Generation of Synthetic Copolymer Libraries by Combinatorial

Review pubs.acs.org/acscombsci

Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates Dehui Kong, Wayland Yeung, and Ryan Hili* Department of Chemistry, University of Georgia, 140 Cedar Street, Athens, Georgia 30602, United States ABSTRACT: Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acidtemplated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function. KEYWORDS: synthetic copolymer libraries, combinatorial assembly, modified nucleic acids, sequence-controlled, molecular evolution, aptamer

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INTRODUCTION Nature’s sequence-defined monodispersed copolymers are generated through nucleic acid-templated copolymerizations of libraries of monomers (Figure 1).1 The three most prevalent nucleic acid-templated copolymerization processes used in nature are DNA replication, transcription, and translation. DNA replication is the sequence-defined DNA-templated copolymerization of deoxynucleoside triphosphates (dNTPs) to yield a new complementary DNA copolymer. In a similar manner, transcription is the DNA-templated polymerasecatalyzed copolymerization of nucleoside triphosphates (NTPs) to yield a new complementary RNA copolymer. Lastly, translation is formally the RNA-templated copolymerization of amino acids to yield polypeptides, which is mediated by tRNAs and catalyzed by the ribosome. These copolymerization processes are templated with near-flawless fidelity, generating biopolymers with exquisite control down to single-monomer resolution. The ability to generate a library of biopolymer phenotypes from a library of template genotypes enables the Darwinian evolution of molecular function, both in nature and in the laboratory.2 This very process has given rise to functional biopolymers ranging from macromolecular receptors to catalysts.3 In contrast to nature’s proteinogenic and nucleic acid polymers, synthetic polymers are not generated in a manner that provides precise control over sequence and length. Despite the recent surge in progress toward controlled polymerization,1 the generation of sequence-defined monodispersed synthetic polymers has remained an elusive challenge. Due mainly to this lack of sequence control, synthetic polymers have primarily served as bulk materials rather than discretely folded macromolecules with defined function−a realm primarily dominated © XXXX American Chemical Society

by biopolymers. Chemists have sought to emulate nature’s approach to copolymer synthesis; the ability to generate synthetic copolymers from replicable genotypes is anticipated to enable the evolution of polymers comprising functional groups beyond the scope of nature, and possibly evolve new molecular function. One particular attractive emulative strategy that has come to the fore is to use nucleic acid polymers to template the polymerization of synthetic monomers. In this Review, we will describe various nucleic acid-templated polymerization strategies used to generate synthetic copolymers. In particular, this review will focus on technologies that have enabled the copolymerization of a comonomer libraries along a libraries of of nucleic acid templates. Thus, outside the scope of this Review are methods that generate polymers using stepwise DNA-templated synthesis.4

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APPROACHES TO NUCLEIC ACID-TEMPLATED COPOLYMERIZATION This Review will cover three main strategies in the nucleic acidtemplated copolymerization of comonomers. The first approach hinges upon the ability of both natural and evolved RNA and DNA polymerases to accommodate small modifications on the nucleoside triphosphate monomer (Figure 2a). In principle, up to four chemical modifications can be incorporated into the new synthetic nucleic acid polymer; theoretically, greater numbers of modifications could be incorporated with an expanded genetic alphabet.5 The second approach implements nucleic acidtemplated ligation of modified oligonucleotides to incorporate Received: April 21, 2016 Revised: May 26, 2016

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DOI: 10.1021/acscombsci.6b00059 ACS Comb. Sci. XXXX, XXX, XXX−XXX

Review

ACS Combinatorial Science

Figure 1. Generation of sequence-defined monodispersed polymers in nature. (a) Polymerase-catalyzed nucleic acid-templated copolymerization of nucleoside triphosphates. (b) Ribosome-catalyzed, mRNA-templated copolymerization of amino acids via assembly of aminoacylated tRNAs.

Figure 2. Strategies in the nucleic acid-templated copolymerization of monomers. (a) Polymerase-catalyzed copolymerization of modified nucleotide triphosphates. (b) Ligase-catalyzed copolymerization of modified oligonucleotides. (c) Generation of copolymer structurally unrelated to nucleic acids by templated polymerization.

Figure 3. Numbering nomenclature for nucleoside triphosphates.

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modifications throughout the newly synthesized nucleic acid polymer (Figure 2b). While this strategy enables greater than four modifications to be incorporated simultaneously throughout the polymer, the density of the modifications is far less than that achievable through the polymerase approach. The third approach, which emulates ribosomal peptide synthesis, involves the nucleic acid-templated assembly of oligonucleotides outfitted with an additional non-nucleic acid oligomer (Figure 2c). The assembly then undergoes a templated polymerization of the nonnucleic acid portion to generate a sequence-defined block copolymer.6 All three approaches present unique advantages and shortcomings, which will be discussed in detail in the sections to follow.

NUCLEIC ACID-TEMPLATED COPOLYMERIZATION OF MODIFIED NUCLEOTIDES

Single-stranded nucleic acid polymers can adopt complex threedimensional structures that are governed by its nucleotide sequence.7 Because of their ability to serve as both a replicable polymer and a functional polymer, nucleic acid polymers uniquely represent both genotype and phenotype. This molecular duality enables researchers to readily evolve and select the molecular structure of large libraries of nucleic acid polymers for a wide variety of functions including molecular recognition and catalysis.8 Independently discovered by the Szostak, Joyce, and Gold laboratories in 1990,9−11 this process is termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX has since been used extensively as a powerful approach to not only study structure−function relationships of nucleic acid B


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and RNA using primer extension reactions.13−15 As a result of these efforts, nucleic acid polymers containing multiple sequence-defined instances of a single chemical modification continue to be a mainstay for SELEX experiments. Most notably, slow off-rate modified aptamers (SOMAmers), which are typically RNA aptamers containing hydrophobic modifications through the 5-position of uracil, have proven to be extremely effective at targeting protein surfaces, and have been used to generate high-affinity reagents to >3000 human proteins.19 These modified nucleic acid polymers have enabled access to hydrophobic surface binding, which contrasts sharply with the polar interaction typically observed between canonical nucleic acid aptamers and their protein target.20 The incorporation of functional groups by templated copolymerization of modified nucleoside triphosphates has also enabled researchers to expand the potential of nucleic acids as catalytically active biopolymers, moving beyond the inherent limitations of the native polymer, including metal ion dependence.21,22 In their efforts to develop DNA enzymes that can mimic the catalytic function of ribonuclease enzymes, Joyce, Barbas, and co-workers first reported the polymerase-catalyzed incorporation of C5-imidazole-functionalized dUTP in place of thymidine within ssDNA libraries, and subsequent in vitro selection for ribonuclease activity in the presence of Zn2+.23 A small 32-nt imidazole-functionalized DNA enzyme was identified from the selection, which catalyzed RNA cleavage with rates of 4 min−1 in the presence of micromolar concentrations of Zn2+. A 12 nt stem-loop region was identified to be the catalytic domain, which contained three indispensable imidazole groups, which the authors hypothesized to be involved with binding Zn2+ and engaging in catalysis (Figure 5). The catalytic rate of the DNA

