Templated Self-Assembly of Dynamic Peptide Nucleic Acids

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Templated Self-Assembly of Dynamic Peptide Nucleic Acids John M Beierle, Yasuyuki Ura, M. Reza Ghadiri, and Luke J. Leman Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00656 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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Templated Self-Assembly of Dynamic Peptide Nucleic Acids John M. Beierle, Yasuyuki Ura, M. Reza Ghadiri, and Luke J. Leman*

Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037

ACS Paragon Plus Environment

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ABSTRACT: Template directed macromolecule synthesis is a hallmark of living systems. Inspired by this natural process, several fundamentally novel mechanisms for template-directed assembly of nucleic acid analogs have been developed. Although these approaches have broad significance, including potential applications in biotechnology and implications for the origins of life, there are unresolved challenges in how to characterize in detail the complex assembly equilibria associated with dynamic templated reactions. Here we describe mechanistic studies of template-directed dynamic assembly for thioester peptide nucleic acid (tPNA), an informational polymer that responds to selection pressures under enzyme-free conditions. To overcome some of the inherent challenges in mechanistic studies of dynamic oligomers, we designed, synthesized, and implemented tPNA-DNA conjugates. The DNA primer region affords a high level of control over the location and register of the tPNA backbone in relation to the template strand. We characterized the degree and kinetics of dynamic nucleobase mismatch correction at defined backbone positions. Furthermore, we report the fidelity of dynamic assembly in tPNA as a function of position along the peptide backbone. Finally, we present theoretical studies that explore the level of fidelity that can be expected for an oligomer having a given hybridization affinity in dynamic templated reactions, and provide guidance for the future development of sequence self-editing polymers and materials. As our results demonstrate, the use of molecular conjugates of constitutionally static and dynamic polymers establishes a new methodology to expedite characterization of the complex chemical equilibria that underlie the assembly of dynamic informational polymers.


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INTRODUCTION Several fundamentally novel approaches for the template-directed synthesis of nucleic acid analogs have recently been demonstrated.1-15 These approaches break from the paradigm used in biology, where enzymes sequentially add monomeric units to extend a growing oligonucleotide backbone. The same basic strategy has often been followed in the laboratory for the templatedirected synthesis of biopolymers: a series of backbone extension reactions that irreversibly couples monomers or fragments together to form an oligomer.16-34 In contrast, recently described alternative approaches involve reversible covalent bonds1-5,11-15 (giving rise to dynamic combinatorial oligomers35-40) and/or attachment of the nucleobase units onto a preformed backbone6-10 (a so-called “nucleobase-filling” reaction). These new approaches hold considerable promise for the design of improved sequence-adaptive or self-healing materials.41,42 A nucleobase-filling mechanism offers a number of potential advantages compared to the traditional backbone elongation mechanism of template-directed synthesis. From a practical perspective, the nucleobase filling approach is inherently more modular than backbone elongation, because diverse oligomers can serve as the anchoring backbones for the same set of informational monomer units (as opposed to synthesizing a new set of monomers for each desired backbone architecture). Also, the nucleobase-filling mechanism could potentially be a more cooperative assembly process than templated polymerizations, because an unsubstituted site in the base-filling approach yields a polymer with an abasic site, whereas a missing linkage in the traditional approach leaves two shorter fragments (which is more detrimental to binding). It has further been suggested8 that the occurrence of non-templated side reactions might be lower because the informational units in a nucleobase filling reaction are not themselves


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polymerizable, and that the sequence fidelity of the templated reaction may be higher because the reaction site is closer to the site of the informational recognition. Likewise, the use of reversible covalent bonds in the synthesis of nucleic acid analogs affords them some unique characteristics. For instance, dynamic macromolecules assembled via reversible covalent reactions possess the ability to sample many different sequences from a relatively small pool of building blocks. By simply mixing the nucleobase units and the backbone under appropriate conditions, a dynamic combinatorial library of informational oligomers36,38,43,44 self-assembles spontaneously. In the presence of an oligonucleotide template (or other thermodynamic driving force), the constitution of the library can be shifted in a directed fashion, selectively amplifying those oligomers that are thermodynamically favored. Despite the apparent potential of dynamic nucleic acid analogs, their characterization and optimization have been limited by challenges inherent in studying complex chemical systems. Improved mechanistic understandings of these processes would help establish their advantages and limitations, and could point to strategies by which they could be improved. Here we report our efforts to address some of these issues by investigating base pair fidelity, mismatch correction, and DNA template-directed nucleobase filling reactions in the context of thioester peptide nucleic acid (tPNA)7 (Figure 1). This polymer self-assembles via reversible transthioesterification reactions45-47 that link nucleobase units onto simple repeating polycysteine (poly-Cys) peptide backbones. In this process there are several simultaneous chemical equilibria that give rise to many interconverting oligomer sequences. Therefore, to facilitate a detailed mechanistic understanding, we designed, synthesized, and implemented a family of tPNA-DNA chimeric constructs (Figure 1). The constitutionally fixed DNA region of the chimera (the DNA “primer”) preorganizes the peptide backbone to interact with the DNA


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template in a specified orientation, register, and location in a one-to-one ratio. This approach also obviates other complicating issues such as nucleation, facilitating characterization of various steps of the dynamic self-assembly process.

Figure 1. a) Reversible covalent assembly of tPNA oligomers proceeds via anchoring of thioester-derived nucleobase units (adenine is shown) onto an oligopeptide backbone. b) Schematic representation of the tPNA-DNA chimera acting in dynamic template-directed synthesis. The assembly of tPNA oligomers involves reversible transthioesterification reactions that anchor nucleobase monomer units onto poly-Cys peptide backbones. The DNA "primer" region of the chimera preorganizes the peptide backbone to interact with the DNA template in a specified orientation, allows substitutions to be introduced in the template at known positions relative to the peptide, and circumvents nucleation steps of tPNA assembly.

