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Nucleic acid conjugation methodologies involve linking the nucleic acid sequence to other (bio)molecules covalently. This typically allows for nucleic acid property enhancement whether it be for therapeutic purposes, biosensing, etc. Here, we report a streamlined, aqueous compatible, on-column conjugation methodology using nucleic acids containing a site-specific amino-modifier. Both monophosphates and carboxylates were amenable to the conjugation strategy, allowing for the introduction of a variety of useful handles including azide, aryl, and hydrophobic groups in DNA. We find that an on-column approach is superior to post-synthetic template-directed synthesis, mainly with respect to product purification and recovery., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Nonenzymatic genome replication is thought to be an important process for primitive lifeforms, but this has yet to be demonstrated experimentally. Recent studies on the nonenzymatic primer extension mechanism mediated by nucleoside 5'-monophosphates (NMPs) activated with 2-aminoimidazole have revealed that imidazolium-bridged dinucleotide intermediates (N*N) account for the majority of the chemical copying process. As a result, an efficacious synthetic pathway for producing substrates activated with an imidazoyl moiety is desirable. This article provides a detailed protocol for the standard dehydrative redox reaction between NMPs and 2-aminoimidazole to produce nucleotide phosphoroimidazolides. In addition, we describe a similar synthetic pathway to produce N*N in high yields for homodimers. Finally, a simple reversed-phase cation exchange step is described to increase NMP solubility, which significantly increases yields for certain substrates. This approach allows for an efficient and cost-effective methodology to prepare high-quality substrates utilized in origins-of-life studies. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2-aminoimidazolephosphoroimidazolide-activated cytidine Basic Protocol 2: Synthesis of 2-aminoimidazolium-bridged dicytidyl intermediate Basic Protocol 3: Cation exchange of guanosine 5'-monophosphate disodium salt Alternate Protocol: Synthesis of cytidine 5'-phosphoroimidazolide or 2-aminoimidazolium-bridged dicytidyl from cytidine 5'-monophosphate disodium salt., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2'-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

2-Thiouridine (s 2 U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s 2 U:s 2 U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s 2 U:s 2 U base pair. The thermodynamic stability of the s 2 U:s 2 U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s 2 U:s 2 U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s 2 U base pair outcompetes the slightly weaker s 2 U:s 2 U base pair. In addition, even if an s 2 U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s 2 U. Our findings suggest that s 2 U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically relevant processes in a narrow range of nonfluctuating environmental conditions. Here we propose that membrane phase transitions, driven by environmental fluctuations, enabled the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures and the concomitant release of protocellular content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent nonfunctional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally driven tunable prebiotic process, which supports the release and reshuffling of oligonucleotides and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally driven disassembly/assembly cycle proposed herein would have plausibly supported protocellular content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early life forms.

Substitution of exocyclic oxygen with sulfur was shown to substantially influence the properties of RNA/DNA bases, which are crucial for prebiotic chemistry and photodynamic therapies. Upon UV irradiation, thionucleobases were shown to efficiently populate triplet excited states and can be involved in characteristic photochemistry or generation of singlet oxygen. Here, we show that the photochemistry of a thionucleobase can be considerably modified in a nucleoside, that is, by the presence of ribose. Our transient absorption spectroscopy experiments demonstrate that thiocytosine exhibits 5 times longer excited-state lifetime and different excited-state absorption features than thiocytidine. On the basis of accurate quantum chemical simulations, we assign these differences to the dominant population of a shorter-lived triplet nπ* state in the nucleoside and longer-lived triplet ππ* states in the nucleobase. This explains the distinctive photoanomerziation of thiocytidine and indicates that the nucleoside will be a less efficient phototherapeutic agent with regard to singlet oxygen generation.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent advances in prebiotic chemistry are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the standard ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity? Here we focus on nonenzymatic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymatic copying served as a chemical selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixture of prebiotically synthesized nucleotides and oligonucleotides.

Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.

