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We determined the influence of chirality on the behavior of oligonucleotides inside human cells, revealing that mirror-image l-oligonucleotides have the potential to be highly cytotoxic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has resulted in an ongoing global pandemic that has already claimed more than 4 million lives. While most antiviral efforts have focused on essential SARS-CoV-2 proteins, RNA structural elements within the viral genome are also compelling targets. In this study, we identified high-affinity l-DNA aptamers against a SARS-CoV-2 stem-loop II-like motif (s2m), a highly conserved RNA structure with promising diagnostic and therapeutic potential. Optimized l-C1t and l-C3t aptamers bind selectively to s2m RNA with Kd values in the nanomolar range, and are capable of differentiating the monomeric s2m stem-loop from the proposed homodimer duplex. This structure-specific mode of recognition also allows l-C1t and l-C3t to discriminate between s2m RNAs from SARS-CoV-2 and SARS-CoV-1, which differ by just two nucleotides. Finally, we show that l-C1t and l-C3t induce dramatic conformational changes in s2m structure upon binding, and thus, have the potential to block protein–s2m interactions. Overall, these results demonstrate the feasibility of targeting SARS-CoV-2 RNA using l-aptamers, which has important implications in the diagnosis and treatment of COVID-19. Moreover, the high affinity and selectivity of l-C1t and l-C3t, coupled with the intrinsic nuclease resistance of l-DNA, present an opportunity for generating new tools and probes for interrogating s2m function in SARS-CoV-2 and related viruses., In vitro selection was used to identify L-DNA aptamers with high affinity and selectivity for the s2m RNA structure in the SARS-CoV-2 genome.

Aptamers composed of mirror-image L-(deoxy)ribose nucleic acids, referred to as L-aptamers, are a promising class of RNA-binding reagents. Yet, the selectivity of cross-chiral interactions between L-aptamers and their RNA targets remain poorly characterized, limiting the potential utility of this approach for applications in biological systems. Herein, we carried out the first comprehensive analysis of cross-chiral L-aptamer selectivity using a newly developed "inverse" in vitro selection approach that exploits the genetic nature of the D-RNA ligand. By employing a library of more than a million target-derived sequences, we determined the RNA sequence and structural preference of a model L-aptamer and revealed previously unidentified and potentially broad off-target RNA binding behaviors. These results provide valuable information for assessing the likelihood and consequences of potential off-target interactions and reveal strategies to mitigate these effects. Thus, inverse in vitro selection provides several opportunities to advance L-aptamer technology.

Human cyclophilin B (CypB) is oversecreted by pancreatic cancer cells, making it a potential biomarker for early-stage disease diagnosis. Our group is motivated to develop aptamer-based assays to measure CypB levels in biofluids. However, human cyclophilins have been postulated to have collateral nuclease activity, which could impede the use of aptamers for CypB detection. To establish if CypB can hydrolyze electrode-bound nucleic acids, we used ultrasensitive electrochemical sensors to measure CypB's hydrolytic activity. Our sensors use ssDNA and dsDNA in the biologically predominant d-DNA form, and in the nuclease resistant l-DNA form. Challenging such sensors with CypB and control proteins, we unequivocally demonstrate that CypB can cleave nucleic acids. To our knowledge, this is the first study to use electrochemical biosensors to reveal the hydrolytic activity of a protein that is not known to be a nuclease. Future development of CypB bioassays will require the use of nuclease-resistant aptamer sequences.