MATERIALS/EXPERIMENTAL DETAILS


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General experimental procedures. All solvents and reagents were purchased from commercial sources and used without further purification. All silica gel chromatography was performed using 60 A silica gel (SiliCycle), and thin layer chromatography was carried out with F254 silica gel plates (EMD). Buffer A is defined as 50 mM phosphate buffer, 1 M NaCl, 3 mM TCEP, pH = 7.0. Buffer B is defined as 50 mM phosphate buffer, 1 M NaCl, 3 mM iodoacetic acid, pH = 6.0. The matrix for all MALDI-TOF analyses was saturated sinapic acid in ACN:H2O:TFA (1:1:0.001) or trishydroxyacetophenone (18 mg) and sodium citrate (7 mg) in ACN:H2O (1 mL, 1:1). Streptavidin-sepharose High Performance beads were purchased from GE Healthcare. HPLC conditions and normalization. Analytical reverse-phase HPLC was performed at 260 nm using Phenomenex Jupiter Proteo or Zorbax 300-SB C-18 columns connected to a Hitachi D7000 HPLC system at a flow rate of 1.5 ml/min. Solvent system: binary gradients of solvent HPLC A1 (99% H2O, 0.9% acetonitrile, 0.1% TFA) and solvent HPLC B1 (90% acetonitrile, 9.9% H2O, 0.07% TFA). Integrated HPLC peak areas were normalized using the following measured extinction coefficients (pH ~2) for the nucleobase amides (produced by aminolysis cleavage of nucleobases from the peptide backbone using isobutylamine): adenine amide = 11,259 M-1 cm-1, cytosine amide = 7,191 M-1 cm-1, thymine amide = 9,295 M-1 cm-1, 7deazaguanine amide = 15,236 M-1 cm-1, guanine amide = 11,441 M-1 cm-1, acetamidobenzoic amide = 19,341 M-1 cm-1 and nucleobase acids (produced by hydrolysis) adenine acid = 10,365 M-1 cm-1, 7-deazaguanine acid = 14,833 M-1 cm-1. Synthesis of Cys-modified phosphoramidite. 5’-azidothymidine. Thymidine (5.0 g, 20.6 mmol, 1 eq), triphenylphosphine (5.4 g, 20.6 mmol, 1 eq), and sodium azide (6.7 g, 103 mmol, 5 eq) were dissolved in dry DMF (100 mL) under Ar. Carbon tetrabromide (5.2 g, 20.6 mmol, 1


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eq) was added portionwise over 10 min and the reaction was allowed to stir at ambient temperature and pressure under Ar atmosphere overnight. After 16 h MeOH (10 mL) was added in one portion and reaction was allowed to stir for an additional 1 h. The reaction was then evaporated to dryness, followed by absorption onto silica and purification by column chromatography (flash, silica, 2.5–10 % MeOH/DCM) to yield a white solid. (40 % yield). Compound characterization data matched those found in the literature.48 5’-aminothymidine. 5’-Azidothymidine (2.0 g, 8.3 mmol, 1 eq) was dissolved in EtOH (20 mL) with some heating in a round bottom flask charged with Ar. Pd/C (0.32 g, ~10 mol%) was added in one portion, followed by bubbling of pressurized H2. The flask was periodically vented as the reaction ran for 4 h at ambient temperature, monitored by TLC. The reaction was filtered over Celite, which was washed with 200 mL EtOH. The organic portions were combined and concentrated in vacuo to yield a dry white solid that was used immediately in the next step. Fmoc-Cys(tButhio)-5’-aminothymidine. Crude 5’-aminothymidine (1.59 g, 7.42 mmol, 1.05 eq) was dissolved in DMF (70 mL), and DIEA (3.7 mL, 21.2 mmol, 3 eq) was added. FmocCys(tButhio)-OH (3.05 g, 7.07 mmol, 1 eq) was added in one portion and the reaction was cooled to 0 °C. HATU (2.69 g, 7.07 mmol, 1 eq) was added. After 10 min at 0 °C the reaction was warmed to room temperature and stirred overnight at ambient temperature and pressure. DMF was removed in vacuo with heat (~40 °C), followed by column chromatography purification (silica, flash, 2%-5% MeOH/DCM) to yield a white solid. 3.0 g (66% yield from 5’azidothymidine). Characterization matched that of Becker et al.49 Fmoc-Cys(tButhio)-5’-aminothymidine

phosphoramidite.

Fmoc-Cys(tButhio)-5’-

aminothymidine (175 mg, 0.275 mmol, 1 eq) was dissolved in dry THF under Ar. 2-cyanoethyl N,N-diisopropylchloro-phosphoramidite was added in one portion (184 µL, 0.825 mmol, 3 eq),


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followed by dropwise addition of dry DIEA (239 µL, 1.38 mmol, 5 eq). The reaction was left at ambient temperature and pressure for 2 h. THF was removed in vacuo, and the oily residue was taken up in a minimal amount of DCM followed by precipitation with hexanes. Centrifugation followed by additional trituration with hexanes gave the phosphoramidite as a gummy solid. (When desired, purification of the phosphoramidite can be performed via column chromatography (SiO2, EtOAc/hexanes, flash) to yield the phosphoramidite as a mixture of diastereomers.) 1H NMR (400 MHz, CDCl3) for mixture of diastereomers (see Figure S1 for 1H NMR spectra): 7.76 (d, 2H), 7.59 (d, 2H), 7.39 (t, 2H), 7.30 (d, 2H), 7.14-7.10 (m, 2H), 6.075.94 (m, 1H), 5.84-5.75 (m, 1H), 4.45-4.22 (m, 7H), 3.89-3.84 (m, 1H), 3.74-3.57 (m, 3H), 3.10 (br m, 2H), 2.84-2.79 (m, 1H), 2.67-2.56 (m, 4H), 2.42-2.33 (m, 1H), 1.89 (m, 3H), 1.30 (s, 9H), 1.19 (m, 12H). ESI-MS observed for phosphate derivative, due to hydrolysis of the phosphoramidite during LCMS analysis [(m/z) 735.11 [M+H]+ (MWcalcd = 735.19). General synthesis of 5’-Cys-modified oligonucleotides. The Fmoc-Cys(tButhio)-5’aminothymidine phosphoramidite was transferred to a vessel equipped for automated DNA synthesis and was dried under vacuum overnight. The compound was then dissolved in dry ACN (0.25 M, based on phosphoramidite starting material) and used in standard solid phase oligonucleotide synthesis on an ABI 394 automated DNA synthesizer with an extended coupling time (10 min). Standard conditions for cleavage of the oligonucleotide from the CPG (concentrated ammonium hydroxide, 1 h, 57 °C) yielded the 5’-Cys-modified oligonucleotide. Analysis by HPLC showed that the Cys-modified oligonucleotide was >90 % pure. General synthesis of peptide thioesters. The peptides were prepared by standard Fmoc solid phase synthesis protocols with an Advanced Chemtech Apex 396 peptide synthesizer. A typical synthesis was performed on 0.07 mmol scale using 0.5-0.8 mmol/g 2-chlorotrityl chloride resin