The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot nonenzymatic RNA chemical copying of templates with mixed-sequences is insufficient to generate strand products long enough to encode useful function. The use of alternate (RNA-like) genetic polymers may overcome hurdles associated with RNA copying, and further our understanding of nonenzymatic copying chemistry. This protocol describes the nonenzymatic copying of RNA templates into N3'→P5' phosphoramidate DNA (3'-NP-DNA). We describe, in detail, the synthesis of 3'-amino-2',3'-dideoxyribonucleotide monomers activated with 2-aminoimidazole (3'-NH 2 -2AIpddN), and their use in template-directed polymerization., Competing Interests: Competing interestsThe authors declare no competing financial interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3'-amino group at the 3'-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Achieving multiple cycles of RNA replication within a model protocell would be a critical step toward demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an "RNA world" must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an end point for RNA replication. Instead, 3'-phosphate-terminated RNA strands can participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. The presence of free magnesium cations results in cleavage of the pyrophosphate bond within minutes. However, we found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. We show that, under these conditions, pyrophosphate-linked RNA can act as a template for the polymerization of ribonucleotides into canonical 3'-5' phosphodiester-linked RNA. We suggest that primer extension of 3'-phosphate-terminated RNA followed by template-directed copying represents a plausible nonenzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

Non-enzymatic RNA self-replication is integral to the emergence of the 'RNA World'. Despite considerable progress in non-enzymatic template copying, demonstrating a full replication cycle remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible approach to strand displacement synthesis in which short 'invader' oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic primer extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued primer extension with strand displacement by employing activated 3'-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been facilitated by short RNA oligonucleotides., Competing Interests: LZ, SK, KH, DO, CG, TW, JS No competing interests declared, (© 2019, Zhou et al.)

Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3'→P5' phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Efficiently copying mixed-sequence oligonucleotide templates nonenzymatically is a long-standing problem both with respect to the origin of life, and with regard to bottom up efforts to synthesize artificial living systems. Here we report an efficient and sequence-general nonenzymatic process in which RNA templates direct the synthesis of a complementary strand composed of N3'→P5' phosphoramidate DNA (3'-NP-DNA) using 3'-amino-2',3'-dideoxyribonucleotides activated with 2-aminoimidazole. Using only the four canonical nucleobases (A, G, C, and T) of modern DNA, we demonstrate the chemical copying of a variety of mixed-sequence RNA templates, both in solution and within model protocells, into complementary 3'-NP-DNA strands. Templates up to 25 nucleotides long were chemically transcribed with an average stepwise yield of 96-97%. The nonenzymatic template-directed generation of primer extension products long enough to encode active ribozymes and/or aptamers inside model protocells suggests possible routes to the synthesis of evolving cellular systems.

The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although considerable progress has been made toward potentially prebiotic syntheses of the pyrimidine nucleotides (C and U) and their 2-thio variants, efficient routes to the canonical purine nucleotides (A and G) remain elusive. Reported syntheses are low yielding and generate a large number of undesired side products. Recently, a potentially prebiotic pathway to 8-oxo-adenosine and 8-oxo-inosine has been demonstrated, raising the question of the suitability of the 8-oxo-purines as substrates for prebiotic RNA replication. Here we show that the 8-oxo-purine nucleotides are poor substrates for nonenzymatic RNA primer extension, both as activated monomers and when present in the template strand; their presence at the end of a primer also strongly reduces the rate and fidelity of primer extension. To provide a proper comparison with 8-oxo-inosine, we also examined primer extension reactions with inosine, and found that inosine exhibits surprisingly rapid and accurate nonenzymatic RNA copying. We propose that inosine, which can be derived from adenosine by deamination, could have acted as a surrogate for G in the earliest stages of the emergence of life., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