polymers but also to generate functional nucleic acid polymers for use in medicine.12 Despite their ability to fold into sequence-defined macromolecular structures with binding or catalytic activities, there is a clear deficit of chemical functionality available in nucleic acid polymers, particularly in comparison to the rich chemical diversity seen in proteins. This dichotomy likely explains the hegemony of proteins as receptors and catalysts in nature. Thus, researchers have speculated that increasing the chemical diversity of nucleic acid polymers might result in synthetic polymers with activities that exceed canonical nucleic acid polymers. Approaches toward the incorporation of modified nucleoside triphosphates by polymerases have been recently reviewed.13−16 Thus, here we will focus on essential concepts and seminal examples, along with some recent advances in the area. There are three general sites for the incorporation of chemical functionality on the nucleoside triphosphate monomer. Modification of the nucleobase can be achieved at various positions, but are most often installed at position 7 or 8 of purines and position 5 of pyrimidines, due mainly to synthetic tractability of these derivatives (Figure 3). Modification on the sugar backbone is also possible; for instance, substitution at the 2′-position of the ribose ring is regularly employed to generate nuclease resistant nucleic acid derivatives. Replacement of the ribose backbone with natural and non-natural backbones has also been demonstrated, allowing access to novel conformational landscapes, as well as nuclease resistance (Figure 4).

Figure 4. Examples of nucleic acid polymers with synthetic backbones. Figure 5. Modified DNA enzyme with ribonuclease activity. (a) 12-nt stem-loop minimal catalytic motif. “U” on the DNA strand represents the sites of modification. (b) Hypothesized catalytic role of the three essential imidazole dU residues in the DNA enzyme.

Copolymerization of Nucleobase Modified Nucleoside Triphosphates. Early examples of sequence-controlled templated copolymerization of nucleobase modified nucleoside triphosphate by commercially available polymerases focused on having one of the bases modified, while leaving the other three bases unmodified. This enabled multiple instances of sequencespecific incorporation of a single chemical modification throughout the nucleic acid polymer. Seminal works by the Eaton lab17 demonstrated that uridine triphosphates (UTP) could be readily modified at the 5-position with various small functional groups, while maintaining good transcriptional efficiency when using T7 RNA polymerase. Toole and coworkers18 also demonstrated that DNA polymerases could incorporate dUTP with small alkyl modifications at the 5position; the study culminated in the application of this polymerization system to SELEX and the discovery of modified aptamers against human thrombin protein. Since these initial reports, many groups have contributed to the rapid development of the sequence-defined incorporation of modifications in DNA

enzyme is approximately 5-fold faster than that of other nonfunctionalized nucleic acid enzymes of similar size under identical conditions, but about 4 orders of magnitude slower than that of the proteinogenic enzyme, RNase A.23 The gap in rate between the functionalized DNA enzyme and RNase A is likely due to the impact of hetereomultivalency of catalytic residues within the RNase A active site. Several catalytic residues within the active site of RNase A, including two histidines, a lysine, and an aspartate, function in concert to perform the cleavage reaction.24 Of significant importance is that unmodified nucleic acid enzymes have been evolved to catalyze RNA cleavage with comparable rates to the imidazole-modified DNA enzyme, albeit under different divalent metal conditions.25 It has been speculated26 that a nucleic acid enzyme with a kcat of 4 min−1 may represent an upper limit due to practical limitations inherent within the selection cycle, namely the roughly 2 min that are C


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Figure 6. Heteromultivalency in DNA enzymes with ribonuclease activity. (a) Modified nucleoside triphosphates used during the templated polymerization. (b) Minimal catalytic hairpin motif. U and A represent sites of modifications.

required to isolate the active catalyst from other inactive library members by affinity purification. Thus, novel selection methods that can select enzymes with kcat > 4 min−1 may be required to observe the influence of modifications on nuclease activity. A particularly intriguing and high-potential class of modified nucleic acid polymers are those with multiple sequence-defined instances of unique modifications, which more effectively emulate the side-chain heteromultivalency observed in proteins.27 To this end, the Perrin lab sought to generate combinatorial ssDNA enzyme libraries bearing two types of functional groups present within the active site of RNase A, including nitrogenous cations and general acids/bases.26,28 They hypothesized that multiple base modifications working in concert might lift the requirements for carefully controlled amounts of divalent metals during nucleic acid catalysts, and in so doing, more clearly evaluate the benefits of nucleobase modification on nucleic acid function and increase the applicable scope of the enzyme. Thus, imidazole-modified dATP and primary amine-modified dUTP, along with unmodified dCTP and dGTP, were used to generate copolymer libraries of modified DNA, which were subsequently used in selections for ribonuclease activity. Despite the lack of divalent metals, Perrin and co-workers were able to isolate an active DNA enzyme consisting of ten essential nucleobase modifications (Figure 6) and a first-order rate constant for the intramolecular RNA cleavage of 0.044 min−1: 4-fold greater than unmodified DNA enzymes.29 While the DNA enzyme was selected in the absence of Mg2+ and Ca2+, the enzymes still functioned when they were present at physiological concentrations, which uniquely positioned this class of enzyme for potential applications in vivo. Building from their previous studies, the Perrin lab pushed the limits of in vitro selection to accommodate ssDNA copolymer libraries generated by the DNA-templated Sequenace polymerase-catalyzed copolymerization of guanidinium-modified dCTP, ammonium-modified dUTP, imodizole-modified dATP, and unmodified dGTP.30 Divalent metal-independent modified DNA enzymes with ribonuclease activity against an all-RNA target were isolated, with rates of 0.06 min−1. This represents the fastest divalent metal-independent cleavage of an all-RNA substrate by a DNA enzyme and opens potential therapeutic applications to neutralizing viral and oncogenic mRNAs. In an attempt to address the functional group deficit of nucleic acid polymers, Famulok and co-workers identified naturally occurring family B polymerases that were able to effect the simultaneous copolymerization of four modified dNTPs, generating combinatorial libraries of DNA polymers with high density functionalization (Figure 7).31 Importantly, these polymerases were found to efficiently catalyze the DNAtemplated copolymerization during primer extensions and during polymerase chain reaction (PCR). One noted limitation of this method was observed when sequencing primer extensions

Figure 7. Fully modified set of dNTP comonomers used during polymerase-catalyzed primer extension.

derived from C- and G-rich templates. While sequence fidelity was high, the authors found that sequence information was lost after 14−15 bases from the 3′-primer site, which correlated with poor efficiency for extension using these templates. The authors thus concluded that efficiency and sequence maintenance strongly depends upon sequence contexts. These limitations have since precluded application of a tetra-modified nucleotide set in SELEX systems. Notwithstanding, family B polymerases hold great promise to expanding the functional group repertoire of DNA. Their ability to accommodate consecutive incorporations of modified dNTPs is likely a result of the fact that family B polymerases binds B-form dsDNA, rather than A-form dsDNA, which family A polymerases bind.32 As B-form DNA is elongated compared to A-form DNA, this might favor consecutive incorporations.33 Furthermore, family B polymerases, such as KOD XL DNA polymerase, bind dsDNA phosphate backbone above the minor groove, whereas family A polymerases interact with the phosphate backbone by extending into the major groove. As modifications at the 5-position of pyrimidines and the 7-position of purines extend into the major groove, steric clash could explain the lower polymerization efficiencies of family A polymerases. It is worth noting that the Hocek lab, who has spent considerable effort studying and applying base modified dCTP and dATP in vitro, has observed that various family A and family B polymerases preferentially incorporate 7-aryl-7-deazaadenine 2′-deoxyribonucleoside triphosphates over unmodified dATP.34 Modified nucleic acid enzymes have been pushed the boundaries of catalytic potential for nucleic acid polymers. One particularly challenging catalytic reaction is amide hydrolysis. The Silverman lab has studied DNA-catalyzed amide bond hydrolysis, and initially reported that in vitro selection enriched D