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(Novabiochem). The peptide was cleaved using AcOH/TFE/CH2Cl2 for 2 h to provide the side chain-protected C-terminal acid. Hexanes was added to the cleavage solution (to act as an azeotrope with AcOH) and the mixture was concentrated under reduced pressure and dried overnight. The peptide (70 mg, 0.03 mmol, 1 eq) was dissolved in a 1:1 mixture of DCM:DMF (4 mL). DIC (23 µL, 0.150 mmol, 5 eq), HOBt (20 mg, 0.150 mmol, 5 eq), and DIEA (52 µL, 0.30 mmol, 10 eq) were then added to the solution, followed by stirring at ambient temperature for 10 min. N-Acetylcysteamine (SNAc, 80 µL, 0.75 mmol, 25 eq) was added in one portion and the reaction was left overnight (16 h). The solution was then concentrated to an oily residue in vacuo, followed by side chain deprotection with 3 mL TFA:TIS:EDT:H2O (90:4:4:2) for 2 h at ambient temperature and precipitation with Et2O to yield the crude peptide thioester, which was purified by reverse-phase preparative HPLC. Ac[Glu(tBu)Cys(Trt)] 4Glu(tBu)-SNAc. MALDI-TOF (m/z) = 1217.6 (MWcalcd = 1219.4) Ac[ArgCysGlyCys] 2Gly-SNAc. MALDI-TOF (m/z) = 1057.4 (MWcalcd = 1058.2) Ac[GluCys] 9Glu-SNAc. MALDI-TOF (m/z) = 2375.3 (MWcalcd = 2378.2) General method for native chemical ligation of peptides to oligonucleotides. Peptide thioester (4 µmol, 10 eq) was dissolved in 750 µL of buffer (1 M TRIS, 200 mM NaCl, 0.175 M TCEP, 3 % w/v SNAc, pH = 7.5) to which the 5’-Cys-modified oligonucleotide (0.4 µmol, 1 eq) was added in one portion. The reaction was covered with foil and allowed to react at room temperature for 4 h. Completion was monitored by HPLC and MALDI-TOF. The reaction mixture was diluted two-fold and purified by anion exchange on FPLC (gradient: 0-75% FPLC B, 45 min). Ac[ArgCysGlyCys] 2GlyCys-TCAGCACCTA (1). MALDI-TOF (m/z) = 4009.3 (MWcalcd = 4010.3).


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Ac[ArgCysGlyCys] 2GlyCys-TCAGC (2). MALDI-TOF (m/z) = 2500.4 (MWcalcd = 2504.2). Ac[GluCys] 5-TCAGCACCTA (3). MALDI-TOF (m/z) = 4167.9 (MWcalcd = 4171.4). Ac[GluCys] 10-TCAGCACCTA (4). MALDI-TOF (m/z) = 5332.0 (MWcalcd = 5329.1). Oligonucleotide template synthesis and characterization. DNA template strands were obtained from Sigma or e-oligos (www.e-oligos.com) or synthesized on an ABI 394 automated oligonucleotide synthesizer using standard protocols. All reagents for oligonucleotide synthesis were obtained from Glen Research. Nucleobase protecting groups for solid phase synthesis were Pac-dA, Ac-dC, and iPr-Pac-dG. Biotin was incorporated using a biotin phosphoramidite (Glen Research) with an increased coupling time of 15 min. Cleavage and deprotection were carried out by treating the CPG with concentrated ammonium hydroxide at 50 °C for 4 h. The cleavage solution was filtered and concentrated by rotary evaporation. In some cases, the DNA strands were HPLC purified on a semipreparative C18 column (Vydac, #218TP510) (1.0 cm x 25 cm) using a binary gradient of solvent A3 (100 mM triethylammonium acetate, pH 7.0, 0.0125 % sodium azide) and solvent B3 (100 mM triethylammonium acetate, pH 7.0, 80 % ACN, 0.0125 % sodium azide). DNA strands were then desalted using C18 Sep-Pak cartridges (Waters) and lyophilized or concentrated by rotary evaporation. Synthesis of nucleobase thioesters. Nucleobase thioesters A, T, 7-deazaG, and C were prepared as described in Ura et al.7 Acetamidobenzoic acid thioester. Synthesis of the acetamidobenzoic thioester proceeded by treating acetamidobezoic acid (200 mg, 1.12 mmol, 1 eq) with EDC•HCl (257 mg, 1.34 mmol, 1.2 eq), HOBt (188 mg, 1.23 mmol, 1.1 eq), and DIEA (585 µL, 3.36 mmol, 3 eq) in DCM (10 mL). After 10 min, methylthioglycolate (200 µL, 2.24 mmol, 2 eq) was added in one portion. The reaction was left to stir at ambient temperature and pressure overnight. The reaction was


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taken up in H2O and extracted with DCM (3x, 20 mL each). The organics were combined and successively washed with 5 % NaHCO3 (30 mL), 5 % KHSO4 (30 mL) and brine (30 mL). The organic portion was then dried over MgSO4 and evaporated to a white solid. 215 mg (80 % yield). 1H NMR (400 MHz, DMSO-d6): 10.37 (br s, 1H), 7.91-7.74 (m, 4H), 3.95 (s, 2H), 3.63 (s, 3H), 2.09 (s, 3H) ppm. ESI-MS [(m/z) 290.05 [M+Na]+ (MWcalcd = 290.05). Guanine thioester. The O-Bn guanine acetic acid was prepared by a reported procedure. 50 Dry DMF (10 mL) was added to a mixture of O-Bn guanine acetic acid (171 mg, 0.57 mmol), EDC·HCl (121 mg, 0.63 mmol), and HOSu (73 mg, 0.63 mmol) under Ar, and the reaction mixture was stirred at room temperature for 2 h. After cooling to 0 °C, methyl thioglycolate (66 µL, 0.74 mmol) and DIEA (129 µL, 0.74 mmol) were added and the reaction mixture was warmed to room temperature. After 2 h, AcOH (49 µL, 0.86 mmol) was added and solvent was removed under reduced pressure. The residue was taken up in EtOAc and water and extracted. The organic layer was washed with water and brine, and was dried over Na2SO4. Filtration and removal of solvent gave a white solid, which was then triturated with Et2O. The solid was collected by suction filtration and was again washed with Et2O. After drying under vacuum, the O-Bn thioester was obtained in 78% yield (173 mg, 0.45 mmol). 1H NMR (500 MHz, DMSOd6): δ 7.90 (s, 1H), 7.53-7.34 (m, 5H), 6.56 (s, 2H), 5.51 (s, 2H), 5.17 (s, 2H), 3.82 (s, 2H), 3.63 (s, 3H). ESI-MS (m/z) 388.1073 [M+H]+ (MWcalcd = 388.1074). Removal of the O-Bn protecting group was accomplished by introducing HF (ca. 8 mL) to a mixture of the O-Bn thioester (128 mg, 0.33 mmol) and anisole (400 µL) at –78 °C, and allowing the reaction mixture to stir at 0 °C for 1 h. HF was evaporated by a stream of N2. The residue was taken up in water and Et2O and the aqueous layer was removed. The organic layer, which contained a white solid, was filtered by suction filtration, and the collected solid was washed with Et2O. After drying under vacuum, the