O 6-Alkylguanine DNA alkyltransferases (AGTs) are proteins found in most organisms whose role is to remove alkylation damage from the O6- and O4-positions of 2'-deoxyguanosine (dG) and thymidine (dT), respectively. Variations in active site residues between AGTs from different organisms leads to differences in repair proficiency: The human variant (hAGT) has a proclivity for removal of alkyl groups at the O6-position of guanine and the E. coli OGT protein has activity towards the O4-position of thymine. A chimeric protein (hOGT) that our laboratory has engineered with twenty of the active site residues mutated in hAGT to those found in OGT, exhibited activity towards a broader range of substrates relative to native OGT. Among the substrates that the hOGT protein was found to act upon was interstrand cross-linked DNA connected by an alkylene linkage at the O6-position of dG to the complementary strand. In the present study the activity of hOGT towards DNA containing alkylene intrastrand cross-links (IaCL) at the O6- and O4-positions respectively of dG and dT, which lack a phosphodiester linkage between the connected residues, was evaluated. The hOGT protein exhibited proficiency at removal of an alkylene linkage at the O6-atom of dG but the O4-position of dT was refractory to protein activity. The activity of the chimeric hOGT protein towards these IaCLs to prepare well defined DNA-protein cross-linked conjugates will enable mechanistic and high resolution structural studies to address the differences observed in the repair adeptness of O4-alkylated dT by the OGT protein relative to other AGT variants.

Non-enzymatic RNA replication may have been one of the processes involved in the appearance of life on Earth. Attempts to recreate this process in a laboratory setting have not been successful thus far, highlighting a critical need for finding prebiotic conditions that increase the rate and the yield. Now a highly parallel assay for template directed RNA synthesis is presented that relies on the intrinsic fluorescence of a 2-aminopurine modified G-quadruplex. The application of the assay to examine the combined influence of multiple variables including pH, divalent metal concentrations and ribonucleotide concentrations on the copying of RNA sequences is demonstrated. The assay enables a direct survey of physical and chemical conditions, potentially prebiotic, which could enable the chemical replication of RNA., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The chemical replication of RNA inside fatty acid vesicles is a plausible step in the emergence of cellular life. On the primitive Earth, simple protocells with the ability to import nucleotides and short oligomers from their environment could potentially have replicated and retained larger genomic RNA oligonucleotides within a spatially defined compartment. We have previously shown that short 5'-phosphoroimidazolide-activated "helper" RNA oligomers enable the nonenzymatic copying of mixed-sequence templates in solution, using 5'-phosphoroimidazolide-activated mononucleotides. Here, we report that citrate-chelated Mg 2+ , a catalyst of nonenzymatic primer extension, enhances fatty acid membrane permeability to such short RNA oligomers up to the size of tetramers, without disrupting vesicle membranes. In addition, selective permeability of short, but not long, oligomers can be further enhanced by elevating the temperature. The ability to increase the permeability of fatty acid membranes to short oligonucleotides allows for the nonenzymatic copying of RNA templates containing all four nucleotides inside vesicles, bringing us one step closer to the goal of building a protocell capable of Darwinian evolution.

Oligonucleotides containing various adducts, including ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl groups, at the O 4 atom of 5-fluoro-O 4 -alkyl-2'-deoxyuridine were prepared by solid-phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5-fluoro-2'-deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B-form DNA structure. O 6 -Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5-fluoro-O 4 -benzyl-2'-deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O 4 -ethyl and benzyl adducts of 5-fluoro-2-deoxyuridine. Computational assessment of N1-methyl analogues of the O 4 -alkylated nucleobases revealed that the C5-fluorine modification had an influence on reducing the electron density of the O 4 -C α bond, relative to thymine (C5-methyl) and uracil (C5-hydrogen). These results reveal the positive influence of the C5-fluorine atom on the repair of larger O 4 -alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA oligomers containing dimethylene and trimethylene intrastrand crosslinks (IaCLs) between the O4 and O6 atoms of neighboring thymidine (T) and 2'-deoxyguanosine (dG) residues were prepared by solid-phase synthesis. UV thermal denaturation (T m ) experiments revealed that these IaCLs had a destabilizing effect on the DNA duplex relative to the control. Circular dichroism spectroscopy suggested these IaCLs induced minimal structural distortions. Susceptibility to dealkylation by reaction with various O 6 -alkylguanine DNA alkyltransferases (AGTs) from human and Escherichia coli was evaluated. It was revealed that only human AGT displayed activity towards the IaCL DNA, with reduced efficiency as the IaCL shortened (from four to two methylene linkages). Changing the site of attachment of the ethylene linkage at the 5'-end of the IaCL to the N3 atom of T had minimal influence on duplex stability and structure, and was refractory to AGT activity., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A working model of nonenzymatic RNA primer extension could illuminate how prebiotic chemistry transitioned to biology. All currently known experimental reconstructions of nonenzymatic RNA primer extension yield a mixture of 2'-5' and 3'-5' internucleotide linkages. Although long seen as a major problem, the causes of the poor regioselectivity of the reaction are unknown. We used a combination of different leaving groups, nucleobases, and templating sequences to uncover the factors that yield selective formation of 3'-5' internucleotide linkages. We found that fast and high yielding reactions selectively form 3'-5' linkages. Additionally, in all cases with high 3'-5' regioselectivity, Watson-Crick base pairing between the RNA monomers and the template is observed at the extension site and at the adjacent downstream position. Mismatched base-pairs and other factors that would perturb the geometry of the imidazolium bridged intermediate lower both the rate and regioselectivity of the reaction.

