrane cell membrane penetrationpenetration [28]. [28 Similarly,]. Similarly, new new classes classes of of chiral chiral PNAs PNAs (α, (α ,and and ɣ--GPNA)GPNA) havehave beenbeen recently designed by recently designed bymerging merging the the recognition recognition ability ability of PNA of PNA with thewith uptake the uptake features features of cationic of guanidiniumcationic groups [29,30]. guanidinium groupsThese [29, studies30]. These showed studies that showed GPNAs thatwith GPNAsα-d and / withor - l α-backbone-D and/or configuration ɣ-L-backbone exhibited improved configuration exhibitedcellular improved uptake, cellular increased uptake, binding increased affinity binding for DNA affinity and RNA,for DNA and and enhanced RNA, and thermal stability of the enhanced thermal stabilityresulting of hybrids.the resulting These hybrids. favorable These properties favorable were properties ascribable were to ascribable the conformational to the preorganization conformational preorganizationof chiral PNAs of andchiral the PNAs electrostatic and the interactions electrostatic between interactions cationic between guanidine cationic and phosphate groups. guanidine and phosphateIn particular, groups. they demonstrated In particular that, the they backbone demonstrated stereochemistry that the contributed backbone more with respect to stereochemistry contributedthe electrostatic more with forces respect in stabilizing to the theelectrostatic hybrid duplex. forces in stabilizing the hybrid duplex. In this regard, nucleopeptides represent alternative chemotypes in which nucleobases are combined In this regard,with nucleopeptides a peptide structure represent [31– 33 alternative]. This fascinating chemotypes class in of moleculeswhich nucleobases are characterized are by the presence combined with a peptideof amino structure acids-bearing [31–33]. nucleobasesThis fascinating as side class chains of molecules that promote are characterized nucleic acid by recognition through the presence of aminoboth acids base-bearing pairing nucleobases and stacking as interactions.side chains that In nucleopeptides, promote nucleic the acid nucleobase-functionalized recognition amino through both baseacids pairing are alternated and stacking with underivatized interactions. amino In nucleopeptides, acids at defined the positions nucleobase in the- peptide backbone. functionalized aminoDiederichsen acids are alternated et al. [34 with] and underivatized Mihara et al. [amino15,35,36 acids] reported at defined the synthesispositions ofin αtheand β-peptides with peptide backbone. Diederichsennucleobase-functionalized et al. [34] and sideMihara chains et al. and [35 the,36, use15] ofreportedl-α-amino- the synthesis-nucleobase-butyric of α and acid to assemble β-peptides with nucleobaseRNA binding-functionalized molecules and side functional chains and peptides, the use respectively. of L-α-amino More-ɣ-nucleobase recently, we- have reported new butyric acid to assemblesynthetic RNA strategies binding to obtain molecules both homo- and functionaland hetero-sequences peptides, of respectively. cationic nucleopeptides More that exhibited recently, we have reportedadvantageous new synthetic properties strategies in terms ofto binding,obtain both specificity, homo- celland permeability hetero-sequences and delivery of for RNA, DNA cationic nucleopeptidesand PNAthat exhibited molecules advantageous (Figure1)[ 37 properties,38]. Although in terms the therapeuticof binding, specific potentiality, of cell nucleopeptides is less permeability and deliveryknown with for RNA, the respect DNA to and the parentPNA molecules PNA molecules, (Figure they 1) [are37,38 under]. Although investigation the for their ability to therapeutic potentialinterfere of nucleopeptides with the Reverse is less Transcriptaseknown with the of respect the Human to the Immunodeficiency parent PNA molecules, Virus (RT-HIV), which is they are under investigationfundamental for their for HIVability infection to interfere [39]. with Additionally, the Reverse nucleopeptides Transcriptase haveof the been Human successfully employed Immunodeficiency in Virus combination (RT-HIV), with which Doxorubicin, is fundamental to improve for HIV its e ffi infectioncacy in Multidrug[39]. Additionally, Resistant Cancer (MRD) by nucleopeptides havesequestering been successfully adenosine employed triphosphate in combination (ATP) [40 ].with Doxorubicin, to improve its efficacy in Multidrug Resistant Cancer (MRD) by sequestering adenosine triphosphate (ATP) [40].

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FigureFigure 1. 1.StereogenicStereogenic sites sites in in DNA, DNA, PNA, PNA, and Arginine-based and Arginine- nucleopeptidebased nucleopeptide building blocks. building blocks. Herein, we describe the use of our synthetic approach to introduce specific structural modifications, d l suchHerein, as the incorporation we describe of the- and use/ or of -amino our synthetic acids, in approach nucleopeptides to introduce with the specific aim to investigate structural the role of backbone stereochemistry in determining the formation of DNA and RNA hybrids. CD modifications, such as the incorporation of D- and/or L-amino acids, in nucleopeptides with the aim spectroscopyto investigate and the UV-thermalrole of backbone stability stereochemistry investigations in provided determining evidence the offormation the impact of DNA of the and backbone RNA stereochemistryhybrids. CD spectroscopy on the ability and of nucleopeptidesUV-thermal stability to hybridize investigations DNA and provided RNA. Finally, evidence the interactionof the impact of theof the best backbone performing stereochemistry chiral nucleopeptide on the was ability studied of nucleopeptides by molecular dynamics to hybridize (MD) DNA simulations and RNA. to achieveFinally, a the new interaction insight on ofthe the structure best pe ofrforming the resulting chiral complexes nucleopeptide with RNA was complementary studied by molecular strands. 2.dynamics Materials (MD) and simulations Methods to achieve a new insight on the structure of the resulting complexes with RNA complementary strands. All solvents for peptide synthesis, reagent grade HPLC solvents (water and ACN), Fmoc-Rink amide-Am2. Materials resin, and Methods 1-hydroxybenzotriazole hydrate (HOBt) (purity > 97% dry weight, water 12%), ≈ triisopropylsilane (TIS) (purity 98%), acetic anhydride (Ac O, purity > 98%), anhydrous All solvents for peptide synthesis, reagent grade HPLC solvents2 (water and ACN), Fmoc-Rink N,N’-dimethylformamide (DMF), (1H-7-Azabenzotriazol-1-yl-oxy)tris-pyrrolidinophosphonium amide-Am resin, 1-hydroxybenzotriazole hydrate (HOBt) (purity > 97% dry weight, water ≈ 12%), hexafluorophosphate (PyAOP) (purity 96%), 1-hydroxy-7-azabenzotriazole (HOAt, purity 96%), and triisopropylsilane (TIS) (purity 98%), acetic anhydride (Ac2O, purity > 98%), anhydrous N,N’- anhydrous dichloromethane (DCM) were purchased from commercial sources (Sigma-Aldrich, Milano, dimethylformamide (DMF), (1H-7-Azabenzotriazol-1-yl-oxy)tris-pyrrolidinophosphonium Italy) and used without any additional purification. The following reagents were obtained from IRIS Biotech hexafluorophosphate (PyAOP) (purity 96%), 1-hydroxy-7-azabenzotriazole (HOAt, purity 96%), and (Marktredwitz, Germany): O-benzotriazole-N,N,N’,N’-tetra-methyl-uroniumhexafluorophosphate anhydrous dichloromethane (DCM) were purchased from commercial sources (Sigma-Aldrich, (HBTU, purity 99%), N,N-diisopropylethylamine (DIEA, purity 99%), piperidine, trifluoroacetic Milano, Italy) and used without any additional purification. The following reagents were obtained acid (TFA, purity 99%), and Nα-Fmoc-protected amino acids. from Sigma-Aldrich (Milano, Italy). from IRIS Biotech (Marktredwitz, Germany): O-benzotriazole-N,N,N’,N’-tetra-methyl- Nucleopeptides were purified by preparative HPLC (Shimadzu HPLC system) by using a C18-bounded uroniumhexafluorophosphate (HBTU, purity 99%), N,N-diisopropylethylamine (DIEA, purity 99%), preparative RP-HPLC column (Phenomenex Kinetex 21.2 mm 150 mm, 5 µM). The analyses were piperidine, trifluoroacetic acid (TFA, purity 99%), and Nα-Fmoc×-protected amino acids. from Sigma- accomplished by analytical HPLC (Shimadzu Prominence analytic HPLC system) on a C18-bounded Aldrich (Milano, Italy). Nucleopeptides were purified by preparative HPLC (Shimadzu HPLC analytical RP-HPLC column (Phenomenex Kinetex, 4.6 mm 150 mm, 5 µM), using a gradient elution system) by using a C18-bounded preparative RP-HPLC column× (Phenomenex Kinetex 21.2 mm × 150 from 10 to 90% ACN in water (0.1% TFA) over 20 min, a flow rate of 1.0 mL/min and detecting mm, 5 μM). The analyses were accomplished by analytical HPLC (Shimadzu Prominence analytic by an UV diode array. Molecular weights of nucleopeptides were assessed by mass spectrometry HPLC system) on a C18-bounded analytical RP-HPLC column (Phenomenex Kinetex, 4.6 mm × 150 analyses carried out by matrix assisted laser desorption ionization time of-flight (MALDI-TOF) mass mm, 5 μM), using a gradient elution from 10 to 90% ACN in water (0.1% TFA) over 20 min, a flow spectrometer implemented with a pulsed nitrogen laser (k = 337 nm). rate of 1.0 mL/min and detecting by an UV diode array. Molecular weights of nucleopeptides were 2.1.assessed Synthesis by m ofass Nucleopeptides spectrometry np-1 analyses/4 carried out by matrix assisted laser desorption ionization time of-flight (MALDI-TOF) mass spectrometer implemented with a pulsed nitrogen laser (k = 337 nm). Rink Amide AM-PS resin (60 mg, 0.033 mmol, 0.55 mmol/g) was suspended for 30 min in DCM/DMF 1:1 and then washed with DCM (3 1 min) and DMF (3 1 min). The Fmoc deprotection 2.1. Synthesis of Nucleopeptides np-1/4 × × was carried out by treatment with a solution of 20% (v/v) piperidine in DMF (1 5 min; 1 25 min). × × The succeedingRink Amide amide AM-PS couplings resin (60 were mg, performed 0.033 mmol, using 0.55 4 mmol/g) equivalents was (accordingsuspended tofor the 30 original min in loadingDCM/DMF of the 1:1 resin) and then of the washed Fmoc-protected with DCMl (3or ×d 1-aminoacids. min) and DMF To a(3 DMF × 1 min). (1.2 mL)The Fmoc solution deprotection of Fmoc-l (orwasd )-Arg(Pbf)–OHcarried out by treatment (84,5 mg, 0.13with mmol, a solution 4 equivalents), of 20% (v/v) HBTU piperidine (50 mg, in 0.13 DMF mmol, (1 × 5 4 min; equivalents) 1 × 25 min). and HOBtThe succeeding (19 mg, 0.13 amide mmol, couplings 4 equivalents) were DIPEAperformed (45 µ usingL, 0.26 4 mmol, equiv 8alents equivalents) (according was to added the original and the resultingloading of mixture the resin) was of added the Fmoc to the-protected support. L The or D mixture-aminoacids. was shaken To a DMF for 2 h(1.2 at roommL) solution temperature of Fmoc and- L (or D)-Arg(Pbf)–OH (84,5 mg, 0.13 mmol, 4 equivalents), HBTU (50 mg, 0.13 mmol, 4 equivalents) and HOBt (19 mg, 0.13 mmol, 4 equivalents) DIPEA (45 µL, 0.26 mmol, 8 equivalents) was added and