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guanine thioester was obtained in 79% yield (77 mg, 0.26 mmol). The removed aqueous layer was washed with Et2O and was lyophilized to give additional amount of the product (20 mg, 0.068 µmol, 20%). 1H NMR (300 MHz, DMSO-d6): δ 10.64 (s, 1H), 7.73 (s, 1H), 6.55 (s, 2H), 5.10 (s, 2H), 3.82 (s, 2H), 3.63 (s, 3H). ESI-MS (m/z) 298.0600 [M+H]+ (MWcalcd = 298.0604). Preparation of stock solutions for oligonucleotides and nucleobase thioesters. Oligonucleotide stock solutions (typically 1 mM) were prepared by dissolving the strands in water or TE buffer (10 mM TRIS, 1 mM EDTA) and were stored at 4 °C. Oligonucleotide stock concentrations were determined by UV absorbance at 260 nm using extinction coefficients calculated online with OligoAnalyzer version 3.1 (www.idtdna.com). Adenine and cytosine thioester stock solutions were prepared fresh daily in concentrations of 15-25 mM using water. Thymine and 7-deazaguanine stocks were prepared in concentrations of 30-50 mM using DMF and were kept for up to one week. Stock concentrations were determined by UV absorbance at 260 nm using the following measured extinction coefficients (pH ~6) for the nucleobase thioesters: adenine = 10,900 M-1 cm-1, cytosine = 7,790 M-1 cm-1, thymine = 9,252 M-1 cm-1, 7deazaguanine = 15,663 M-1 cm-1, guanine = 12,306 M-1 cm-1, acetamidobenzoic acid = 13,722 M1

cm-1. General procedure for thermal denaturation experiments. The nucleobase thioester (200

µM, 2 eq/Cys) was incubated with the peptide-DNA chimera (10 µM) and the DNA template (10 µM) for 30 min in buffer A (total volume 50 µL). A 5 µL aliquot was removed and subjected to MALDI-TOF analysis to ensure full substitution. The remaining solution was diluted to a total volume of 500 µL and subjected to UV monitored thermal denaturation (25–65 °C, 2 °C/min). Controls were run compensating the missing reaction components with equivalent amounts of H2O where appropriate. This procedure was repeated for a number of combinations of monomer,


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chimera, and template as detailed in Table 1. In all cases, the nucleobase thioester was used at a concentration of 2 eq/Cys and full substitution was confirmed by MALDI-TOF analysis. In cases where chimera 2 was employed, a thermal annealing step (heating from 25–65 °C, 2 °C/min) was carried out on the diluted reaction solution immediately prior to analysis. General procedure for template directed synthesis competition reactions. Ac[GluCys]10TCAGCACCTA (chimera 4, 25 µM) was incubated with two, three, or four thioester monomer compounds (monomer concentration ranged from 1–2 mM and were chosen such that equal amounts of each monomer were anchored to the peptide in the absence of a template) and methylthioglycolate (1 mM) for 10 min at room temperature in Buffer A. This master solution was then split to multiple vials, to each of which a different biotinylated DNA template strand (50 µM, annealed at 40 °C for 10 min) was added. The final concentration of thioester monomers in the reactions was 0.5–1 mM. After 2 h at 0 °C the reactions were transferred to vials containing Streptavidin-sepharose High Performance beads that had been washed five times with 400 µL buffer A. The reactions were incubated an additional hour at 0 °C with the beads. The beads were then transferred to centrifuge filter tubes and were spun down and washed twice with buffer A at 22 °C (200 µL each). The beads were then transferred to eppendorf vials using H2O (100 µL). After centrifugation and decanting, the beads were frozen and lyophilized to remove excess water. Following lyophilization, the thioester nucleobases were hydrolyzed with 0.5 M NaOH or subjected to aminolysis by treatment with neat isobutylamine (depending on which treatment yielded the best resolution of products in the subsequent HPLC analysis). For the hydrolysis treatment, after 10 min at 22 °C, TFA was added to acidify the beads to pH = ~1 (5.3 µL) and the supernatant was used for HPLC analysis. For the aminolysis treatment, after 10 min at 22 °C, the excess amine was removed in vacuo and the nucleobase amides were taken up in


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HPLC solvent A1 (100 µL) for analysis. The presence or absence of beads had no observed effect on the ratio of monomers anchored to the peptide, nor did the time at which the beads were added. In a control reaction involving only beads, almost no nucleobases were observed, indicating a low level of non-specific binding of the nucleobases. Additional figures are shown in the Supporting Information for reactions involving various DNA templates and different final concentrations of thioester monomers. HPLC gradient: 100% solvent A1 for 3 min followed by a linear ramp to 50 % solvent A1 over a period of 25 min. Fidelity as a function of position experiments. Ac[GluCys]10-TCAGCACCTA (chimera 4, 12.5 µM) was incubated with A thioester (0.45 mM) and dG thioester (0.5 mM), and ABA thioester (0.5 mM) along with methylthioglycolate (1 mM) for 10 min at room temperature in Buffer A. The solution was then split to six vials containing Streptavidin-sepharose High Performance beads that had been washed five times with 400 µL buffer A. A different biotinylated DNA template strand was added to each vial (25 µM of each, annealed at 40 °C for 10 min before adding). The reactions were then incubated for 2 h at 0 °C. The beads were then transferred to centrifuge filter tubes spun down and washed two times with buffer A at 22 °C (200 µL). The washed beads were next transferred to an eppendorf tube using H2O (100 µL). The water was decanted and the beads were treated with neat isobutylamine (50 µL, 22 °C) to subject nucleobase thioesters to aminolysis. The excess solvent was removed under vacuum. The dried beads were then taken up in HPLC solvent system 1 (100 µL) and sonicated for 10 min to ensure dissolution of the nucleobase amides. Removal of the beads by centrifugation and analysis of the supernatant by HPLC was then performed to determine the ratio of adenine-N9acetic