O ⁶-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O ⁶-atom of guanine and O ⁴-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O ⁶-adducts of guanine and O ⁴-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O ⁶-atoms of guanine in a DNA duplex. When compared to the human AGT protein (hAGT), the S134P OGT variant displayed reduced activity towards O ⁶-alkylation but a much broader substrate range for O ⁴-alkylation damage reversal. The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O ⁶-position of guanine. Finally, a method to generate a covalent conjugate between hAGT and a model nucleoside using a single-stranded oligonucleotide substrate is demonstrated., Competing Interests: The authors declare no conflict of interest.

DNA interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O 6 -Alkylguanine DNA alkyltransferases (AGTs) are capable of removing alkylation damage from the O 6 -atom of 2'-deoxyguanosine and the O 4 -atom of thymidine. Human AGT (hAGT) has demonstrated the ability to repair an interstrand cross-linked duplex where two O 6 -atoms of 2'-deoxyguanosine were tethered by a butylene (XLGG4) or heptylene (XLGG7) linkage. However, the analogous ICL between the O 4 -atoms of thymidine was found to evade repair. ICL duplexes connecting the O 4 -atoms of 2'-deoxyuridine by a butylene (XLUU4) or heptylene (XLUU7) linkage have been prepared to examine the influence of the C5-methyl group on AGT-mediated repair. Both XLUU4 and XLUU7 were refractory to repair by human and E. coli (OGT and Ada-C) AGTs with comparably low μM dissociation constants for 2 : 1 or 4 : 1 AGT/DNA stoichiometries. The solution structures of two heptylene linked DNA duplexes (CGAAAYTTTCG) 2 , XLUU7 (Y = dU) and XLGG7 (Y = dG), were solved and the global structures were virtually identical with a RMSD of 1.22 Å. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4-O 4 bond for XLUU7 whereas a Z conformation about the C6-O 6 bond was observed for XLGG7. This E versus Z conformation may partially account for hAGTs discrimination towards the repair of these ICL, supported by the crystal structures of hAGT with various substrates which have been observed to adopt a Z conformation. In addition, a higher mobility at the ICL site for XLUU7 is observed relative to XLGG7 that may play a role in repair by hAGT. Taken together, these findings provide insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.

Achieving efficient nonenzymatic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymatic copying of templates containing mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addition as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences.

A methodology is reported to conjugate human O 6 -alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O 4 -thymidine-alkylene-O 4 -thymidine and the (5'→3') O 6 -2'-deoxyguanosine-alkylene-O 4 -thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5'→3') O 4 -thymidine-alkylene-O 6 -2'-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.

DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O 6 -dG-derived ICLs. Although less abundant, O 6 -alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O 6 -2'-deoxyguanosine-butylene-O 6 -2'-deoxyguanosine (O 6 -dG-C4-O 6 -dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O 6 -dG-C4-O 6 -dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol κ is completely inhibited by O 6 -dG-C4-O 6 -dG ICLs. Moderate bypass activities are observed for human pol η and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol η exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol η in O 6 -dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol η. Together, this study provides the first set of biochemical evidence for the mutagenicity of O 6 -dG-derived ICLs.