Symmetry 2019, 11 FOR PEER REVIEW 4 the resulting mixture was added to the support. TheSymmetry mixture2019 was, 11, 567shaken for 2 h at room temperature 4 of 16 and then washed with DMF (3 × 1 min) and DCM (3 × 1 min). Next, the unreacted free amine groups were acetylated by treatment of the resin with 1 mL of DMF capping solution containing of Ac2O and then washed with DMF (3 1 min) and DCM (3 1 min). Next, the unreacted free amine groups were of DIPEA (2 and 3 equivalents relative to the initial loading of the resin, respectively)× and shaken ×for acetylated by treatment of the resin with 1 mL of DMF capping solution containing of Ac2O and of 5 min. The completion of the peptide sequences was achieved by repeating the cycles of the Fmoc DIPEA (2 and 3 equivalents relative to the initial loading of the resin, respectively) and shaken for deprotection and coupling reaction with Fmoc-L (or D)-Lys(Alloc)-OH and Fmoc-L (or D)-Arg(Pbf)- 5 min. The completion of the peptide sequences was achieved by repeating the cycles of the Fmoc OH as above reported. These steps were monitored by Kaiser ninhydrine and TNBS tests and deprotection and coupling reaction with Fmoc-l (or d)-Lys(Alloc)-OH and Fmoc-l (or d)-Arg(Pbf)-OH ascertained by quantitative UV spectroscopic measurements of Fmoc chromophore. After the last as above reported. These steps were monitored by Kaiser ninhydrine and TNBS tests and ascertained Fmoc removal, the treatment with a mixture of Ac2O (6.2 μL, 2 equiv) and DIPEA (17.3 μL, 3 by quantitative UV spectroscopic measurements of Fmoc chromophore. After the last Fmoc removal, equivalents) in DMF (1 mL) for 10 min, allowed N-terminal acetylation of the peptides. the treatment with a mixture of Ac2O (6.2 µL, 2 equiv) and DIPEA (17.3 µL, 3 equivalents) in DMF The Alloc groups were removed by treating the supported full-protected peptide twice with a (1 mL) for 10 min, allowed N-terminal acetylation of the peptides. solution of Tetrakis(triphenylphosphine)palladium (0) (11 mg, 0.01 mmol, 5% mol relative to each The Alloc groups were removed by treating the supported full-protected peptide twice with a Alloc group) and DMBA (123 mg, 0.0.79 mmol, 4 equivalents relative to each Alloc protective group) solution of Tetrakis(triphenylphosphine)palladium (0) (11 mg, 0.01 mmol, 5% mol relative to each in dry DMF/DCM (1:1) for 1 h. The resulting solution was drained off and the resin was washed (3 × Alloc group) and DMBA (123 mg, 0.0.79 mmol, 4 equivalents relative to each Alloc protective group) 1 min) with DMF and with DCM. The catalyst traces were eliminated by treating the support with a in dry DMF/DCM (1:1) for 1 h. The resulting solution was drained oﬀ and the resin was washed DMF solution of potassium N,N-diethylcarbamodithioate (0.06 M , 1.5 mL) for 1 h. To obtain the (3 1 min) with DMF and with DCM. The catalyst traces were eliminated by treating the support with thyminyl-functionalized nucleopeptides, Thymin-1-×yl-acetic acid (108,1 mg, 0.59 mmol, 3 equivalents a DMF solution of potassium N,N-diethylcarbamodithioate (0.06 M, 1.5 mL) for 1 h. To obtain the for each amine group), HOAt (87 mg, 0.59 mmol) and PyAOP (308 mg, 0.59 mmol), DIPEA (205 µL, thyminyl-functionalized nucleopeptides, Thymin-1-yl-acetic acid (108,1 mg, 0.59 mmol, 3 equivalents 1.08 mmol, 6 equivalents for each amine group) were dissolved in DMF (2 mL) and then added to for each amine group), HOAt (87 mg, 0.59 mmol) and PyAOP (308 mg, 0.59 mmol), DIPEA (205 µL, resin for reacting over 8 h at room temperature. This latter step was repeated once. 1.08 mmol, 6 equivalents for each amine group) were dissolved in DMF (2 mL) and then added to resin for reacting over 8 h at room temperature. This latter step was repeated once. 2.2. Cleavage, HPLC Purification and Characterization of Nucleopeptides np-1/4