isobutylamide,

7-deazaguanine-N1-acetic

isobutylamide,

and

acetamidobenzoic


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isobutylamide. HPLC gradient: 100% solvent A1 for 3 min followed by a linear ramp to 50 % solvent B1 over a period of 25 min. This procedure was performed in duplicate. Kinetic analysis of mismatch correction. Ac[GluCys]10-TCAGCACCTA (chimera 4, 12.5 µM) was incubated with 7-deazaG thioester (1 mM) and ABA thioester (1 mM) with methylthioglycolate (1 mM) for 10 min at room temperature in Buffer A. The solution was then split to five vials containing Streptavidin-sepharose High Performance beads that had been washed five times with 400 µL buffer A. A different biotinylated DNA template strand was added to each vial (25 µM of each, annealed at 40 °C for 10 min before adding). The reactions were incubated at 0 °C for an additional 20 min, after which a prechilled aliquot of A thioester (0.9 mM) was dispensed to each vial. Aliquots for time points were removed at 1, 3, 5, 10, 15, and 30 min. The aliquots were treated as follows: The beads were immediately transferred to centrifuge filter tubes containing 200 µL of Buffer B to quench the reaction, spun down, and washed two more times with buffer B at 22 °C (200 µL). The washed beads were next transferred to an eppendorf tube using H2O (100 µL). The water was decanted and the beads were frozen and lyophilized. After subjecting the beads to aminolysis by reacting 10 min at 22 °C with neat isobutylamine, the excess solvent was removed under vacuum. The dried beads were then taken up in HPLC solvent A1 (100 µL) and sonicated for 10 min to ensure dissolution of the nucleobase amides. Removal of the beads by centrifugation and analysis of the supernatant by HPLC was then performed to determine the ratio of adenine-N9-acetic isobutylamide, 7deazaguanine-N1-acetic isobutylamide, and acetamidobenzoic isobutylamide. HPLC gradient: 100% solvent A1 for 3 min followed by a linear ramp to 50 % solvent A1 over a period of 25 min. This procedure was performed in duplicate.


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Page 16 of 49

Equilibrium simulations. For each simulated reaction, the sequences of all possible oligomers (dependent on the number of dynamic positions and the number of nucleobases present) were first generated using a Python script. ∆G values for hybridization of each sequence to the DNA template

were

calculated

using

the

DINAMelt

two-state

hybridization

server

(http://dinamelt.bioinfo.rpi.edu/twostate.php) at 0 °C with 1 M Na+, 0 M Mg2+, and strand concentrations of 10 µM. The output ∆G values from DINAMelt (kcal) were converted to kJ for input into the DCLSim program.51 Simulations were carried out using the Single Library module of DCLSim at 0 °C, which outputs the concentration of every sequence at equilibrium, given ∆G values for binding to the template. Each simulated reaction contained a chimera ("p") containing either five or one dynamic positions, a template ("t"), and 2–4 monomers ("a", "b", "c", or "d", which represent a, g, c, and t nucleobases, respectively). Typical concentrations of building blocks used in the simulations were p=1, t=2, and monomers=40 (eight-fold eq relative to the dynamic positions). An equilibrium constant of 10 was used for the formation of each oligomer from the chimera and monomer building blocks, based on the high efficiency of tPNA assembly. To scale the ∆G value contributed by the dynamic region of the chimera, we used the following equation: ∆Gscaled = ∆Gprimer + (scale factor)(∆GDNA − ∆Gprimer) where ∆Gscaled is the ∆G value for hybridization of the scaled chimera to the template, ∆Gprimer is the ∆G value contributed by the primer region of the chimera, and ∆GDNA is the ∆G value calculated for DNA/DNA hybridization. The term (∆GDNA − ∆Gprimer) represents the ∆G value contributed by the dynamic region of the chimera. ∆Gprimer was determined from the DINAMelt ∆G values for sequences having no complementary base pairing in the template region. Sample DCLSim input and output files are shown in Table S1 and Table S2.


16

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Biochemistry

RESULTS AND DISCUSSION Design and synthesis of DNA-tPNA chimeras. To facilitate our mechanistic studies, we synthesized a family of tPNA-DNA chimeras (Figure 2). In designing an appropriate linker to connect the tPNA peptide backbone to a DNA primer, we considered several factors. First, the backbone repeats of DNA and tPNA both contain six bonds (Figure 2a), so we targeted a linker structure that would retain six backbone bonds between the last nucleobase of the DNA primer and the first nucleobase of the tPNA peptide. Second, we favored a linkage between the Cterminus of the peptide backbone and the 5'-end of the DNA primer. This design aspect was based on the preferred pairing arrangement of traditional PNA hybridized to DNA (5'-end of DNA aligned with C-terminus of PNA),52 and the idealized pairing conformation of dipeptidebased informational oligomers proposed by Eschenmoser, Krishnamurthy, and coworkers (5'-end of oligonucleotides correspond to N-terminus of dipeptides).53-55 Finally, we required an efficient, modular, chemically orthogonal method to ligate the peptide backbone to DNA so that we could combine various DNA and peptide fragments to produce a family of chimeras. Although several peptide-oligonucleotide ligation techniques have been described,56-64 conjugation via native chemical ligation65-68 met all the above criteria, and seemed especially appropriate because the tPNA backbone is a poly-Cys peptide. For the native chemical ligation reactions, we required a collection DNA oligonucleotides modified at the 5’ terminus with a Cys residue and peptides modified at the C-terminus with a thioester. Accordingly, we prepared an Fmoc-Cys(StBu)-modified thymine phosphoramidite following the method of Becker et al49 for incorporation at the 5' end of synthetic DNA strands during solid-phase oligonucleotide synthesis (Figure 2c). Briefly, the phosphoramidite synthesis


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Page 18 of 49

involved conversion of thymidine to 5’-aminothymidine via the intermediacy of 5’azidothymidine. Subsequent coupling of Fmoc-Cys(StBu)-OH to the 5’-amino group and phosphoramidation yielded the desired building block. The peptide C-terminal thioesters were prepared using standard solid-phase peptide synthesis methodologies. The corresponding fully deprotected DNA and peptide fragments could be coupled efficiently under neutral aqueous conditions (Figure 2c), allowing us access to a number of tPNA-DNA chimeras varying in peptide sequence and DNA primer length.

Figure 2. a) A comparison of DNA, tPNA, and the tPNA-DNA chimera backbones. The linker region in the chimera is highlighted. b) Structures of nucleobase thioester monomers used. 7deazaG = 7-deazaguanine, ABA = acetamidobenzoic acid. c) The tPNA-DNA conjugates were prepared from a set of suitably derivatized peptide and DNA fragments using native chemical ligation. The 5'-terminally modified DNA fragments were prepared using a Cys-appended thymine phosphoramidite (inset).