This unit describes the preparation O(6) -2'-deoxyguanosine-butylene-O(6) -2'-deoxyguanosine dimer phosphoramidites and precursors for incorporation of site-specific intrastrand cross-links (IaCL) into DNA oligonucleotides. Protected 2'-deoxyguanosine dimers are produced using the Mitsunobu reaction. IaCL DNA containing the intradimer phosphodiester are first chemically phosphorylated, followed by a ring-closing reaction using the condensing reagent 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole. Phosphoramidites are incorporated into oligonucleotides by solid-phase synthesis and standard deprotection and cleavage protocols are employed. This approach allows for the preparation of IaCL DNA substrates in amounts and purity amenable for biophysical characterization, and biochemical studies as substrates to investigate DNA repair and bypass pathways. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O 4 -Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4- O 4 bond on processing by human DNA polymerase η (hPol η ) was studied for oligonucleotides containing O 4 -methylthymidine, O 4 -ethylthymidine, and analogs restricting the O 4 -methylene group in an anti -orientation. Primer extension assays revealed that the O 4 -alkyl orientation influences hPol η bypass. Crystal structures of hPol η •DNA•dNTP ternary complexes with O 4 -methyl- or O 4 -ethylthymidine in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn - O 4 -methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O 4 -methylthymidine with hPol η , inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O 4 MedT and O 4 EtdT when acted upon by hPol η .

Oligonucleotides containing an alkylene intrastrand cross-link (IaCL) between the O(6) -atoms of two consecutive 2'-deoxyguanosines (dG) were prepared by solid-phase synthesis. UV thermal denaturation studies of duplexes containing butylene and heptylene IaCL revealed a 20 °C reduction in stability compared to the unmodified duplexes. Circular dichroism profiles of these IaCL DNA duplexes exhibited signatures consistent with B-form DNA. Human O(6) -alkylguanine DNA alkyltransferase (hAGT) was capable of repairing both IaCL containing duplexes with slightly greater efficiency towards the heptylene analog. Interestingly, repair efficiencies of hAGT towards these IaCL were lower compared to O(6) -alkylene linked IaCL lacking the 5'-3'-phosphodiester linkage between the connected 2'-deoxyguanosine residues. These results demonstrate that the proficiency of hAGT activity towards IaCL at the O(6) -atom of dG is influenced by the backbone phosphodiester linkage between the cross-linked residues., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrastrand cross-links (IaCL) connecting two purine nucleobases in DNA pose a challenge to high-fidelity replication in the cell. Various repair pathways or polymerase bypass can cope with these lesions. The influence of the phosphodiester linkage between two neighboring 2'-deoxyguanosine (dG) residues attached through the O(6) atoms by an alkylene linker on bypass with human DNA polymerase η (hPol η) was explored in vitro. Steady-state kinetics and mass spectrometric analysis of products from nucleotide incorporation revealed that although hPol η is capable of bypassing the 3'-dG in a mostly error-free fashion, significant misinsertion was observed for the 5'-dG of the IaCL containing a butylene or heptylene linker. The lack of the phosphodiester linkage triggered an important increase in frameshift adduct formation across the 5'-dG by hPol η, in comparison to the 5'-dG of IaCL DNA containing the phosphodiester group.

Oligonucleotides tethered by an alkylene linkage between the O(6) -atoms of two consecutive 2'-deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross-linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene- and heptylene-linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B-form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O(6) -alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada-C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This protocol describes the preparation of O(4)-thymidine-alkylene-O(4)-thymidine dimer bis-phosphoramidites and precursors for incorporation into DNA sequences to produce site-specific DNA interstrand cross-links. Linkers are introduced at the 4-position of thymidine by reacting the sodium salt of a diol with a pyrimidinyl-convertible nucleoside to produce mono-adducts, which then undergo reaction with a stoichiometric equivalent of a pyrimidinyl-convertible nucleoside under basic conditions to form O(4)-thymidine-alkylene-O(4)-thymidine dimers. Bis-phosphoramidites are incorporated into oligonucleotides by solid-phase synthesis, and mild conditions for deprotection and cleavage from the solid support are employed to prevent degradation of the thymidine modifications. Purification of these cross-linked oligonucleotides is performed by denaturing polyacrylamide gel electrophoresis. This approach allows for the preparation of cross-linked DNA substrates in quantities and purity sufficient for a wide range of biophysical experiments and biochemical studies as substrates to investigate DNA repair pathways., (Copyright © 2014 John Wiley & Sons, Inc.)