After washing with DMF (3 × 1 min), DCM2.2. (3 Cleavage,× 1 min), HPLCand Et Purification2O (3 × 1 min) and Characterization the supported of Nucleopeptides np-1/4 nucleopeptides np-1/4 After washing with DMF (3 1 min), DCM (3 1 min), and Et O (3 1 min) the supported × × 2 × were then dried exhaustively. The np-1/4 werenucleopeptides released fromnp-1 the /solid4 were support then dried by 3 exhaustively.h treatment The np-1/4 were released from the solid support with TFA/TIS (95:5, 1.5 mL) at room temperatureby. 3Then, h treatment the resin with was TFA filtered/TIS (95:5, off 1.5and mL) the at crude room temperature. Then, the resin was filtered off and nucleopeptides were precipitated from cold Et2O (15 mL) and then centrifuged (6000 rpm × 15 min). the crude nucleopeptides were precipitated from cold Et2O (15 mL) and then centrifuged (6000 rpm After removal of supernatant, the precipitate was suspended once in Et2O, centrifuged and the × 15 min). After removal of supernatant, the precipitate was suspended once in Et2O, centrifuged and supernatant removed. The resulting wet solid wasthe dried supernatant under removed.vacuum, dissolved The resulting in water/ wet solidACN was dried under vacuum, dissolved in water/ACN (9:1) and purified by RP-HPLC (solvent A: water +(9:1) 0.1% and TFA; purified solvent by B: RP-HPLC acetonitrile (solvent + 0.1% A: TFA; water from+ 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; from 10 to 70% of solvent B over 25 min, flow rate: 10 mL min−1). The collected fractions were evaporated 1 10 to 70% of solvent B over 25 min, flow rate: 10 mL min− ). The collected fractions were evaporated from organic solvents under reduced pressure, frofromzen and organic then solvents lyophilized. under The reduced final products pressure, were frozen and then lyophilized. The final products were analyzed by analytical RP-HPLC (10–90% acetonitrileanalyzed in water by analytical (0.1% TFA) RP-HPLC over 20 (10–90%min, flow acetonitrile rate of in water (0.1% TFA) over 20 min, flow rate of 1.0 mL/min) and characterized by MALDI-TOF spectrometry1.0 mL/min) (Table and characterized 1). Yield, purity, by MALDI-TOF retention times, spectrometry (Table1). Yield, purity, retention times, and analytical data are reported in Table 1. and analytical data are reported in Table1.

Table 1. Yield, purity, retention times, and analytical dataTable of the 1. Yield, described purity, nucleopeptides retention times,. and analytical data of the described nucleopeptides.

R ID Yield (%) Purity t (min) Mass CalcdIDMass Yield Found (%) [M+H]+ Purity tR (min) Mass Calcd Mass Found [M+H]+ np-1 76% ≥95% 11.90 2761.45np-1 76%2762.30 95% 11.90 2761.45 2762.30 ≥ np-2 64% ≥95% 13.60 2761.45np-2 64%2762.65 95% 13.60 2761.45 2762.65 ≥ np-3 68% ≥95% 12.70 2761.45np-3 68%2762.62 95% 12.70 2761.45 2762.62 ≥ np-4 71% 95% 12.50 2761.45 2762.72 np-4 71% ≥95% 12.50 2761.45 2762.72 ≥

2.3. UV and CD procedures 2.3. UV and CD procedures UV measurements were performed by a spectrophotometerUV measurements equipped were with performed a Peltier temperature by a spectrophotometer equipped with a Peltier temperature controller. Nucleopeptide stock solutions were preparedcontroller. using Nucleopeptide doubly distilled stock solutionswater and were RNAse prepared using doubly distilled water and RNAse free free water. Their concentration was calculated bywater. UV measurements Their concentration at 260 wasnm at calculated 90 °C by by using UV measurementsthe at 260 nm at 90 ◦C by using the following following molar extinction coefficient: Ɛ 260 = 13.7molar mL/(μ extinctionmol × cm) coe forﬃ A,cient: and Ɛ 260260 == 8.813.7 mL/( mLμ/(molµmol cm) for A, and 260 = 8.8 mL/(µmol cm) × × × cm) for T. CD spectra and melting curves were acquiredfor T. CD in spectra 1 cm path and- meltinglength quartz curves cell, were on acquired a Jasco in 1 cm path-length quartz cell, on a Jasco J-810 J-810 Circular Dichroism Spectrometer equippedCircular with a Pelt Dichroismier temperature Spectrometer programmer. equipped Samples with a Peltier temperature programmer. Samples were were lyophilized for 16 h. Duplexes were formedlyophilized by mixing equimolar for 16 h. Duplexes concentrations were formedof np-1/4 by (10 mixing equimolar concentrations of np-1/4 (10 µM each base) and single stranded complementary polyA (M.W. average 100–500 kDa) and polydA (M.W.

Symmetry 2019, 11, 567 5 of 16 average 85–170 kDa) (10 µM each base) in a 10 mM phosphate buﬀer, 100 mM NaCl, pH 7.0 solution. The samples were annealed by incubating at 90 ◦C for 5 min, slowly cooling down at room temperature and then kept at 4 ◦C overnight. UV and CD melting was monitored at λ = 260 nm (for UV) and at at λ = 267 nm for the polyA containing hybrids and at λ = 247 nm for polydA containing hybrids, with a 1 scan rate of 1 ◦C min− in the 5–85 ◦C temperature range. Melting temperatures (Tm) were extrapolated by the ﬁrst derivatives of CD melting curves. CD spectra were performed in the 210–300 nm spectral range, keeping the temperature constant at 20 ◦C.

2.4. CD Measurements of Apparent Kd Determination of np-1/polyA and np1/polydA The titration experiment was carried out by the addition of increasing concentrations of np-1 (0, 1.87, 3.76, 5.60, 7.47, 9.33, 11.20, 13.07, 14.93, 16.8, 18.67 10 µM each base) to a solution containing the single stranded complementary polyA (10 µM each base) and polydA (10 µM each base) in a 10 mM phosphate buﬀer, 100 mM NaCl, pH 7.0 solution. CD spectra were acquired at 20 ◦C, monitoring the signal decrements at λ = 260 nm for the polyA and at λ = 216 nm for polydA. The detected changes in CD intensity were plotted to generate saturation isotherm curves as a function of added np-1 amounts. Assuming the formation of 1:1 complex for each nucleobase, we calculated the apparent Kd values by using the following equation:

Θ = Θmin [(Θmin Θmax)/(2cA)] {Kd+cT+cA [(Kd + cT + cA)2 4cAcT]1/2} − − × − − where Θ is the CD intensity of the characteristic hybrid wavelength, Θmin and Θmax are the minimum and maximum value of CD intensity, respectively, and cA and cT are the Adenine and Thymine nucleobase concentrations, respectively.

2.5. Building of np-1/RNA Complex Maestro [41,42] was used to build cp-1, the complex structure, using a double-stranded B-DNA hexamer of complementary A-T bases as a template. The phosphofuranose backbone chain of the Thymine-containing single-strand was removed and a nucleopeptide chain, consisting of 6 L-Arg interpolated with six L-Lys residues (np-1), was constructed in its place in an antiparallel fashion, while keeping the A-T base paring fixed. Finally, hydroxyl groups with R configuration were added to the 2’ carbon atoms of the remaining furanose rings, and the C- and N-terminal ends of np-1 was capped with an amino and an acetyl group respectively. The initial structure of cp-1 was constructed base on the experimentally determined 2:1 stoichiometric interaction of two nucleopeptide molecules with a single 12 mer RNA oligonucleotide [37] To finally assemble the structure of cp-1 composed of a single-stranded 12 mer Adenine RNA oligonucleotide (A12 RNA ss) and two molecules of nucleopeptide np-1, the previously obtained cp-1 complex was duplicated and the RNA chains were linked covalently. The union was made by setting one copy of cp-1 on top of the other and connecting the 3’ end of one RNA strands with the 5’ end of the other through a newly added phosphate group.