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Biochemistry

Characterization of tPNA-DNA chimeras. To confirm that the linker design was appropriate to allow tPNA hybridization to the template strand, we first characterized chimeras 1–3 (Table 1), which vary in the length of DNA primer region or amino acid sequence in the tPNA backbone. The peptide sequence contains Cys residues at every other position to anchor nucleobase units via transthioesterification, interspersed by other amino acids to provide the appropriate six-atom backbone repeat distance between nucleobases to allow hybridization to oligonucleotides. Incubation of the chimeras with two equivalents (relative to Cys) of nucleobase thioester (see Figure 2b for nucleobase thioester structures) at room temperature for 30 min led to quantitative substitution at the Cys residues as determined by MALDI-TOF mass spectrometry. Next, we carried out UV-monitored thermal denaturation analyses to confirm that the primer region of the chimeras would localize the tPNA adjacent to a DNA template, and that anchoring of nucleobase thioesters complementary to the tPNA template region would increase the stability of the hybridized structure (Table 1; see Supporting Information Figure S2 for example melting curves). We were pleased to observe increases in melting temperature when chimera 1 (DNA primer region of 10 bases, [Arg-Cys-Gly-Cys]2Gly-Cys peptide) was reacted with G, A, or C nucleobase monomers and incubated with complementary DNA templates (Table 1). The T nucleobase thioester did not offer any observable additional stability to the hybridized assembly with a poly-A template region, consistent with previous studies using nonprimed tPNA oligomers.7 Using chimera 2, which had a shortened DNA primer region of only 5 bases, no denaturation curves were observed in the absence of nucleobase thioesters (as would be expected for such a short primer), but prominent sigmoidal melting transitions were observed at 20 °C or 29 °C in the presence of the A or G monomers, respectively, with the appropriate templates (Table 1). Chimera 3 (DNA template region of 10 bases, [Glu-Cys]5 peptide) was


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Page 20 of 49

prepared using a peptide sequence that lacks Arg residues, to reduce the propensity of this chimera to aggregate nonspecifically with the DNA primer or template due to electrostatic interactions. With 3, we carried out three reactions involving anchoring of the G monomer, followed by thermal denaturation with DNA strands having either a poly-C, poly-T, or poly-A template region. As expected, the melting temperatures for these complexes increased only in the case of the

Table 1. Observed changes in melting temperature for tPNA–DNA chimeras 1–3 in the presence of nucleobase thioester monomers.

Chimera

[a]

Template

[b]

Tm1 Tm2 DNA/DNA [f] [c] [d] Monomer (°C) (°C) ∆Tm ∆Tm[g]

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGACCCCC TCAGCACCTA-3' (1)

G

16

30

14

45

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGATTTTT TCAGCACCTA-3' (1)

A

16

22

6

29

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGAGGGGG TCAGCACCTA-3' (1)

C

19

22

3

45

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGAAAAAA TCAGCACCTA-3' (1)

T

19

18

–1

29

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGACCCCC TCAGC-3' (2)

G

n.o.[e]

29

≥ 29

53

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGATTTTT TCAGC-3' (2)

A

n.o.

20

≥ 20

35

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGAGGGGG TCAGC-3' (2)

C

n.o.

n.o.

~

53

Ac(ArgCysGlyCys)2GlyCys– 5'-GCTGAAAAAA TCAGC-3' (2)

T

n.o.

n.o.

~

36

Ac(GluCys)5– TCAGCACCTA-3' (3)

5'-TAGGTGCTGACCCCC

G

43

49

6

23


5'-TAGGTGCTGATTTTT

G

44

44

0

0


20

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Biochemistry


5'-TAGGTGCTGAAAAAA

G

45

45

0

0

[a]

Amino acids and nucleobases are abbreviated using their three-letter and one-letter codes, respectively. [b] All DNA templates contain a primer region (complementary to the DNA sequence in the chimera) and a tPNA template region; the template region is shown in bold type. [c] Tm1 is the average of two UV denaturation experiments conducted at equimolar DNA and chimera concentrations of 10 µM (chimeras 1 and 2) or 1 µM (chimera 3) in the absence of nucleobase thioesters. All melting curves were measured in neutral aqueous phosphate buffer containing 1 M NaCl. [d] Tm2 is the average of two denaturation experiments conducted at equimolar DNA and chimera concentrations of 10 µM (chimeras 1 and 2) or 1 µM (chimera 3) in the presence of the indicated nucleobase thioesters (4 eq/Cys). [e] "n.o." indicates a sigmoidal melting transition was not observed. [f] ∆Tm is the difference between Tm1 and Tm2 in °C, which represents the increase in duplex stability afforded by the nucleobase-substituted tPNA region of the chimera. [g] DNA/DNA ∆Tm is the difference in Tm values that would be expected (as calculated by nearest-neighbor methods) if the chimera were a fully complementary DNA strand.

complementary poly-C template region (complete base filling was confirmed by MALDI-TOF for all reactions) (Table 1). To further validate the chimeras, we next established that they could be employed in pull-down competition reactions involving two or more nucleobase thioesters (Figure 3). Establishing the relative ratios of anchored nucleobases captured by various biotinylated template sequences provides an indication of the selectivity of templated tPNA assembly. We carried out three reactions involving competitions between the A and 7-deazaguanine (7-deazaG), the A and G, or the C and T monomers. 7-Deazaguanine was used in place of guanine in some reactions to avoid the possibility of forming of G-quadruplex type structures, but still retain the Watson-Crick specificity of guanine. For each reaction, the appropriate monomers were incubated with tPNADNA chimera 4 (Ac-[Glu-Cys]10–TCAGCACCTA-3’) at room temperature in neutral buffer (10 min) followed by splitting the reaction to three vessels. To each vessel, a different biotinylated DNA template strand was added. After 2 h at 0 °C, streptavidin beads were added and the reactions were left for one additional hour on ice. The beads were then filtered, washed at room


21

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Page 22 of 49

temperature, and treated with base to determine by HPLC analysis the ratio of nucleobases anchored to 4. For the competition reaction involving the A and 7-deazaG monomers, the DNA sequences having poly-T or poly-C tPNA template regions led to enrichment of the complementary nucleobase thioester of around 75% (Table 2). In a similar competition experiment involving the A and G monomers, three of the four templates (including the control poly-A template) produced enrichments of the G-monomer (Table 2). When the conditions of the reaction were changed to reduce the concentration of the G thioester, we observed the expected enrichments of complementary nucleobase thioester of 82-91% (Table 2).

Figure 3. Schematic illustration of protocol for template directed dynamic competition reactions. A mixture of 2–4 nucleobase monomers was incubated with chimera 4 and a sepharose-bound DNA template. After washing and cleavage of the bound tPNA nucleobases, analysis by HPLC allowed us to establish the relative ratios of anchored nucleobases captured by each template sequence as an indication of the selectivity of templated tPNA assembly.