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Figure 8. DNA-catalyzed amide hydrolysis with functionalized ssDNA. (a) Modified dUTP used during copolymerization. (b) Architecture used during the selection of modified ssDNA enzymes.

unmodified ssDNAs capable of hydrolyzing aromatic amides;35 ostensibly, hydrolysis of aliphatic amides was beyond their observed catalytic ability. In their efforts to yield DNA enzymes capable of catalyzing the hydrolysis of aliphatic amides, Silverman and co-workers performed SELEX on combinatorial libraries of functionalized ssDNA.36 The ssDNA libraries either incorporated amine-modified dU, carboxylate-modified dU, or hydroxyl-modified dU, and were generated with the family B polymerase, KOD XL (Figure 8). Amine-modified DNA enzymes were isolated that were able to catalyze aliphatic amide-bond hydrolysis with rates of up to 0.11 h−1. The DNA enzymes contained multiple instances of amino groups within its sequence, two were found to be indispensable for high catalytic activity. Single mutants (unmodified dT) yieldied 3-fold lower activity, while double mutants abolished activity altogether. Active DNA amidases were also isolated from the carboxylatemodified dU library, which yielded DNA catalysts possessing catalytic rates of 0.3−0.4 h−1; similar to the amine-modified library, the carboxylates were also essential to catalytic function. For selection on the libraries containing hydroxyl-modified dU, active DNA amidases were isolated with catalytic rates of 0.1−0.2 h−1. In an ironic twist of fate, it was found that the unmodified DNA catalyst retained substantial activity (0.03 h−1); nonetheless, combinatorial DNA libraries adorned with chemical functionality yielded DNA catalysts with improved scope and activity compared to unmodified DNA. Future efforts in this area are certain to demonstrate the enormous potential of this class of evolvable polymer. The ability to scaffold large chemical modification on DNA has widespread utility ranging from materials to medical applications.37 However, the sequence-defined incorporation of large chemical modifications throughout a nucleic acid polymer presents numerous challenges, not the least of which is the accommodation of these large modifications by the polymerase active site. The Marx lab spent considerable effort expanding the scope of polymerase-mediated incorporations of large modifications in part by using structural data garnered from crystal structures of polymerases complexed with primer-template DNA and modified nucleotides.38 Using 9°Nm polymerase, Marx and co-workers demonstrated the sequence-specific incorporation of linear polyethylene glycol monomethyl ethers and branched polyamido dendrons with varying terminal groups when appended through the 5-position of dUTP (Figure 9).38 These polymer-modified dUTPs were incorporated with remarkable efficiency through primer extension, achieving >10 consecutive incorporations of the modification could be achieved with ease. Its combinatorial distribution throughout a library of comonomers (dATP, dCTP and dGTP) was also efficient. Building from these preliminary data, Marx and co-workers demonstrated the incorporation of dNTPs modified with long polynucleotide sequences.39 Using KlenTaq polymerase, a truncated mutant of

Figure 9. dUTP monomers functionlized with polymers or dendrimers.

Thermus aquaticus (Taq) polymerase, enabled efficient copolymerizations involving the incorporation of dNTPs bearing oligonucleotide modifications of up to 40 nt is length. Furthermore, up to seven consecutive incorporations was demonstrated with dNTPs modified with a 20 nt ssDNA. This approach was later applied to the sequence-specific incorporation of G-quadruplex-derived DNA enzymes along a dsDNA scaffold.40 Structural studies on the molecular basis by which polymerases can accommodate large modifications revealed that these modifications extend outside the enzyme, provided that the linker was long enough.41 The composition of the linker was also found to be important, as DNA polymerases better tolerated linkers that engaged in hydrogen bonding with the enzyme. An effective approach to facilitate the sequence-defined incorporation of large modifications throughout a nucleic acid polymer is through bioconjugation of postprimer extension products. This approach hinges upon the sequence-specific incorporation of dNTPs containing a clickable handle, such as an alkyne, which can be derivatized downstream of the primer extension process. This would allow the incorporation of various groups that would have otherwise not been tolerated during the primer extension step. Also, this strategy circumvents issues with consecutive incorporations of large modifications, which can greatly reduce the efficiency of primer extension.42 One notable example of this approach is the SELMA (Selection with Modified Aptamers) method,43−46 which was recently developed by the Krauss lab (Figure 10). SELMA enables the evolution of aptamers with large modifications, such as oligosaccharides, but could potentially be applied to a wide scope of other synthetic or biological oligomers. In their efforts toward the development of an HIV vaccine, the Krauss lab used SELMA to evolved a ssDNAscaffolded glycocluster capable of mimicking the HIV envelope glycoprotein, gp120. The aim was to induce 2G12 (HIV neutralizing antibody) creation against this glycocluster mimetic. In SELMA, each library member contains a hairpin loop (constant region), ssDNA random region, and primer (constant region).43 Polymerase extension generates alkynl modified dsDNA by using EdUTP(alkynye-bearing dUTP) instead of dTTP. Using Cu(I)-catalyzed alkyne−azide 1,3-dipolar cycloaddition (CuAAC) chemistry, glycan azide modifications can be E


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Figure 10. General approach to the combinatorial generation and selection of ssDNA-scaffolded glycoclusters using the selection of modified aptamers (SELMA) method.

nucleic acid backbone analogues have taken place. Among the first demonstrated examples of this class of polymer was the generation of sequence-defined 2′-OMe-RNA copolymers from 2′-OMe NTPs monomers (Figure 3). Building off early studies by the Ellington lab,47 which focused on the incorporation of 2′OMe NTPs (A, C, and U, not G) using T7 RNA polymerase mutants, Keefe and co-workers were able to optimize the transcription process to generate RNA polymers comprising a fully modified 2′-OMe backbone.48 Some notable findings of this research were that an all-purine leader sequence greatly increased transcription yields of 2′-OMe-G containing polymers. The authors attributed this result to the leader region enabling the polymerase to adopt an elongation conformation, which facilitated 2′-OMe GTP incorporation. Also, when transcription conditions were augmented with small quantities of 2′-OH GTP, initiation was greatly facilitated, and higher yields of full-length product were observed. Two main concerns surrounding the templated copolymerization of unnatural monomers are (i) fidelity of templated monomer incorporation and (ii) compositional bias when the polymerization operates at low yield. Sequencing analysis showed that the error rate, which comprises both transcription and reverse transcription of the modified RNA, was markedly higher when compared to unmodified RNA. While there was no significant increase in error by nucleotide deletion, error by insertion and substation rose considerably, summing to an overall error rate of approximately 2%. Keefe and co-workers integrated this 2′-OMe RNA transcription/reverse transcription process into a SELEX system, and raised an aptamer against vascular endothelial growth factor (VEGF) with a Kd of 2 nM.48 Notably, as the RNA polymer has a synthetic