2.6. Molecular Dynamics Simulation Cp-1 complex was initially minimized with MacroModel using an OPLS 2005 force ﬁeld in water and a Polak-Ribiere Conjugate Gradient minimization protocol of 100,000 maximum iterations and a convergence threshold of 0.001. A constrain in the base paring distances of A-T residues was used to ﬁx the double-strand conformation during the process. When satisfactory minimized structures were obtained (i.e. no anomalous bond angles, amide dihedral angles, bond distances, etc.), molecular dynamic (MD) simulations were launched using Desmond as implemented in the Schrodinger suite [43–45]. The model system was created using the System Builder tool of Desmond with an explicit TIP3P water solvent model. The positive charge of the complex was neutralized by 1 chloride ion, and additional ions up to a total concentration of 0.15 M KCl were added, all embedded into a rectangular box of 15 Å in each direction. Relaxation of the model system before performing the MD Symmetry 2019, 11 FOR PEER REVIEW 4

the resulting mixture was added to the support. The mixture was shaken for 2 h at room temperature and then washed with DMF (3 × 1 min) and DCM (3 × 1 min). Next, the unreacted free amine groups were acetylated by treatment of the resin with 1 mL of DMF capping solution containing of Ac2O and of DIPEA (2 and 3 equivalents relative to the initial loading of the resin, respectively) and shaken for 5 min. The completion of the peptide sequences was achieved by repeating the cycles of the Fmoc deprotection and coupling reaction with Fmoc-L (or D)-Lys(Alloc)-OH and Fmoc-L (or D)-Arg(Pbf)- OH as above reported. These steps were monitored by Kaiser ninhydrine and TNBS tests and ascertained by quantitative UV spectroscopic measurements of Fmoc chromophore. After the last Fmoc removal, the treatment with a mixture of Ac2O (6.2 μL, 2 equiv) and DIPEA (17.3 μL, 3 equivalents) in DMF (1 mL) for 10 min, allowed N-terminal acetylation of the peptides. The Alloc groups were removed by treating the supported full-protected peptide twice with a solution of Tetrakis(triphenylphosphine)palladium (0) (11 mg, 0.01 mmol, 5% mol relative to each Alloc group) and DMBA (123 mg, 0.0.79 mmol, 4 equivalents relative to each Alloc protective group) in dry DMF/DCM (1:1) for 1 h. The resulting solution was drained off and the resin was washed (3 × 1 min) with DMF and with DCM. The catalyst traces were eliminated by treating the support with a DMF solution of potassium N,N-diethylcarbamodithioate (0.06 M , 1.5 mL) for 1 h. To obtain the thyminyl-functionalized nucleopeptides, Thymin-1-yl-acetic acid (108,1 mg, 0.59 mmol, 3 equivalents Symmetry 2019, 11, 567 for each amine group), HOAt (87 mg, 0.59 mmol) and PyAOP (308 mg, 0.59 mmol), DIPEA6 of16 (205 µL, 1.08 mmol, 6 equivalents for each amine group) were dissolved in DMF (2 mL) and then added to resin for reacting over 8 h at room temperature. This latter step was repeated once. simulation was carried out with the default Desmond parameters, followed by a 200 ns unrestrained 2.2. Cleavage, HPLC Purification and Characterization of Nucleopeptides np-1/4 MD simulation with an NPT ensemble (300 K, 1.013 bar). An integration time-step of 2 fs was used After washing with DMF (3 × 1 min), DCM (3 × 1 min), and Et2O (3 × 1 min) the supported for the multistep protocol with an interpolating function for electrostatic interactions (cut-off radius nucleopeptides np-1/4 of 9.0 Å). Steady temperaturewere and then pressure dried exhaustively. values were The np maintained-1/4 were released using from the the Nose–Hoover solid support by chain3 h treatment thermostat [45]. with TFA/TIS (95:5, 1.5 mL) at room temperature. Then, the resin was filtered off and the crude nucleopeptides were precipitated from cold Et2O (15 mL) and then centrifuged (6000 rpm × 15 min). 2.7. RMSD Clustering AnalysisAfter removal of supernatant, the precipitate was suspended once in Et2O, centrifuged and the supernatant removed. The resulting wet solid was dried under vacuum, dissolved in water/ACN The clustering analysis(9:1) and was purified carried by RP out-HPLC using (solvent the A: MD water movie + 0.1% ToolTFA; solvent in UCSF B: acetonitrile Chimera + 0.1% 1.10.1 TFA; from (University of California, San10 to Francisco, 70% of solvent CA, B USA)over 25 [min,45] asflow follows: rate: 10 mL the min final−1). MDThe collected simulation fractions frames were were evaporated exported with Maestro (Schrödinger,from organic solvents Cambridge, under reduced MA, pressure, USA) to fr separateozen and then pdb lyophilized. files and The opened final products with were analyzed by analytical RP-HPLC (10–90% acetonitrile in water (0.1% TFA) over 20 min, flow rate of UCSF Chimera MD movie1.0 Tool. mL/min) A cluster and characterized analysis wasby MALDI next- carriedTOF spectrometry out, setting (Table frame 1). Yield, 1 as purity, the startingretention times, one, and a step size of 150.and The analytical remaining data are options reported were in Table set 1. as default. The final cluster-representative structures given by Chimera were minimized with MacroModel (Schrödinger, Cambridge, MA, USA) Table 1. Yield, purity, retention times, and analytical data of the described nucleopeptides. with an OPLS 2005 force field in a Polak-Ribier Conjugate Gradient method for a maximum of 5000 iterations and a convergence thresholdID ofYield 0.05 in(%) water. Purity tR (min) Mass Calcd Mass Found [M+H]+ np-1 76% ≥95% 11.90 2761.45 2762.30 3. Results and Discussion np-2 64% ≥95% 13.60 2761.45 2762.65 np-3 68% ≥95% 12.70 2761.45 2762.62 np-4 71% ≥95% 12.50 2761.45 2762.72 3.1. Synthesis The synthesis of the2.3. nucleopeptides UV and CD procedures set was performed by a general synthetic path, that was developed by using AllyloxycarbonylUV measurements [46–48 were] (Alloc) performed as temporary by a spectrophotometer protection equipped strategy with for a Peltier the amino temperature group connecting the nucleobase.controller. TheNucleopeptide Alloc approach stock solutions allowed were using prepared commercially using doubly available distilled water building and RNAse free water. Their concentration was calculated by UV measurements at 260 nm at 90 °C by using the blocks avoiding the solutionfollow phaseing molar synthetic extinction steps coefficient: required Ɛ 260for = 13.7 the mL/( preparationμmol × cm) for of A, the and N Ɛ -protected260 = 8.8 mL/(μmol derivatives or nucleoamino× cm) acids for T.needed CD spectra for and the melting subsequent curves were assembly acquired ofin 1 nucleopeptide cm path-length quartz sequences cell, on a Jasco on the solid support. TheJ- use810 Circular of Alloc Dichroism protecting Spectrometer group isequipped widely with employed a Peltier totemperature preserve programmer. amino acid Samples side chains, including Lysinewere lyophilized and Ornitine, for 16 h. those Duplexes are were required formed to by be mixing selectively equimolar released concentrations for direct of np-1/4 (10 functionalization on solid support (i.e. FITC, fatty acid etc) or lactam bridged cyclization [45,46]. Interestingly, in spite of the application of an Alloc-protecting strategy for single amino acid being consolidated, few investigations have been reported for the protection of multiple sites. Thus, we aimed to explore its use also for oligomers containing numerous amino groups, those can be simultaneously released for further on-line derivatization, including the introduction of nucleobases in the case of nucleopeptides. As described in Scheme1, the 12 mer amino acidic backbone of the nucleopeptides consisting of alternation of l-Arg and l-Lys (np-1) or d-Arg and l-Lys (np-2) or l-Arg and d-Lys (np-3) or d-Arg and d-Lys (np-4) was assembled using a Rink-amide-aminomethyl polystyrene (AM-PS) resin. After acetylation of the N-terminal function, all the Lys side chains were concurrently deprotected by a DMF/DCM solution of Pd(PPh3)4 and N,N-Dimethylbarbituric acid (DMBA) as scavenger. The treatment was reiterated three times to assure the complete removal of Alloc protecting groups as confirmed by HPLC-MS analyses. Next, the Thymine-1-acetic acids (T-CH2COOH) were placed by one-pot reaction by a 3 h reaction in the presence of HBTU/HOBt as activating/additive agents. All the nucleopeptides (np-1/4) were eventually released from the resin, purified by RP-HPLC and characterized by MALDI-TOF MS. Symmetry 2019, 11, 567 7 of 16 SymmetrySymmetry 20192019,, 1111 FORFOR PEERPEER REVIEWREVIEW 77