This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases., (Copyright © 2014 John Wiley & Sons, Inc.)

The bisalkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), used in cancer chemotherapy to hinder cellular proliferation, forms lethal interstrand cross-links (ICLs) in DNA. BCNU generates an ethylene linkage connecting the two DNA strands at the N1 atom of 2'-deoxyguanosine and N3 atom of 2'-deoxycytidine, which is a synthetically challenging probe to prepare. To this end, an ICL duplex linking the N1 atom of 2'-deoxyinosine to the N3 atom of thymidine via an ethylene linker was devised as a mimic. We have solved the structure of this ICL duplex by a combination of molecular dynamics and high-field NMR experiments. The ethylene linker is well-accommodated in the duplex with minimal global and local perturbations relative to the unmodified duplex. These results may account for the substantial stabilization of the ICL duplex observed by UV thermal denaturation experiments and provides structural insights of a probe that may be useful for DNA repair studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA duplexes containing a directly opposed O(4)-2'-deoxythymidine-alkyl-O(4)-2'-deoxythymidine (O(4)-dT-alkyl-O(4)-dT) interstrand cross-link (ICL) have been prepared by the synthesis of cross-linked nucleoside dimers which were converted to phosphoramidites to produce site specific ICL. ICL duplexes containing alkyl chains of four and seven methylene groups were prepared and characterized by mass spectrometry and nuclease digests. Thermal denaturation experiments revealed four and seven methylene containing ICL increased the T(m) of the duplex with respect to the non-cross-linked control with an observed decrease in enthalpy based on thermodynamic analysis of the denaturation curves. Circular dichroism experiments on the ICL duplexes indicated minimal difference from B-form DNA structure. These ICL were used for DNA repair studies with O(6)-alkylguanine DNA alkyltransferase (AGT) proteins from human (hAGT) and E. coli (Ada-C and OGT), whose purpose is to remove O(6)-alkylguanine and in some cases O(4)-alkylthymine lesions. It has been previously shown that hAGT can repair O(6)-2'-deoxyguanosine-alkyl-O(6)-2'-deoxyguanosine ICL. The O(4)-dT-alkyl-O(4)-dT ICL prepared in this study were found to evade repair by hAGT, OGT and Ada-C. Electromobility shift assay (EMSA) results indicated that the absence of any repair by hAGT was not a result of binding. OGT was the only AGT to show activity in the repair of oligonucleotides containing the mono-adducts O(4)-butyl-4-ol-2'-deoxythymidine and O(4)-heptyl-7-ol-2'-deoxythymidine. Binding experiments conducted with hAGT demonstrated that the protein bound O(4)-alkylthymine lesions with similar affinities to O(6)-methylguanine, which hAGT repairs efficiently, suggesting the lack of O(4)-alkylthymine repair by hAGT is not a function of recognition.

Oligonucleotides containing modified 2'-deoxyguanosines bearing a seven carbon linker at the O(6)- atom with either a terminal hydroxyl or 2'- deoxyguanosine group have been synthesized as potential intermediates formed during repair of interstrand cross-linked DNA. Repair of these substrates with Escherichia coli OGT was investigated with an assay involving cleavage of the unmodified duplex with the restriction endonuclease PvuII followed by analysis of the products by denaturing polyacrylamide gel electrophoresis. Duplexes containing these modifications were repaired by OGT suggesting that direct repair may play a role, in combination with other repair pathways, in reversing interstrand crosslink DNA damage.