readily coupled to the alkyne positions along the DNA library. Following strand displacement and extension with natural dNTPs, the ssDNA-scaffolded glycans will be able to fold into a library of glycoclusters. After selecting for 2G12 affinity, successful genotypes are amplified by PCR, and the hairpin template is restored for subsequent rounds of selection. The first reported in vitro selections to yield gp120 mimetics resulted in an aptamer bearing ten glycosylation sites of Man4 fragments.43 Further optimization revealed that the absence of glycosylation sites in the template primer region, decreased frequency of dA in the randomized template region, and using the full Man9 oligosaccharide rather than the Man4 fragment allowed SELMA to select for aptamers with significantly lower valency and higher affinity.44 In a separate study, the selection temperature was increased from room temperature to 37 °C.45 This selection yielded fewer glycosylation sites, and resulted in aptamers with Kd of 1.7−16 nM as determined by nitrocellulose filter binding assays. At room temperature, the selected aptamers had a Kd of ∼300 nM. It was observed that the higher temperature mitigated the trend of increasing glycosylation as the selection progressed. The authors theorized that at room temperature, the sheer number of multivalent members with moderate Kd outcompeted the few low-valent, ultralow Kd members. However, at higher temperatures, multivalent members pay a higher entropic penalty compared to members containing fewer glycosylated sites, which drives the increased survival of these library members. Copolymerization of Nucleoside Triphosphates with Unnatural Backbones. In recent years, significant advances in the nucleic acid-templated copolymerization of fully unnatural F


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Figure 11. Unnatural hydrogen-bonding nucleobases used in expanded DNA genetic alphabet within the Benner Lab.

replication fidelity dropped to 96.4% replication fidelity. The authors noted that 90% of the errors resulted from G → C transversions. Detailed analysis of the mutational profile revealed that this transversion could be suppressed to just 3% when G was preceded by A in the template library, which yielded replication fidelities of 99%. A breakthrough in the generation of synthetic genetic polymers (XNA) by templated copolymerization was recently reported by Holliger and co-workers.53 Using a selection strategy called compartmentalized self-tagging (CST),54 the researchers were able to discover XNA polymerases from a library of TgoT DNA polymerase mutants that were capable of copolymerization of XNA nucleoside triphosphates along DNA templates. The identified polymerases were able to transcribe 1,5-anhydrohexitol nucleic acids (HNAs), cyclohexenyl nucleic acids (CeNAs), 2′-O,4′-C-methylene-b-D-ribonucleic acids [locked nucleic acids (LNAs)], arabinonucleic acids (ANAs), 2′-fluoro-arabinonucleic acids (FANAs), and TNA from DNA templates (see Figure 4 for backbone structures). Aggregate fidelities of DNA template polymerization and reverse transcription ranged from 93.7% for LNA to 98.5% for CeNA, which are sufficient to support Darwinian evolution of molecular function for these polymers. To demonstrate this, Holliger and co-workers subjected HNA to eight rounds of SELEX against TAR RNA motif (sTAR), and successfully isolated HNA aptamers that bound specifically to sTAR with Kd as low as 28 nM. In a separate SELEX experiment, the authors report isolating an HNA aptamer against hen egg lysozyme with Kd of 107 nM. Holliger and co-workers recently expanded the functional utility of XNA copolymers to catalysis. Using the templated replication system previously described,53 they sought to discover various XNA enzymes with RNA endonuclease activity using SELEX.55 Nucleic acid polymer enzymes comprising backbones of FANA, ANA, HNA, and CeNA were discovered with catalytic rates ranging from 0.06−0.0001 min−1 for the intramolecular (in cis) RNA cleavage at 25 °C. Interestingly, only the FANA polymer yielded enzymes with comparable activity to those previously discovered from RNA and DNA pools, with the other synthetic biocopolymers yielding catalytic rates 20−600 fold lower. The FANA catalyst was also found to operate in trans,

backbone, the aptamer exhibited remarkable plasma stability, with no detectable degradation at 96 h. More recently, other fully modified nucleic acid polymers have been generated using templated copolymerization. α-L-threofuranosyl nucleic acid (TNA) (Figure 4), which has generated considerable interest as a possible RNA world progenitor,49 is a particular interesting example. Research spanning more than a decade culminated in a high fidelity TNA transcription system reported by the Chaput lab.50 Early work on TNA library transcription from a library of DNA templates resulted in poor efficiencies.51 Using commercially available therminator DNA polymerase (9°N exo-: A485L), Chaput and co-workers undertook a thorough investigation into various transcription parameters. Guided by previous studies by Eschenmoser,52 which established that the diaminopurine modification significantly enhances the thermodynamic stability of TNA/TNA and TNA/DNA duplexes, they replaced ATP for 2-amino-ATP in the comonomer pool. Chaput co-workers also found that short Grepeats on the DNA template caused the therminator DNA polymerase to pause during transcription, resulting in failure during transcription. By removing G from the DNA template library, thus resulting in a library comprising only A, C, and T, 60% yield of full-length TNA polymer was observed. When G was incorporated into the DNA template library at low levels (12.5% frequency), full-length polymer yield dropped to 30%. Because of the limited efficiency of reverse transcription of TNA,51 Chaput used DNA display SELEX, whereby the phenotype (TNA) was covalently attached to its encoding genotype (DNA).51 Despite the lack of cytidine within the TNA polymers, the team was able to raise TNA aptamers against thrombin protein, with Kd as low as 200 nM. While the error rate for the templated enzymatic polymerization of the three nucleotide TNA genetic code was not reported, earlier reports suggest therminator DNA polymerase can effect DNA-templated TNA synthesis with >99% fidelity, albeit in a simpler context.51 In later studies, the Chaput lab measured the aggregate fidelity of a complete TNA replication cycle (DNA → TNA → DNA), and found that template libraries comprising A, C, and T resulted in 99.6% replication fidelity. When the complexity of the library was increased, such that templates comprised A, C, G, and T, G


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Figure 12. Examples of hydrophobic nucleoitide pairing developed in the Romesberg Lab.