SchemeSchemeScheme 1.1. 1. SyntheticSyntheticSynthetic pathpath path toto nucleopeptidesnucleopeptidesnp-1 npnp/--4.1/4.1/4.

3.2.3.2. CDCD spectroscopicspectroscopic Spectroscopic studiesstudies Studies CDCD studies studies were were carried carried out out with with npnp-1np--1/41/4/4 andand with with the the hybrids hybrids obtained obtained by by combining combining the the synthesized nucleopeptides np-1np-1/4/4 withwith polyA polyA and and polydA polydA ss ss targets.targets. All All the CD analyses were synthesized nucleopeptides np-1/4 with polyA and polydA ss targets. All the CD analyses were performedperformed usingusing 1010 10 μMμMµM concentrationconcentration concentration ofof of npnpnp-1--1/41/4/ 4 ininin 1010 10 mMmM mM phosphatephosphate phosphate bufferbuffer buﬀ er(pH(pH (pH == 7.0)7.0)= 7.0) atat 2020 at °C.°C. 20 As◦AsC. depictedAsdepicted depicted inin Figure Figurein Figure 2,2, 2 all all, all the the the single single single stranded stranded stranded nucleopeptides nucleopeptides nucleopeptides ( ( (npnpnp-1--1/4)1/4)/4 )exhibitedexhibited exhibited the the the distinctive distinctive CD CD spectraspectra of of linear linear cationic cationic peptides peptides inin anan unordered unordered statestate endowedendowed withwithwith aaa singlesinglesingle bandbandband belowbelowbelow 200200200 nm.nm.nm.

Figure 2. CD spectra of nucleopeptides np-1/4. FigureFigure 2.2. CDCD spectraspectra ofof nucleopeptidesnucleopeptides npnp--1/4.1/4.

Symmetry 2019, 11 FOR PEER REVIEW 8 Symmetry 2019, 11 FOR PEER REVIEW 8 Symmetry 2019, 11, 567 8 of 16 As expected, enantiomeric nucleopeptides containing L-Arg/L-Lys and D-Arg/D-Lys (np-1 and - As expected, enantiomeric nucleopeptides containing L-Arg/L-Lys and D-Arg/D-Lys (np-1 and - 4) and D-Arg/L-Lys and L-Arg/D-Lys (np-2 and -3) exhibited mirror CD spectra. Next, we investigated 4) andAs D expected,-Arg/L-Lys enantiomeric and L-Arg/D nucleopeptides-Lys (np-2 and - containing3) exhibitedl-Arg mirror/l-Lys CD andspectra.d-Arg Next,/d-Lys we ( np-1investigatedand -4) the spectroscopic behavior of the hybrids composed of nucleopeptides np-1/4 with PolyA and andthe d spectroscopic-Arg/l-Lys and behaviorl-Arg/d -Lys of the (np-2 hybridsand -3 ) composed exhibited mirror of nucleopeptides CD spectra. Next,np-1/4 we with investigated PolyA and the PolydA (1:1 ratio in nucleobases). Long oligomers, such as polyA and polydA, were chosen for the spectroscopicPolydA (1:1 ratio behavior in nucleobases). of the hybrids Long composed oligomers, of such nucleopeptides as polyA andnp-1 polydA,/4 with were PolyA chosen and PolydA for the CD experiments to amplify the resulting binding effects in CD spectra and to take advantage from (1:1CD ratioexperiments in nucleobases). to amplify Longthe resulting oligomers, binding such effects as polyA in CD and spectra polydA, and were to take chosen advantage for the from CD “the flanking base effect”, that allows the nucleobases next to 5’ and 3’ termini to stabilize the experiments“the flanking to base amplify effect”, the resulting that allows binding the nucleobases effects in CD next spectra to 5 and’ and to 3take’ termini advantage to stabilize from “the the conformation of inner bases even if they do not contribute to the base pairing. Compared to polyA flankingconformation base e ffofect”, inner that bases allows even the if nucleobases they do not next contribute to 5’ and to 3’the termini base pa toiring. stabilize Compared the conformation to polyA and polydA ss, the CD spectra of np-1/4/polyA (Figure 3a) and np-1/4/polydA (Figure 3b) hybrids ofand inner polydA bases ss, even the if CD they spectra do not contributeof np-1/4/polyA to the base(Figure pairing. 3a) and Compared np-1/4/polydA to polyA (F andigure polydA 3b) hybrids ss, the exhibited hypochromic effects that could be ascribable to the cationic character of the side chain of CDexhibited spectra hypochromic of np-1/4/polyA effects (Figure that3 a)could and benp-1 ascribable/4/polydA to (Figurethe cationic3b) hybrids character exhibited of the hypochromic side chain of Arginine residues. eAffrginineects that residues. could be ascribable to the cationic character of the side chain of Arginine residues.

Figure 3. (a) CD spectra of polyA ss and np-1/4/polyA hybrids; (b) CD spectra of polydA ss FigureFigure 3. 3. ((aa)) CDCD spectra spectra of of polyA polyA ss ss and andnp-1 np/-41/4/polyA/polyA hybrids; hybrids; (b) ( CDb) CD spectra spectra of polydA of polydA ss and ss and np-1/4/polydA hybrids. np-1and/ 4np/polydA-1/4/polydA hybrids. hybrids.

ToTo assessassess the the interaction interaction between between nucleopeptides nucleopeptides and and nucleic nucleic acids, acids, CD bindingCD binding experiments experiments were To assess the interaction between nucleopeptides and nucleic acids, CD binding experiments performedwere performed in tandem in tandem cells, collectingcells, collecting “sum” ‘‘sum’’ CD spectra CD spectra relative relative to the separatedto the separatednp-1/4 npand-1/4 ss polyAand ss were performed in tandem cells, collecting ‘‘sum’’ CD spectra relative to the separated np-1/4 and ss andpolyA polydA, and polydA, and “mix” and CD ‘‘mix’’ spectra, CD acquiredspectra, acquired after cell mixingafter cell and m annealingixing and ofannealingnp-1/4 with of np polyA-1/4 with and polyA and polydA, and ‘‘mix’’ CD spectra, acquired after cell mixing and annealing of np-1/4 with polydApolyA and ss. polydA The diﬀ erencesss. The differences observed comparing observed comparing the “sum” the and ‘‘sum’’ “mix” and CD spectra‘‘mix’’ CD strongly spectra suggested strongly polyA and polydA ss. The differences observed comparing the ‘‘sum’’ and ‘‘mix’’ CD spectra strongly thesuggested occurrence the occurrence of the hybridization of the hybridization event in both event cases in (Figureboth cases4a,b). (Figure 4a and 4b). suggested the occurrence of the hybridization event in both cases (Figure 4a and 4b).