22

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Biochemistry

In the reactions involving the C and T monomers, the poly-G template strand enriched C monomer anchoring to 68%, somewhat lower than the 75-90% enrichment levels observed for the A, 7-deazaG, or G thioesters. The lower enrichment observed for C thioester anchoring could result from self-aggregation of the template containing the poly-G tail, which would reduce its availability to template tPNA formation. The T monomer was not enriched in the presence of a poly-A template region (Table 2). In contrast, in a series of template directed synthesis experiments involving mixed DNA template sequences (as opposed to homopolymeric templates) with the A, C, or T nucleobase monomers and chimera 4, the ratio of each of these three nucleobases (including the T monomer) increased similarly in the presence of the corresponding complementary template (see Figures S3 and S4). There are at

Table 2. Percent incorporation of nucleobase monomers onto chimera 4 in various templated competition reactions.[a] A

7-deazaG

G

C

T

(%)

(%)

(%)

(%)

(%)

A10 (control) 49

51

~

~

~

C10

24

76

~

~

~

T10

73

27

~

~

~

A10 (control) 34

~

66

~

~

A vs. G

C10

4

~

96

~

~

1:1

T10

69

~

31

~

~

G10

20

~

80

~

~

A10 (control) 61

~

39

~

~

Entry A vs. 7deazaG 1:1

A vs. G

Template Region


23

Biochemistry

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1 : 0.5

Page 24 of 49

C10

9

~

91

~

~

T10

82

~

18

~

~

G10

37

~

63

~

~

T10 (control)

~

~

~

51

49

G10

~

~

~

68

32

A10

~

~

~

47

53

C vs. T 1:1 [a]

Monomers were incubated in the ratios shown (where "1" represents 8 equivalents relative to Cys) with chimera 4 (25 µM) and a DNA template (50 µM) in neutral phosphate buffer containing TCEP (3 mM), methylthioglycolate (1 mM), and NaCl (1 M) for ~2 h at 0 °C in the presence of streptavidin beads. After filtering and washing, the beads were analyzed by HPLC to determine the ratio of anchored thioesters. The DNA templates = Biotin-5'-TAGGTGCTGABBBBBBBBBB. Chimera 4 = Ac-(GluCys)10-TCAGCACCTA-3'. Standard error for nucleobase incorporation reactions was ≤3%.

least two possible reasons for this discrepancy. First, the observed small/absent melting temperature changes and poor levels of templated synthesis for the pyrimidine nucleobases described above may arise from unique issues associated with the corresponding homopolymeric DNA template sequences, such as unusual secondary structure. Second, dynamic combinatorial library members assembled from heteromeric constituents have a competitive entropic assembly advantage over homomeric library members,44,69-71 so it is understandable that the templated synthesis of heteromeric sequences could proceed more readily than for homomeric oligomers. Even so, the increased library sizes associated with heteromeric libraries offer their own complications, as discussed below. The above results suggest that base stacking interactions are an important factor in the dynamic base filling mechanism, and could help explain the observed higher fidelity of templated synthesis for the purine nucleobases compared to the pyrimidines in the tPNA oligomer.


24

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Biochemistry

Fidelity of nucleobase anchoring. An important question is how the fidelity of reversible nucleobase anchoring varies as a function of position along the peptide backbone. A key feature of the tPNA-DNA chimera is that the fixed DNA region preorganizes the tPNA backbone to interact with the DNA template in a specified orientation and location, making it possible to determine the fidelity of dynamic nucleobase incorporation at desired positions within the tPNA backbone. We carried out reactions in which chimera 4 was incubated with a pool of nucleobase monomers along with one of several DNA templates, each having a TT substitution at a different position along the length of a poly-C sequence (Figure 4). We could then compare the fidelity of complementary nucleobase incorporation as a function of the location of the TT substitution in the template. In these experiments, we employed an approximately stoichiometric ratio of A and 7-deazaG nucleobase thioesters, along with a “nonsense” thioester monomer derived from

Figure 4. Fidelity of nucleobase incorporation as a function of position against template. Incorporation fidelity was determined by incubating the A, 7-deazaG, and ABA monomers with chimera 4 and a DNA template in neutral buffer for 2 h at 0 °C, after which the tPNA-anchored


25

Biochemistry

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Page 26 of 49

nucleobases were analyzed by HPLC. The ABA thioester was included as a control to establish the level of background, nonspecific thioester anchoring. Error bars represent the standard deviation from two independent experiments.

acetamidobenzoic acid (ABA), which was included to establish the level of non-specific thioester anchoring. HPLC analyses of the reactions indicated that incorporation of the A monomer was increased for all the TT substitution templates relative to a poly-C control sequence, although the fidelity of incorporation decreased as the substitution site approached the terminus of the duplex. In contrast, incorporation of the ABA thioester did not vary depending on the template (observed incorporation of 15±1% across all templates tested), as would be expected for a nonspecific interaction. Similar results were obtained in analogous experiments conducted without the nonsense thioester ABA (Figure S5). The decrease in fidelity for anchoring of the A monomer as the substitution site approached the terminus likely derived from frayed edges in the hybridized structure due to reduced base stacking energetics compared to internal base pairs.72,73 Another possibility is that the tPNA and DNA backbones fall out-ofregister as they progress farther from the duplexed primer region, although our previous studies with longer non-primed 16-mer and 20-mer tPNA strands did not indicate any issues involving backbone registry.7 Kinetics of nucleobase exchange and mismatch correction. We next carried out an investigation to better understand the kinetics of nucleobase exchange and mismatch correction in the tPNA oligomers. Dynamic combinatorial libraries such as tPNA are usually studied at thermodynamic equilibrium. However, the kinetic factors associated with the assembly of dynamic oligomers can also impact the properties of sequence-adaptive polymers. A priori, it


26

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Biochemistry

was unclear how the rate of reversible nucleobase substitution reactions at “internal” positions (Cys thiol moieties within a lengthy stretch of substituted Cys residues) would compare to those at a terminus of the oligomer. More importantly, in the presence of an oligonucleotide template, the surrounding duplex regions might significantly retard or enhance the dynamic exchange rate of buried mismatches, which could affect the opportunity for the sequences to correct incorporation errors. To gain insight, we preincubated chimera 4 with the 7-deazaG and ABA thioesters to fully load the backbone. The ABA monomer was included to serve as an internal standard for background, nonspecific anchoring. The adenine thioester was then added (time 0), and the incorporation of adenine thioester onto the backbone was monitored in the presence of different DNA templates (Figure 5). Because the chimera was preincubated with the 7-deazaG and ABA monomers, the