with catalytic rates of 0.026 min−1 and with multiple turnovers. The catalyst is also Mg2+-dependent and pH-dependent, with highest catalytic rates occurring at pH 9.25. Using a similar approach, Holliger and co-workers also evolved a FANA enzyme capable of templated 3′-5′ RNA ligation at rates of 2 × 10−4 min−1 at 25 °C. While slow, these rates are comparable to DNA enzymes discovered for specific 3′-5′ RNA ligation,56 albeit considerably slower than those observed for in vitro evolved RNA catalysts which have surpassed 10 min−1.57 Copolymerization of Nucleoside Triphosphates from an Expanded Genetic Alphabet. Using four nucleotides that form two unique base pairs, DNA is able to efficiently encode life. As alternative uses for nucleic acids were discovered, researchers acknowledged the practicality of developing an expanded genetic alphabet. With an expanded alphabet, modified DNA could potentially encode more information but could also be used as a functionalized synthetic polymer. While naturally occurring DNA employs hydrogen bonding to form base pairs, alternative methods of duplex formation have included shape complementation, hydrophobicity, and π−π stacking. As a pioneer in the field of expanding the genetic alphabet, the Benner lab developed DNA that incorporated diso-C−disoG58,59 pairing beginning in the late 1980s (Figure 11). The team managed to perform DNA templated copolymerization incorporating the two new nucleotides alongside the four natural nucleotides using Klenow fragment of Escherichia coli polymerase I. However, the polymerization exhibited relatively poor fidelity, low efficiency, and product instability. One reason behind the lower fidelity stemmed from diso-G−dT mispairing because of diso-G tautomerization. Later, the same lab discovered that Klenow could accept deoxyxanthosine (dX) opposite from 2,4diaminopyrimidine (dκ);60 other homologous polymerases failed to accept the novel base pair. In 2004, the researchers addressed the tautomer problem by replacing diso-G with dC7iso-G, which demonstrated a more stable keto-form and improved the fidelity of the system.61 Another more effective strategy was to use d2-thioT to preclude mispairing with dG. The steric bulk and reduced hydrogen bonding properties of the thione moiety in 2-thioT rendered mispairing with the minor tautomer of disoG ineffective. The efforts of Benner and coworkers culminated in a refined six nucleotide system comprising dA, d2-thioT, dG, dC, diso-C, and diso-G. The system uses the Klenow fragment of titanium Taq polymerase and effects the

DNA-templated polymerization of the six comonomers with 98% fidelity.62 While this fidelity is sufficient for most applications, it may be too low for some that require multiple PCR cycles, as after 30 cycles of PCR, 45.5% of the library would contain at least one mutation. In 2007, the Benner lab developed dP−dZ base pairing (Figure 11) using Vent (exo-) and DeepVent (exo-) polymerases with 97.5% fidelity for their incorporation.63 The group later achieved 99.8% fidelity using Taq DNA polymerase.64 Recently, dP−dZ base pairing was applied to in vitro selection against breast cancer cells yielding aptamers with Kd = 30 ± 1 nM in 12 SELEX cycles.65 In 2015, aptamers were evolved using laboratory in vitro evolution (LIVE) to target HepG2 liver cancer cells, with Kd down to 14 ± 3 nM.66 During specificity assays, aptamers exhibited much higher dissociation constants when exposed to untransformed cells. Furthermore, testing confirmed that dP and dZ were essential to binding. Despite PCR disfavoring the incorporation of the new nucleotide pair, selection favored library members with dP and dZ, suggesting that the expanded alphabet led to tighter binding. Two possible explanations for this increase in binding propensity were offered: the nitro moiety in dZ may be a “universal weak binder” or increased information density removes potential ambiguity in folding. In 1999, the Romesberg lab demonstrated that hydrophobicity and packing forces could also drive the formation of stable DNA duplexes (Figure 12). Using Klenow fragment, self-pairing 7propynylisocarbostyril (dPICS) nucleoside was incorporated into a DNA duplex.67 The group later synthesized 37 unique nucleotides with large isocarbostyril, naphthyl, and indole scaffolds.5 While the incorporation of the new nucleotides was successful, extension from the non-natural nucleotide was inefficient. After performing a structure study using NMR and X-ray crystallography, researchers found that the hydrophobic nucleotides were intercalated as opposed to being edge-on-edge in the traditional Watson−Crick pairing;68 the intercalated DNA duplex terminates the extension process. To circumvent the intercalation issue, researchers tested different polymerases for the primer extension. Using d7AI, a hydrophobic nucleotide, researchers performed templated polymerization using mammalian polymerase β (Pol β) and Klenow fragment.69 Pol β is able to extend the primer and allow polymerization to continue, while Klenow would stall at d7AI. In more recent efforts, researchers sought to evolve polymerase that H


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Figure 13. Development of hydrophobic nucleotide pairs in the Hirao Lab.

ization rate is still slower than natural base pairs, making PCR bias of natural nucleotides a confounding issue. Researchers synthesized another pool of nucleotides to screen. The screening effort identified dTPT3−dNaM as a candidate pair that emulates the fidelity and efficiency of natural base pairs.75 DNA containing dTPT3−dNaM underwent 48 cycles of OneTaq PCR with >99% retention and >98.9% fidelity. These data suggest that the dTPT3−dNaM shows promise in applications such as SELEX for evolving synthetic nucleic acid polymers with novel molecular functions. In 1999, the Hirao lab created their own unnatural base pair predicated upon hydrogen bonding and shape complementarity.76 Researchers took advantage of steric hindrance to preclude mispairing. Using T7 RNA polymerase, researchers were able incorporate the dx−dy pair into RNA.77 However, the pair was incapable of PCR amplification as dx had a dimethylamino group that interfered with stacking interactions during DNA duplex formation. To address this problem, researchers created ds which replaced the dimethylamino with a thiophenyl group.78 DNA containing ds−dy was capable of amplification with moderate specificity, however dy−dA mispairing was observed at significant frequencies. This was solved by replacing dy with dz.79 While ds−dz enjoyed increased fidelity, various DNA and RNA polymerases had difficulty recognizing and incorporating dz. In 2003, researchers shifted to developing hydrophobic nucleotides. Inspired by unnatural dQ−dF pair developed in the Kool lab,80 the Hirao group produced dQ−dPa pairing.81 dPa employs an aldehyde as a hydrogen bond acceptor, allowing Klenow fragment and reverse transcriptase to easily recognize the nucleotide. dQ was later replaced by dDs which has a bulky thienyl group that prevents undesired pairing with natural bases.82 The dDs−dPa base pair exhibited >99% fidelity per PCR cycle using Deep Vent (exo+). The PCR protocol requires dDs

were efficient at incorporating hydrophobic base pairs. Using a phage display selection system, the Romesberg lab evolved the Stoffel fragment of Taq DNA polymerase to enable extension of a primer with a terminal hydrophobic nucleotide (dPICS).70 A mutant enzyme resulting from the selection exhibited efficient primer extension from dPICS, while the wild type polymerase showed no detectable enzyme activity with this primer substrate. Another method of addressing the intercalation issue is to synthesize nucleobases with smaller hydrophobic molecular architectures. This brought about the second generation of hydrophobic nucleotides that use benzene, pyridine, and pyridone scaffolds.5 Experimentation confirmed the viability of this approach as smaller scaffolds reduced undesired distortions in duplex structure.71 During the initial studies of this new class of hydrophobic nucleotides, researchers observed that the polymerase required a hydrogen-bond acceptor on the nucleotide to perform primer extension efficiently.72 They approached this problem by screening the efficiency of 3600 possible pairings between 60 hydrophobic nucleotides that were selected from both generations.73 These polymerizations were catalyzed by Klenow (exo-). dSICS−dMMO2 arose as a candidate pair with improved efficiency and fidelity. In dSICS, the thioketone sulfur is larger and more polarizable than a ketone oxygen, which allowed it to retain hydrophobicity while accepting hydrogen bonds; in dMMO2, the ortho-substituted O-methyl accomplishes the same task. These findings culminated in the third generation of hydrophobic nucleotides. Two of the most successful pairs of the third generation were d5SICS−dMMO2 and d5SICS to dNaM. Using Deep Vent and Taq polymerases, researchers achieved high fidelity amplification of DNA containing dNaM−d5SICS with 35-fold amplification at >98% fidelity.74 The same feat was achieved with Deep Vent polymerase on DNA containing d5SICS−dMMO2 pairs with 25-fold amplification at >97% fidelity. However, the polymerI