FigureFigure 4. 4.( a()a CD) CD spectra spectra of np-1of np/4/-polyA1/4/polyA sum andsumnp-1 and/4 np/polyA-1/4/polyA mix; (b) mix; CD spectra (b) CD of spectranp-1/4/polydA of np- Figure 4. (a) CD spectra of np-1/4/polyA sum and np-1/4/polyA mix; (b) CD spectra of np- sum1/4/polydA and np-1 sum/4/polydA and np mix.-1/4/polydA mix. 1/4/polydA sum and np-1/4/polydA mix.

InIn particular,particular, wewe observedobserved dramaticdramatic changeschanges inin thethe CDCD spectraspectra ofof hybridshybrids thatthat could be attributed In particular, we observed dramatic changes in the CD spectra of hybrids that could be attributed toto thethe lossloss ofof nucleicnucleic acidsacids helicityhelicity resultingresulting fromfrom non-speciﬁcnon-specific electrostaticelectrostatic interactionsinteractions withwith cationiccationic to the loss of nucleic acids helicity resulting from non-specific electrostatic interactions with cationic nucleopeptides.nucleopeptides. Intriguingly,Intriguingly, the the strongest strongest hypochromic hypochromic eﬀ effectect was was observed observed for for hybrids hybrids containing containing full nucleopeptides. Intriguingly, the strongest hypochromic effect was observed for hybrids containing L-nucleopeptide,full L-nucleopeptide suggesting, suggesting that besides that the besides electrostatic the electrostaticcontribution, the contribution, backbone stereochemistry the backbone full L-nucleopeptide, suggesting that besides the electrostatic contribution, the backbone playsstereochemistry a main role p inlay determinings a main role the in conformationdetermining the assumed conformation by the hybridassumed duplexes. by the hybrid duplexes. stereochemistry plays a main role in determining the conformation assumed by the hybrid duplexes. TheThe thermalthermal stability (melting(melting temperature, Tm)Tm) ofof hybrids involving nucleopeptides np-1np-1/4/4 andand The thermal stability (melting temperature, Tm) of hybrids involving nucleopeptides np-1/4 and bothboth polyApolyA andand polydApolydA complementscomplements waswas determineddetermined by CDCD andand UVUV experiments,experiments, andand comparedcompared both polyA and polydA complements was determined by CD and UV experiments, and compared withwith TmTm ofof thethe hexathyminehexathymine oligodeoxynucleotideoligodeoxynucleotide (T6)(T6)/PolyA/PolyA andand PolydAPolydA andand ss.ss. (Table(Table2 ).2). with Tm of the hexathymine oligodeoxynucleotide (T6)/PolyA and PolydA and ss. (Table 2).

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Table 2. Tm values of np-1/4 polyA and polydA hybrids. Lowercase indicates D-amino acid; uppercaseTable 2. Tm indicates values of Lnp-1-amino/4 polyA acid. and polydA hybrids. Lowercase indicates d-amino acid; uppercase indicates l-amino acid. Entry Sequence polyA (Tm) polydA (Tm) Entry Sequence polyA (Tm) polydA (Tm) T6 3’-TTTTTT-5’ 35 25 T6 3’-TTTTTT-5’ 35 25 np-1 Ac-K(T)-R-K(T)-R-K(T)-R-K(T)-R-K(T)-R-K(T)-RCONH2 45 35 np-1 Ac-K(T)-R-K(T)-R-K(T)-R-K(T)-R-K(T)-R-K(T)-RCONH2 45 35 np-2 np-2Ac-K(T)Ac-K(T)-r-K(T)-r-K(T)-r-K(T)-r-K(T)-r-K(T)-rCONH-r-K(T)-r-K(T)-r-K(T)-r-K(T)-r-K(T)-rCONH2 2Not DetectedNot Detected Not Not Detected Detected np-3 Ac-k(T)-R-k(T)-R-k(T)-R-k(T)-R-k(T)-R-k(T)-RCONH Not Detected Not Detected np-3 Ac-k(T)-R-k(T)-R-k(T)-R-k(T)-R-k(T)-R-k(T)-RCONH2 2 Not Detected Not Detected np-4 Ac-k(T)-r-k(T)-r-k(T)-r-k(T)-r-k(T)-r-k(T)-rCONH2 Not Detected Not Detected np-4 Ac-k(T)-r-k(T)-r-k(T)-r-k(T)-r-k(T)-r-k(T)-rCONH2 Not Detected Not Detected

NotablyNotably,, the the highest highest detected thermalthermal stabilitystability was was observed observed for fornp-1 np/-polyA1/polyA hybrid hybrid (Tm (Tm= 45 = ◦45C), °C),revealing revealing that thatnp-1 nprepresented-1 represented the most the stabilizingmost stabilizing RNA binder RNA binder even if even compared if compared to np-3 /4 to/polyA np- 3/4and/polyA polydA and hybrids. polydA Moreover, hybrids. Moreover, the melting the profile melting appeared profile as appeared a sigmoid as shape a sigmo for np-1id shape/polyA forwhen np- 1compared/polyA when to meltingcompared profiles to melting of the profiles any other of hybridsthe any (Figureother hybrids5). In particular, (Figure 5). the In melting particular, profiles the meltingof polyA profiles hybrids of in polyA the presence hybrids of inl -Arg the / presenced-Lys and ofd -ArgL-Arg//d-LysD-Lys containing and D-Arg/ nucleopeptidesD-Lys containing (np-2 nucleopeptidesand -3) appeared (np very-2 and complex, -3) appeared suggesting very thecomplex, existence suggesting of more than the existence two species of moreand a thanmultistate two speciesequilibrium and a (Figuremultistate5). equilibrium (Figure 5).

FigureFigure 5. 5. (a) CD(a) melting CD melting profiles profiles of np of-1/4np-1/polyA/4/polyA hybrids; hybrids; (b) CD (b melting) CD melting profiles profiles of np-1- of npnp-1-np-4-4/polydA/polydA hybrids; hybrids; (c) (c UV) UV melting melting profiles profiles of ofnp-1 np/4-/1/4polyA/polyA hybrids; hybrids; (d) UV (d melting) UV profilesmelting of np-1/4/polydA hybrids. profiles of np-1/4/polydA hybrids. The CD melting profiles suggested that the dissociation of np-1/polyA and np-1/polydA was The CD melting profiles suggested that the dissociation of np-1/polyA and np-1/polydA was a a two-state process. Thus, we were able to estimate the apparent Kd values of np-1 to polyA and two-state process. Thus, we were able to estimate the apparent Kd values of np-1 to polyA and polydA by performing CD titration experiment with increasing concentrations of np-1 in the presence polydA by performing CD titration experiment with increasing concentrations of np-1 in the presence of a constant concentration of nucleic acids. As depicted in Figure6, the apparent Kd values further of a constant concentration of nucleic acids. As depicted in Figure 6, the apparent Kd values further corroborated the results achieved by us and other groups [37,39], indicating that cationic nucleopeptides corroborated the results achieved by us and other groups [37,39], indicating that cationic are endowed with higher affinity for RNA (Figure6a) with the respect to DNA molecules (Figure6b). nucleopeptides are endowed with higher affinity for RNA (Figure 6a) with the respect to DNA molecules (Figure 6b).