Figure 5. Kinetics of nucleobase exchange and mismatch correction as a function of position against template. Chimera 4 and one of several DNA templates were pre-incubated with


27

Biochemistry

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Page 28 of 49

equimolar amounts of 7-deazaG and ABA monomers for 20 min at 22 °C. The A monomer was then added to each reaction (designated as time = 0). Aliquots were quenched at various time points and analyzed by HPLC. a) Kinetic traces for the reactions. Error bars represent the standard deviation from two independent experiments. b) The initial rate of incorporation of the A nucleobase was determined from the first 5 min of the reactions.

observation

of

adenine

anchoring

minimally

requires

a

thioester

hydrolysis

or

transthioesterification event to free one of the Cys residues, followed by anchoring of the A monomer. As expected, incorporation of the adenine nucleobase onto chimera 4 was fastest in the presence of the control poly-A template (not complementary to any of the nucleobase monomers present) and slowest for the poly-C template (lacking a TT substitution). The initial rates of adenine incorporation for the three TT mismatch strands were similar, but decreased as the TT substitution was moved closer to the terminus (Figure 5b). These findings suggest that there are no significant steric or other impediments to nucleobase exchange at internal sites compared to positions at the terminus of these relatively short oligomers. The faster initial rates of exchange for the templates with the TT sequence closer to the center of the duplex indicates that the terminus of the tPNA may be frayed due to reduced base stacking,72,73 leading to a reduced thermodynamic driving force for mismatch correction. Increasing number of nucleobase monomers. A critical issue in implementing dynamic combinatorial libraries for template-directed synthesis is whether the driving force promoting the assembly of particular library members is sufficient to produce useful changes in the distribution of products.74 We compared three independent reactions involving chimera 4 that differed in the number of nucleobase monomers in solution, increasing from two (A and 7-deazaG) to three (A,


28

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Biochemistry

7-deazaG, C) to four (A, 7-deazaG, C, ABA). This comparison indicated a marked decrease in overall incorporation of 7-deazaG in response to a C10 template, from 73% incorporation, to 50%, and finally to 40% (Figure 6). The observed decrease in fidelity was likely due to the increasing entropic barrier for assembly of complementary oligomers associated with the larger pools of library members, which increase from a possible 1,024 sequences (210) to 59,049 (310) and finally to 1,048,576 (410) possible oligomers for the (Cys-Glu)10 sequence, assuming complete backbone substitution. These reactions are also complicated by the potential formation of chimeras in which the tPNA regions self-pair via intramolecular or intermolecular associations. Theoretical considerations. To better understand the parameters affecting the assembly and fidelity of dynamic nucleic acid analogs, we simulated a series of libraries using the program DCLSim developed by Otto and coworkers.51 Previous theoretical considerations of dynamic


29

Biochemistry

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Page 30 of 49

Figure 6. Theoretical properties of template-directed dynamic base-filling reactions. a) Equilibrium model employed for our simulations, using the program DCLSim.11 In the model, each substituted chimera (chimerax) interacts with the DNA template according to an equilibrium constant (Kx) calculated from ∆G values of hybridization for corresponding DNA/DNA duplexes. To account for nucleic acid analogs with weaker binding to DNA, we also simulated reactions in which the hybridization ∆G values contributed by the dynamic region of the chimera were systematically decreased. b) Comparison of experimental and theoretical results for assembly reactions with an increasing number of nucleobase monomers. The theoretical results shown correspond to hybridization ∆G values of 20% those of the analogous DNA/DNA


30

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Biochemistry

hybridization. c) Calculated yields of the fully complementary sequence as a function of hybridization ∆G contributed by the dynamic positions (relative to the hybridization ∆G of DNA). Data are shown for several structures, where X denotes dynamic sites. Four nucleobases were used in the simulations unless otherwise noted.

combinatorial libraries (DCLs)51,70,71,74-76 served as a useful starting point for our analysis, while the properties of nucleic acids provided a framework for choosing binding constants to use in our simulations. In our model, the pool of nucleobases reversibly reacts with the chimera backbone to form a library of fully substituted chimeras. Each substituted chimera (chimerax) interacts with the DNA template according to an equilibrium constant (Kx), calculated from ∆G values of hybridization for analogous DNA-DNA duplexes computed using nearest-neighbor methods on the DINAMelt server77,78 (Figure 6a). Every sequence in the model can interconvert into any other sequence; although the actual chemical equilibria are more complex, they may be represented in this way because the distribution of oligomers at equilibrium is mechanism independent. All sequences are present in the same concentration in the absence of template. We first considered a chimeric molecule composed of a 10mer DNA primer region and a 5mer region capable of dynamic base filling reactions. Our idealized libraries involved two (A, G), three (A, G, C), or four (A, G, C, T) nucleobase monomers and ignored the possibilty of library members with abasic sites, yielding libraries of 32 (25), 243 (35), or 1024 (45) oligomers, respectively. To match our experimental conditions, the simulations used a two-fold excess of template and an eight-fold excess of each nucleobase monomer compared to the base-filling backbone. Unless otherwise noted, a C5 template region was used for the simulations. It must be noted that these simulations do not take into account the effects of base stacking of the incoming


31

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nucleobase, which are known to play a role in template-directed nucleic acid synthesis79 and could cause different efficiencies in practice. Our simulations indicated that the yield at equilibrium of the fully complementary dynamic sequence would be 87%, 78%, or 66% in reactions involving two, three, or four nucleobases, respectively, assuming that hybridization interactions at the base-filling sites were energetically identical to corresponding DNA/DNA hybridizations (Figure 6c, see Tables S1–S2 for sample DCLSim input and output files). In the four-nucleobase reaction, this yield represents a 680-fold amplification in concentration compared to the non-templated case. However, given the modified backbones of dynamic nucleic acid analogs that, to date, exhibit lower hybridization energies than corresponding DNA/DNA duplexes,7-9 the actual fidelities in experimental systems will be lower than these calculations. We therefore simulated reactions in which the hybridization energy contributed by the dynamic region of the chimera was systematically decreased. As would be expected for a template-directed synthesis, the equilibrium concentration of the fully complementary sequence dropped with decreasing hybridization energy (Figure 6c). Our experimental data most closely match the simulations using a dynamic nucleic acid region contributing approximately 20% of the binding energy of the corresponding DNA/DNA duplex (Figure 6b). This value is in general agreement with the observed changes in melting temperature for tPNA/DNA duplexes being ~20–30% of the values expected for DNA/DNA pairs (Table 1). Simulations involving other DNA template sequences, such as A5 or CTGAC, gave yields of the complement sequence that were slightly lower (lower by

Templated Self-Assembly of Dynamic Peptide Nucleic Acids

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