Review


Figure 14. (a) General strategy for the T4 DNA ligase-catalyzed copolymerization of modified oligonucleotides. (b) Example of modified comonomer set.

and dA to be used in γ-amidotriphosphate form in order to reduce dDs−dDs and dA−dPa mispairing. Further optimization yielded dPn which replaced the aldehyde in dPa with a nitro group.83 This removed the need for γ-amidotriphosphates. Addition of an alkynylamine group to the dPn group yielded dPx, which further increased the fidelity of the copolymerization.84 Using Deep Vent (exo+) or AccuPrime Pfx, DNA containing dDs−dPx was generated with 99.8% fidelity per cycle of PCR, representing the highest fidelity to date for hydrophobic shapefitting base pairs. In 2013, the Hirao lab performed SELEX on ssDNA libraries containing up to three instances of dDs−dPx base pairs.85 Selection yielded VEGF-165 and IFN-γ aptamers with 0.65 pM and 0.038 nM Kd, respectively.

upon T4 DNA ligase to catalyze DNA-templated phosphodiester bond formation between adjacent 5′-phosphorylated trinucleotides adorned with functional groups attached to the nucleobase (Figure 14a). This method effectively enables the incorporation of a much larger number of modifications, theoretically up to 64 in a trinucleotide codon set, that would not be possible using single nucleotide incorporation. The group studied the polymerization efficiency and fidelity of an eight-membered codon set, using codons having 66% CG-content (Figure 14b). The polymerization of modified trinucleotides was found the be greatly enhanced by molecular crowding reagents, such as PEG 6000, and by bovine serum albumin (BSA), which increased the half-life of T4 DNA ligase activity during lengthy incubations,87 generating full-length products for up to 50 codon templates with >80% yield. Using a chain termination assay, they studied the fidelity of the copolymerization process, demonstrating a high degree of sequence specificity for the incorporation; no quantitative measurement of fidelity was reported. The authors integrated the capabilities of this copolymerization system in a mock in vitro selection. A full cycle of templated copolymerization, selection, and amplification were tested to enrich a modified DNA copolymer containing a known pharmacophore, Gly-Leu4-carboxybenzene-sulfonamide (GLCBS), which binds carbonic anhydrase II (CAII). The template that uniquely encoded the incorporation of the GLCBS was diluted 5.8 × 106-fold into an uncompetitive template library (encoding for non-GLCBS ligands). The GLCBS-encoding genotype was enriched 2.5 × 107 over four rounds of SELEX against CAII target, which demonstrates the potential of the system for de novo discovery of modified ssDNA copolymers with functional properties. Our own laboratory has contributed to this area by expanding the codon set used in oligonucleotide copolymerization beyond eight, and developing high-throughput methods to study the fidelity of the process.88 We have demonstrated that the T4 DNA ligase-catalyzed copolymerization system can work efficiently with a library of functionalized pentnucleotides comprising four degenerate bases (256-membered library). To study the fidelity of this large library copolymerization, we developed a highthroughput DNA duplex sequencing method that could evaluate the fidelity at the pentanucleotide incorporation level. This method relies upon a postpolymerization duplex barcoding

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DNA-TEMPLATED COPOLYMERIZATION OF MODIFIED OLIGONUCELOTIDES Conventional strategies for the sequence-specific incorporation of functional groups throughout a nucleic acid polymer rely upon polymerases to incorporate modified nucleoside triphosphates. This strategy has two main shortcomings with respect to the generation of functional polymers. The first is that the number of unique modifications is limited by the number of nucleobases in the genetic code, which is typically four, albeit efforts to expand the genetic alphabet could enable access beyond this upper limit.5 The number of unique modifications will certainly influence the functional capacity of the polymer, thus expanding beyond these limitations is of particular interest to the aptamer and nucleic acid enzyme communities. The second limitation is that the size and nature of the functional group will be limited to those parameters that are accommodated by the polymerase active site. While the incorporation of large modifications postpolymerization has been successfully demonstrated,46 the ability to directly incorporate various unique large modifications in the polymerization process has numerous advantages. Overcoming these two main limitation requires a strategy beyond conventional polymerase-based approaches. Inspired by the trinucleotide coding system used during the ribosomal translation of mRNA into protein, Liu and co-workers recently developed a T4 DNA ligase-catalyzed DNA-templated copolymerization of modified trinucleotides to generate functionalized nucleic acid polymers.86 The approach relies J


Review


data highlight the potential of the system to enable the discovery of functional ssDNAs with large repertoires of modifications. ssDNA has been widely used as a scaffold for the multivalent display of bioactive oligomeric ligands to generate high-affinity macromolecules with binding properties that rival antibodies.43,45,91−94 One particularly attractive feature of using ssDNA as a scaffold is the ready evolution its three-dimensional structure through SELEX to find the optimal spatial configuration of ligand display for the desired activity. Traditional approaches to ssDNA scaffolding have generally employed homomultivalency rather than hetereomultivalency, as methods to incorporate large libraries of oligomeric ligands have not been available. Recently, our group has employed the ligase-catalyzed DNA-templated oligonucleotide copolymerization process to install libraries of peptide fragments along a library of ssDNAs in a sequence defined manner.95 The ligase-catalyzed copolymerization process was significantly overhauled to accommodate peptide fragments of up to eight amino acids in length on the pentanucleotides. Critical to the success of the polymerization process were the optimization of two main variables, namely ATP concentration and directionality of the polymerization. When ATP was used at the standard 1 mM concentrations, significant truncation products were observed. It was determined that high concentrations of ATP were inhibiting the polymerization by shifting the equilibrium of the reaction to a mixture of 5′-adenylated template and adenylated ligase, effectively shutting down the polymerization (Figure 16). When the ATP concentration was decreased to 25 μM, polymerization was pushed to completion. The directionality of polymerization was also essential to high-efficiency polymerization. When initiating polymerization of peptide-modified oligonucleotides from a 5′phosphorylated primer, only truncation products were observed, without any observable full-length product. However, when extending from a 3′-primer, a highly efficient polymerization was observed, yielding exclusively full-length product in high yield. We hypothesized that this observation was rooted in template annealing of the 3′-hydroxyl nucleophile in the transition state during phosphodiester bond-formation. In the 5′-extension

strategy prior to PCR amplification, which enabled us to identify the template and corresponding polymerized daughter strand (duplex pairs) after high-throughput DNA sequencing.88 Using this approach, we were able to readily calculate the misincorporation frequency of each modified pentanucleotide by T4 DNA ligase. Remarkably, T4 DNA ligase was found to incorporate modified pentanucleotides with >95% fidelity using a 256 codon system. The most remarkable observation was that the fidelity was significantly increased when a modification was present on the pentanucleotide. For example, when using the codon set NTNNN to template the copolymerization of a 5′PNNNAN library, the fidelity increased from 83.7% to 98.4% when the adenosine nucleobase was modified at the 8-position. We believe that this is a result of the nucleobase modification adopting a syn conformation about the N-glycoside bond, rather than the anti conformation, which is preferred in the unmodified case (Figure 15).89,90 This is expected to result in a drop in