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Symmetry 2019, 11 FOR PEER REVIEW 10

FigureFigure 6. 6.Binding Binding curve curve of 10 of uM 10 polyA uM ( polyAa) and polydA(a) and (b polydA) titrated with(b) t increasingitrated with concentration increasing ofconcentrationnp-1. of np-1. 3.3. Molecular Dynamics Simulations 3.3. Molecular Dynamics Simulations To gain structural insights into the binding capabilities of chiral nucleopeptide np-1 toward

RNA,To we gain performed structural molecular insights dynamics into the (MD) binding simulations capabilities of the of single chiral nucleopeptide nucleopeptide consistingnp-1 toward of interpolatedRNA,Figure we performed 6.l-Arg Binding and molecular l curve-Lys residues of dynamics 10 uM covalently polyA(MD) simulations( linkeda) and to polydA Thymine of the (singleb nucleobases.) tit rnucleopeptideated with In increasing this consisting case, np-1 of interpolatedconcentration L-Arg of and np -L1-.Lys residues covalently linked to Thymine nucleobases. In this case, np-1 contained six l-Arg and six l-Lys residues with the Thymine nucleobases linked to the ε-NH2-group of thecontained Lysine side six L chain-Arg throughand six L an-Lys acetyl residues group with (Figure the Thymine6). This polymer nucleobases was chosenlinked asto athe model ε-NH for2-group the MD3.3.of the simulationsMolecular Lysine Dynamicsside due chain to itsSimulations through interesting an acetyl spectroscopic group (Figure behavior 6). towardThis polymer RNA ss.was chosen as a model for the MD simulations due to its interesting spectroscopic behavior toward RNA ss. TheTo gain interactions structural of the insights molecular into the complex bindingcp1 capabilities(Figure7) made of chiral up by nucleopeptide a single-stranded np-1 12toward mer AdenineRNA, we RNA performed oligonucleotide molecular (Adynamics12 RNA (MD) ss) and simulations two nucleopeptide of the singlenp-1 nucleopeptidemolecules were consisting assessed of throughinterpolated MD simulations.L-Arg and L The-Lys initialresidues simulation covalently structure linked was to Thymine constructed nucleobases. based on the In experimentallythis case, np-1 determinedcontained six 2:1 L stoichiometric-Arg and six L interaction-Lys residues of np-1 withwith the aThymine complementary nucleobases single-stranded linked to the 12 merε-NH Adenine2-group RNAof the oligonucleotide, Lysine side chain and through assuming an acetyl an initial group recognition (Figure 6). event This betweenpolymer thewas three chosen macromolecules as a model for guidedthe MD by simulations the base-pairing due to interactionsits interesting of spectroscopic the three species behavior [40] (Figure toward7). RNA ss.

Figure 7. Schematic representation of complex cp-1 submitted to MD.

The interactions of the molecular complex cp1 (Figure 7) made up by a single-stranded 12 mer Adenine RNA oligonucleotide (A12 RNA ss) and two nucleopeptide np-1 molecules were assessed through MD simulations. The initial simulation structure was constructed based on the experimentally determined 2:1 stoichiometric interaction of np-1 with a complementary single- Figure 7. Schematic representation of complex cp-1 submitted to MD. stranded 12 mer AdenineFigure 7. SchematicRNA oligonucleotide, representation and of complex assumingcp-1 ansubmitted initial recognition to MD. event between the three macromolecules guided by the base-pairing interactions of the three species [40] (Figure 7). AfterThe interactions running the of 200 the ns molecular long MD complex simulation cp1 (see (Figure Materials 7) made and up Methods by a single section),-stranded we analyzed 12 mer After running the 200 ns long MD simulation (see Materials and Methods section), we analyzed theAdenine conformational RNA oligonucleotide stability of the (A simulated12 RNA ss) complex and twocp-1 nucleopeptidein terms of the np root-mean-square-1 molecules were deviation assessed the conformational stability of the simulated complex cp-1 in terms of the root-mean-square deviation (RMSD)through of MD all the simulations. non-hydrogen The atoms initial in simulationthe complex structure (Figure8 ). was Overall, constructed the analysis based of the on MD the (RMSD) of all the non-hydrogen atoms in the complex (Figure 8). Overall, the analysis of the MD trajectoriesexperimentally demonstrated determined that 2:1 after stoichiometric an initial equilibration interaction period of np the-1 with complex a complementary reaches a fairly single stable- trajectories demonstrated that after an initial equilibration period the complex reaches a fairly stable conformationstranded 12 mer with Adenine an 8 Å RMSDRNA oligonucleotide, deviation from theand initial assuming input an structure. initial recognition This increase event in RMSDbetween is conformation with an 8 Å RMSD deviation from the initial input structure. This increase in RMSD is followedthe three bymacromolecules a moderate second guided change, by the afterbase- approximatelypairing interactions 40 ns, of tothe reach three a species second [ conformation40] (Figure 7). followed by a moderate second change, after approximately 40 ns, to reach a second conformation with anAfter 11 running Å RMSD the deviation 200 ns long from MD the simulation initial structure. (see M Thisaterials last and conformation Methods section), demonstrates we analyzed to be with an 11 Å RMSD deviation from the initial structure. This last conformation demonstrates to be fairlythe conformation stable all throughal stability the remaining of the simulated part of complex the simulation cp-1 in (160terms ns). of the root-mean-square deviation fairly stable all through the remaining part of the simulation (160 ns). (RMSD) of all the non-hydrogen atoms in the complex (Figure 8). Overall, the analysis of the MD trajectories demonstrated that after an initial equilibration period the complex reaches a fairly stable conformation with an 8 Å RMSD deviation from the initial input structure. This increase in RMSD is followed by a moderate second change, after approximately 40 ns, to reach a second conformation with an 11 Å RMSD deviation from the initial structure. This last conformation demonstrates to be fairly stable all through the remaining part of the simulation (160 ns).

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Figure 8. RMSD plot of MD trajectories from their respective initial frame in complex cp-1. FigureFigure 8. RMSD 8. RMSD plot plot of MD of MD trajectories trajectories from from their respectiverespective initial initial frame frame in complexin complexcp-1 .cp-1. To better determine the conformational behavior of the simulated complex, we conducted an RMSDTo-based betterbetter clustering determine analysis the conformational of the MD 1002 behavior trajectory of theframes simulated obtained complex, after the we MD conducted simulation. an ARMSD-basedRMSD total- basedof three clustering clusters, analysis were obtained of the MD accounting 1002 trajectory for the mostframes frames rep obtained obtainedresentative after after structures the the MD MD simulation. simulation.in the MD simulationA total of three of cp clusters,-1. Of the were three obtained clusters, accounting the last one for stands the most for representativerep theresentative highest number structures of frames in the and MD includessimulation the of structures cp-1cp-1. Of Of the the calculated three three clusters, for the the last last 160 one ns stands of the for simulations. the highest The number most representativeof frames and structureincludes the theof structuresthis structures cluste calculatedr calculatedis depicted for for thein Figure lastthe 160 last 9 nsa 160, ofcorresponding the ns simulations.of the simulations. to Thea wrapped most The representative conformation most representative structure of the complex.ofstructure this cluster of this is depictedcluster is in depicted Figure9a, in corresponding Figure 9a, corresponding to a wrapped to conformation a wrapped conformation of the complex. of the complex.

Figure 9.9.( a()a Representative) Representative structure structure of the of most the populated most populated cluster in cp-1cluster;(b) in Simpliﬁed cp-1; (b representation) Simplified representationFigureof a zenithal 9. (a view) Representative of of a cp-1zenithal. view structure of cp of-1. the most populated cluster in cp-1; (b) Simplified representation of a zenithal view of cp-1. Next, we analyzed the structure depicted in Figure 9a to gather some information about the way np-1 Next,interacts we analyzedwith the RNAthe structure counterpart. depicted As can in Figure be observed 9a to gather in Figure some 9 ainformation, the base-pairing about theof bothway np-1 interacts with the RNA counterpart. As can be observed in Figure 9a, the base-pairing of both