Figure 15. Anti and syn isomerization about the glycosidic bond of a C8modified adenosine base within an oligonucleotide chain.

thermal duplex stability and slow annealing kinetics, thus producing a more discriminating ligation process. This highfidelity copolymerization of modified pentanucleotides was ported into a single round of mock selection for the enrichment of a biotinylated phenotype for binding streptavidin, which resulted in a 350-fold enrichment over the round. Together these

Figure 16. Mechanism for the T4 DNA ligase-catalyzed DNA-templated copolymerization of modified oligonucleotides. Productive polymerization pathway and the high [ATP] termination pathway are shown. K


Review


Figure 17. (a) PNA-DNA duplex formation. (b) General strategy toward the DNA-templated copolymerization of PNA amino aldehydes.

assembled along a DNA template containing a 5′-amine hairpin (Figure 17b). The PNAs assemble in a sequence-specific manner to generate a polyimine PNA polymer, which is subsequently reduced by NaCNBH3 to yield the PNA polymer product. In these early studies using a two-codon tetranucleotide PNA system, high efficiency polymerization was observed for up to ten consecutive incorporations. The fidelity of the system was modest; while the error rate was not directly reported, the level of misincorporation was visible by gel electrophoresis, suggesting that the system might not be capable of supporting copolymerization in a library format and in vitro selection. In later studies, the group moved to a pentanucleotide PNA codon system to access larger libraries with constant pyrimidine content, which was necessary to normalize template melting temperatures and polymerization efficiency.99 Qualitative measurements of fidelity by gel electrophoresis for a twocodon copolymerization showed a marked improvement over the original PNA tetramer system. Expanding on these studies, the group later developed a pentanucleotide codon set for uniform, efficient, and specific copolymerization of pentamer PNA amino aldehydes in a complex library format.100 They sought a codon set that would satisfy the following requirements: (i) constant GC content, (ii) constant pyrimidine content, (iii) preclusion of out-of-frame annealing, and (iv) ready combinatorial synthesis of libraries. Thus, a 12-membered codon set was designed consisting of 5′AYX, where Y represents a degenerate mixture of AC, GT, and TG dinucleotides, and X represents a degenerate mixture of CA, AC, and GT dinucleotides. The fidelity of the 12-codon copolymerization process was qualitatively analyzed using a chain-termination strategy, which established that chain termination could be controlled in a sequence specific manner. While read-through errors from misincorporations were apparent during the analysis, the authors concluded that the fidelity was sufficient for in vitro evolution experiments. To demonstrate the potential of the PNA translation system, it was integrated into a mock in vitro selection system to demonstrate the enrichment of a biotinylated PNA polymer when subjected to iterated rounds of selection pressure based upon streptavidin binding. Since PNA cannot be directly replicated, the selection scheme involved DNA display, whereby the PNA polymer phenotype was displayed on its encoding

process, the 3′-hydroxyl nucleophile is precariously annealed as a pentanucleotide; in contrast, during the 3′-extension process, the 3′hydroxyl nucleophile is fully annealed as the growing strand of a duplex. While the DNA duplex sequencing method88 could not be applied to calculate the fidelity of the copolymerization of peptide-modified oligonucleotides, the fidelity was evaluated using a chain growth inhibition assay, which found the error rate to be below the detection limit of gel electrophoresis and EtBrmediated visualization. To demonstrate the potential of the system to be ported into the SELEX for the discovery of functional nucleic acid polymers based upon DNA-scaffolded peptides, a mock selection for Co2+ binding was performed with a four-membered codon set using a variant of SELMA.46 In this selection, a positive control genotype (encoding for a polyhistidine phenotype), was diluted 1000-fold into an uncompetitive library comprising genotypes that encode for peptides other than poly histidine. The His-tagged phenotype, and its associated genotype, were enriched 190-fold per round of in vitro selection, which open opportunities for the discovery of DNA-scaffolded peptides for molecular recognition. While the ligase-catalyzed DNA template copolymerization of modified oligonucleotides is a promising strategy for the evolution of modified nucleic acid polymers for molecular recognition and catalysis, the system is still in its infancy. The mechanism of templated assembly remains poorly understood as cooperative annealing, step-growth copolymerization, and chaingrowth mechanisms have not been conclusively established. Insight into the mechanistic pathway of polymerization could aid in the optimization of the process and enable generation of more highly substituted scaffolding. Furthermore, trends in codon influence and functional group influence on the fidelity of the polymerization are lacking. These data are needed to develop polymerization systems that not only have high codon fidelity but also low codon bias, a necessary requirement for widespread adoption of the method. Expanding upon their research regarding distance-dependent DNA-templated reductive amination chemistry,96,97 Liu and coworkers developed methods for the sequence-specific DNAtemplated polymerization of peptide nucleic acid amino aldehydes (Figure 17).98 In this method, PNA oligonucleotides, comprising a 5′-amine and a 3′-aldehyde, are combinatorially L


Review


Figure 18. Translation of synthetic polymers from DNA (a) Structure of the macrocylic monomer used during the nonenzymatic translation of DNA into synthetic block copolymers. (b) General strategy for the translation process.

method involved sequence-specific assembly of the macrocycles along a self-priming DNA hairpin template; DNA-templated polymerization of the synthetic strand with concomitant covalent attachment of the polymer to its encoding genotype; and subsequent cleavage of the disulfide linkers to enable folding of the synthetic polymer (Figure 18b). Following extensive optimization of the process, the authors found that the CuAAC reaction was the most effective method for polymerizing the synthetic polymer strand of the templatedassembled monomers. Furthermore, the copolymerization process operated most efficiently in an AA-BB copolymerization format (Figure 18b), as AB monomers were prone to intramolecular cyclization. The translation system generated sequence-defined synthetic block copolymers of up to 26 kDa in size, consisting of PEG, α-(D)-peptide, or β-peptide backbones. Using a chain-termination strategy analogous to that used to assess the fidelity of an earlier PNA polymerization system,100 the researchers established high sequence specificity in a six-codon system. While quantitative analysis of the fidelity was not directly reported, the observed read-through error was qualitatively similar to those reported for the PNA pentamer polymerization previously reported by the same lab, which was found to support mock in vitro selection.100 Indeed, a complete cycle of translation, encoding DNA sequence amplification, DNA hairpin template regeneration, and retranslation was demonstrated, highlighting the potential of this system to be applied to the evolution of functional synthetic polymers.

genotype by using a polymerase-mediated strand displacement approach.101 For the selection, a DNA template genotype encoding for the incorporation for a PNA pentamer containing a biotin tag was diluted 1 000 000-fold into a library comprising 108 noncompetitive genotypes that encoded for nonbiotinylated PNA pentamers. Following six iterative rounds of PNA translation, strand displacement/DNA display, selection for streptavidin binding, elution, amplification, and template regeneration, >1 000 000-fold enrichment was observed for the genotype encoding for the biotinylated PNA polymer.