Symmetry 2019, 11, 567 12 of 16

Symmetry 2019, 11 FOR PEER REVIEW 12 Next, we analyzed the structure depicted in Figure9a to gather some information about the way np-1 interacts with the RNA counterpart. As can be observed in Figure9a, the base-pairing of np-1 molecules with the RNA chain is almost completely lost, with other chemical interactions taking both np-1 molecules with the RNA chain is almost completely lost, with other chemical interactions place in the complex. More importantly, the RNA chain has lost its helical conformation and is taking place in the complex. More importantly, the RNA chain has lost its helical conformation and adopting an almost linear disposition as can be seen in a zenithal view of the complex (Figure 9b). is adopting an almost linear disposition as can be seen in a zenithal view of the complex (Figure9b). Responsible for this loss of helicity are the two np-1 molecules. Of these, the np-1 molecule (np-1- Responsible for this loss of helicity are the two np-1 molecules. Of these, the np-1 molecule (np-1-top) top) originally attached to the 5' end of the complex, uses its three N-terminal Arginines (R1, R2, and originally attached to the 5’ end of the complex, uses its three N-terminal Arginines (R1, R2, and R3) to R3) to form stable ionic interactions with the RNA phosphate backbone at the 3' end. Moreover, np- form stable ionic interactions with the RNA phosphate backbone at the 3’ end. Moreover, np-1-top 1-top also forms H-bonds between its first and second N-terminal Thymine rings (T1 and T2) with also forms H-bonds between its ﬁrst and second N-terminal Thymine rings (T1 and T2) with the two the two middle RNA Adenine (A6 and A7), helping to keep the RNA chain in a near-linear middle RNA Adenine (A6 and A7), helping to keep the RNA chain in a near-linear conformation conformation (Figure 10a). Similarly, important is the H-bond formed by the amide group in (Figure 10a). Similarly, important is the H-bond formed by the amide group in Thymine T3 arm with Thymine T3 arm with Adenine A7, which helps to hold the RNA chain before the turn that takes Adenine A7, which helps to hold the RNA chain before the turn that takes place between Adenine A7 place between Adenine A7 and A8. The ionic interactions, however, are not the only chemical and A8. The ionic interactions, however, are not the only chemical interactions between np-1-top and interactions between np-1-top and RNA. The complex also shows π-stacking interactions among C- RNA. The complex also shows π-stacking interactions among C-terminal Thymine rings (T5 and T6) terminal Thymine rings (T5 and T6) and Adenine A10 and A12. Both T5 and T6 are sandwiched and Adenine A10 and A12. Both T5 and T6 are sandwiched between A10 and A12, holding the top between A10 and A12, holding the top half of the RNA chain with a successive six nucleobases π- half of the RNA chain with a successive six nucleobases π-stacking. stacking.

FigureFigure 10. 10(.a (a)) Simpliﬁed Simplified representation representation of the mostof the important most important interactions interactions between np-1-top betweenand np RNA;-1- (topb) Simpliﬁed and RNA; representation (b) Simplified of representation the most important of the interactions most important between interactionsnp-1-btm and between RNA. RNA np- chain1-btm in and orange, RNA.np-1-top RNA inchain cyan in blue, orange,np-1-btm np-1in-top pink, in andcyan ionic blue, interactions np-1-btm in in purple. pink, and ionic interactions in purple. For the second np-1 molecule (np-1-btm) (Figure 10b), only the two C-terminal Arginines R5 and R6 areFor forming the second stable np ionic-1 molecule interactions (np with-1-btm the) RNA(Figure phosphate 10b), only backbone the two in C the-terminal middle A ofrginines the chain. R5 Theand restR6 are of theformingnp-1-btm stablemolecule, ionic interactions however, with seems the to RNA be interacting phosphate exclusively backbone in at the the middle 3’ end ofof thethe RNA,chain. where The rest the of majority the np- of1-btm the Thymine molecule, rings however, are grouped seems into abe cluster interacting with Adenine exclusively (A2, at A3, the and 3' end A4) byof theπ-stacking RNA, where interactions, the majority participating of the Thymin in thee stabilization rings are grouped of the wrapped in a cluster RNA with conformation. Adenine (A2, A3, and A4) by π-stacking interactions, participating in the stabilization of the wrapped RNA conformation.

Symmetry 2019, 11, 567 13 of 16

Symmetry 2019, 11 FOR PEER REVIEW 13 To account for the importance of the ionic interactions described above, we plotted its distances during theTo account complete for MDthe importance simulation of (Figure the ionic 11 interactions). The results described show above, that all we the plotted interactions its distances are highly conservedduring the during complete the most MD simulation part of the (Figure simulation. 11). The For resultsnp-1-top show, afterthat all an the initial interactions equilibration are highly period of approximatelyconserved during 40 ns, the a fairmost stabilization part of the simulation. of the H-bond For np distances-1-top, after occurs an initial for Argininesequilibration R1, period R2 and R3 of approximately 40 ns, a fair stabilization of the H-bond distances occurs for Arginines R1, R2 and (Figure 11a), while for Thymines T1, T2, and T3 the H-bond distances (two for T1, one for T2 and one R3 (Figure 11a), while for Thymines T1, T2, and T3 the H-bond distances (two for T1, one for T2 and for T3) are quite stable from the beginning of the simulation, particularly those between T1 and A6, one for T3) are quite stable from the beginning of the simulation, particularly those between T1 and andA6, T2 and and A7 T2 and(Figure A7 (Figure11b). As 11 forb). np-1-btm As for np-,1 only-btm, Arginine only Arginine R5 is ableR5 is to able form to stable form stable and signiﬁcant and ionicsignificant interactions ionic with interactions the phosphate with the backbone phosphate after backbone the initial after the 40 nsinitial equilibration 40 ns equilibration period, period, contributing to thecontributing stabilization to the of the stabilization extended of RNA the ext chainended (Figure RNA 11 chainc). These (Figure data 11c). remark These the data importance remark the of the Arginineimportance chains of inthe the Arginine nucleopeptide chains in np-1the nucleopeptideto induce and np maintain-1 to induce an and extended maintain conformation an extended of the RNAconformation chain, as well of the as theRNA role chain, of the as well nucleobase as the role groups, of the notnucleobase only for groups, the initial not recognitiononly for the initial of the RNA chainrecognition but also toof the stabilize RNA chain such but an interactionalso to stabilize with such RNA. an interaction with RNA.

Figure 11. RMSD plots of bond distances from their respective initial frame for Arginines R1, R2 and

R3 (a), Thymine T1, T2 and T3 in np-1-top (b), and Arginines R5 and R6 in np-1-btm (c). Symmetry 2019, 11, 567 14 of 16

4. Conclusions In this study, we report the synthesis of Arginine-based chiral nucleopeptides in which stereochemically-defined nucleobase and underivatized amino acids were alternated in the peptide backbone. With the purpose to investigate the role of backbone stereochemistry in determining the formation of DNA and RNA hybrids, we performed spectroscopic studies. CD experiments highlighted that the nucleopeptide having a fully l-backbone configuration and consisting of interpolated l-Arginine and l-Lysine residues covalently linked to Thymine nucleobases, formed the most intriguing and stable hybrid complexes with RNA molecules. Molecular Dynamics (MD) simulations provided insight into potential structure of the resulting complex of the l-nucleopeptide with RNA complementary strands, pointing out detailed chemical interactions involving both the l-backbone (ionics and H-bonds) and nucleobases (pairing and π-stacking) of the chiral nucleopeptide. In conclusion, we provided the first evidence of the effect of the backbone stereochemistry on the chemical interactions involved in the formation of the nucleopeptide/RNA complexes. These findings added a new piece to the complex nucleopeptide/nucleic interaction puzzle that could contribute to the future design of peptide-based molecules acting as antisense agents or diagnostic probes.

Author Contributions: Conceptualization, A.M.and S.D.M.; Data curation, S.T., F.F.M., R.R., A.M. and S.D.M.; Funding acquisition, S.D.M.; Investigation, S.T., F.F.M., A.M. and S.D.M.; Methodology, S.T., A.M. and S.D.M.; Project administration, A.M. and S.D.M.; Writing–original draft, A.M.; Writing–review & editing, E.N., A.M. and S.D.M. Funding: This research was supported by Scientific Independence of Young Researchers (SIR) 2014 (RBSI142AMA) and University of Campania Luigi Vanvitelli (Valere) to S.D.M. Conflicts of Interest: The authors declare no conflict of interest.

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