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DNA-TEMPLATED COPOLYMERIZATION OF NON-NUCLEIC ACID COPOLYMERS DNA-templated copolymerization approaches that rely upon Watson−Crick base pairing to govern sequence control have traditionally been used to generate synthetic polymers that are structurally related to nucleic acids. The previous sections of this review have highlighted the utility of this approach in generating copolymers comprising modified nucleic acids or nucleic acid analogues. While this approach has enormous potential in generating new classes of high-affinity reagents and catalysts, this requirement imposes major constraints on the structural and functional potential of synthetic polymers generated by these methods. To expand the abilities of DNA-templated copolymerization to encompass the generation of polymers that are structurally unrelated to nucleic acids would have obvious advantages. To this end, Liu and co-workers have explored a nonenzymatic method to “translate” DNA into synthetic copolymers that builds from their earlier work on DNAtemplated combinatorial assembly of PNA pentanucleotides.99,100 Inspired by nature′s approach of using tRNAs to mediate the ribosome-catalyzed RNA-templated copolymerization of amino acids, the team designed macrocyclic monomers comprising a PNA anticodon adapter to engage in Watson− Crick base pairing with the template; a polymer strand with clickable functional groups at the termini; and two cleavable disulfide linkers connecting the anticodon adapter to the synthetic polymer strand (Figure 18a).102 The translation

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FUTURE CHALLENGES AND OUTLOOK This Review has highlighted the varied approaches that chemists have developed to generate libraries of sequence-defined synthetic copolymers from nucleic acid templates. These methods rely upon either hijacking and exploiting existing biomachinery or emulating biomachinery. Many of these approaches have already been ported into in vitro selection systems and have demonstrated that expansion of the functional group repertoire can result in increased fitness of polymer libraries for molecular recognition and catalysis. While research M


Review


(11) Tuerk, C.; Gold, L. Systematic Evolution of Ligands by Exponential Enrichment - Rna Ligands to Bacteriophage-T4 DNAPolymerase. Science 1990, 249, 505−510. (12) Keefe, A. D.; Pai, S.; Ellington, A. Aptamers as therapeutics. Nat. Rev. Drug Discovery 2010, 9, 537−550. (13) Lapa, S. A.; Chudinov, A. V.; Timofeev, E. N. The Toolbox for Modified Aptamers. Mol. Biotechnol. 2016, 50, 237−257. (14) Diafa, S.; Hollenstein, M. Generation of Aptamers with an Expanded Chemical Repertoire. Molecules 2015, 20, 16643−16671. (15) Meek, K. N.; Rangel, A. E.; Heemstra, J. M. Enhancing aptamer function and stability via in vitro selection using modified nucleic acids. Methods 2016, DOI: 10.1016/j.ymeth.2016.03.008. (16) Dadová, J.; Cahová, H.; Hocek, M. In Modified Nucleic Acids; Nakatani, K., Tor, Y., Eds.; Springer International Publishing: Cham, Switzerland, 2016; pp 123−144. (17) Dewey, T. M.; Mundt, A. A.; Crouch, G. J.; Zyzniewski, M. C.; Eaton, B. E. New Uridine Derivatives for Systematic Evolution of Rna Ligands by Exponential Enrichment. J. Am. Chem. Soc. 1995, 117, 8474− 8475. (18) Latham, J. A.; Johnson, R.; Toole, J. J. The Application of a Modified Nucleotide in Aptamer Selection - Novel Thrombin Aptamers Containing 5-(1-Pentynyl)-2′-Deoxyuridine. Nucleic Acids Res. 1994, 22, 2817−2822. (19) Rohloff, J. C.; Gelinas, A. D.; Jarvis, T. C.; Ochsner, U. A.; Schneider, D. J.; Gold, L.; Janjic, N. Nucleic Acid Ligands With Proteinlike Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents. Mol. Ther.–Nucleic Acids 2014, 3, e201. (20) Davies, D. R.; Gelinas, A. D.; Zhang, C.; Rohloff, J. C.; Carter, J. D.; O’Connell, D.; Waugh, S. M.; Wolk, S. K.; Mayfield, W. S.; Burgin, A. B.; Edwards, T. E.; Stewart, L. J.; Gold, L.; Janjic, N.; Jarvis, T. C. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19971−19976. (21) Hollenstein, M. DNA Catalysis: The Chemical Repertoire of DNAzymes. Molecules 2015, 20, 20777−20804. (22) Hollenstein, M.; Hipolito, C. J.; Lam, C. H.; Perrin, D. M. A selfcleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M-2). Nucleic Acids Res. 2009, 37, 1638−1649. (23) Santoro, S. W.; Joyce, G. F.; Sakthivel, K.; Gramatikova, S.; Barbas, C. F., 3rd RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc. 2000, 122, 2433−2439. (24) Raines, R. T. Ribonuclease A. Chem. Rev. 1998, 98, 1045−1066. (25) Santoro, S. W.; Joyce, G. F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 4262−4266. (26) Perrin, D. M.; Garestier, T.; Helene, C. Bridging the gap between proteins and nucleic acids: A metal-independent RNAseA mimic with two protein-like functionalities. J. Am. Chem. Soc. 2001, 123, 1556− 1563. (27) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem., Int. Ed. 1998, 37, 2754−2794. (28) Thomas, J. M.; Yoon, J. K.; Perrin, D. M. Investigation of the Catalytic Mechanism of a Synthetic DNAzyme with Protein-like Functionality: An RNaseA Mimic? J. Am. Chem. Soc. 2009, 131, 5648−5658. (29) Geyer, C. R.; Sen, D. Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme. Chem. Biol. 1997, 4, 579−593. (30) Hollenstein, M.; Hipolito, C. J.; Lam, C. H.; Perrin, D. M. Toward the combinatorial selection of chemically modified DNAzyme RNase A mimics active against all-RNA substrates. ACS Comb. Sci. 2013, 15, 174−182. (31) Jager, S.; Rasched, G.; Kornreich-Leshem, H.; Engeser, M.; Thum, O.; Famulok, M. A versatile toolbox for variable DNA functionalization at high density. J. Am. Chem. Soc. 2005, 127, 15071−15082.

in generating sequence-defined nucleic acid polymers has been ongoing for more than two decades, new polymerization approaches continue to expand the functional utility of this class of biopolymer. Thus, future investigations in this area that focus on expanding the number of modifications throughout nucleic acid polymers without sacrificing the density are of particular interest. Recently evolved polymerases that can handle XNA backbones have greatly expanded the molecular limitations of nucleic acid backbone. Future polymerase engineering that can couple synthetic backbones with modified nucleobases has the potential to generate highly nuclease resistant polymers with dense functionality, greatly increasing their potential to serve as macromolecular receptors or catalysts for applications in medicine. While recent advances in ribosomal engineering103 and reprogramming of the genetic code104 has opened opportunities to generate and evolve polypeptides with multiple instances of new chemical functionalities, robust methods that enable ribosomal synthesis of wholly synthetic polymers have yet to be developed. Current DNA-templated approaches that enable the emulation of ribosomal synthesis of synthetic polymers should allow scientists to begin exploring the evolutionary potential of synthetic macromolecules beyond the scope of current ribosomal translation systems, ranging from βpeptides or peptoids to fully conjugated synthetic polymers.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (DMR 1506667) and the Office for the Vice President of Research, University of Georgia.

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REFERENCES

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