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

Electrochemical aptamer-based (E-AB) sensors are a technology capable of real-time monitoring of drug concentrations directly in the body. These sensors achieve their selectivity from surface-attached aptamers, which alter their conformation upon target binding, thereby causing a change in electron transfer kinetics between aptamer-bound redox reporters and the electrode surface. Because, in theory, aptamers can be selected for nearly any target of interest, E-AB sensors have far-reaching potential for diagnostic and biomedical applications. However, a remaining critical weakness in the platform lies in the time-dependent, spontaneous degradation of the bioelectronic interface. This progressive degradation—seen in part as a continuous drop in faradaic current from aptamer-attached redox reporters—limits the in vivo operational life of E-AB sensors to less than 12 h, prohibiting their long-term application for continuous molecular monitoring in humans. In this work, we study the effects of nuclease action on the signaling lifetime of E-AB sensors, to determine whether the progressive signal loss is caused by hydrolysis of DNA aptamers and thus the loss of signaling moieties from the sensor surface. We continuously interrogate sensors deployed in several undiluted biological fluids at 37 °C and inject nuclease to reach physiologically relevant concentrations. By employing both naturally occurring d-DNA and the nuclease-resistant enantiomer l-DNA, we determine that within the current lifespan of state-of-the-art E-AB sensors, nuclease hydrolysis is not the dominant cause of sensor signal loss under the conditions we tested. Instead, signal loss is driven primarily by the loss of monolayer elements—both blocking alkanethiol and aptamer monolayers—from the electrode surface. While use of l-DNA aptamers may extend the E-AB operational life in the long term, the critical issue of passive monolayer loss must be addressed before those effects can be seen.

Chromatin structures (and modulators thereof) play a central role in genome organization and function. Herein, we report that thymine DNA glycosylase (TDG), an essential enzyme involved in DNA repair and demethylation, has the capacity to alter chromatin structure directly through its physical interactions with DNA. Using chemically defined nucleosome arrays, we demonstrate that TDG induces decompaction of individual chromatin fibers upon binding and promotes self-association of nucleosome arrays into higher-order oligomeric structures (i.e. condensation). Chromatin condensation is mediated by TDG’s disordered polycationic N-terminal domain, whereas its C-terminal domain antagonizes this process. Furthermore, we demonstrate that TDG-mediated chromatin condensation is reversible by growth arrest and DNA damage 45 alpha (GADD45a), implying that TDG cooperates with its binding partners to dynamically control chromatin architecture. Finally, we show that chromatin condensation by TDG is sensitive to the methylation status of the underlying DNA. This new paradigm for TDG has specific implications for associated processes, such as DNA repair, DNA demethylation, and transcription, and general implications for the role of DNA modification ‘readers’ in controlling chromatin organization.

Due to their intrinsic nuclease resistance, l-oligonucleotides are being increasingly utilized in the development of molecular tools and sensors. Yet, it remains challenging to synthesize long l-oligonucleotides, potential limiting future applications. Herein, we report straightforward and versitile approach to assemble long l-RNAs from two or more shorter fragments using T4 RNA ligase 1. We show that this approach is compatible with the assembly of several classes of functional l-RNA, which we highlight by generating a 124-nt l-RNA biosensor that functions in serum.

To overcome technical challenges associated with the use of DNA strand-displacement circuits in vivo, including degradation by cellular nucleases, researchers are increasingly turning to bio-orthogonal l-DNA. Although enhanced stability and improved performance of l-DNA-based circuits within living cells are often implied, direct experimental evidence has not been provided. Herein, we directly compare the functional stability and kinetics of d-DNA and l-DNA strand-displacement in live cells for the first time. We show that l-DNA strand-displacement reaction systems have minimal "leak", fast reaction kinetics, and prolonged stability inside living cells as compared to conventional d-DNA. Furthermore, using "heterochiral" strand-displacement, we demonstrate that biostable l-DNA reaction components can be easily interfaced with native DNA inside cells. Overall, our results strongly support the broader adoption of l-DNA in the field of DNA molecular circuitry, especially for in vivo applications.

Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG’s affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA.

The development of structure-specific RNA binding reagents remains a central challenge in RNA biochemistry and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve l-RNA aptamers that bind tightly to structured d-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of l-DNA, which has several practical advantages compared to l-RNA, remains unknown. Here, we report the discovery and characterization of the first l-DNA aptamers against a structured RNA molecule, precursor microRNA-155, thereby establishing the capacity of DNA and RNA molecules of the opposite handedness to form tight and specific ‘cross-chiral’ interactions with each other. l-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of l-DNA to interact with native d-RNA via Watson–Crick base pairing. Furthermore, l-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of l-DNA aptamers are distinct from previously reported l-RNA aptamers against pre-miR-155, indicating that l-DNA and l-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that l-DNA may be pursued as an alternative to l-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.

Although a functional relationship between active DNA demethylation and chromatin structure is often implied, direct experimental evidence is lacking. We investigated the relationship between chromatin structure and thymine DNA glycosylase (TDG) using chemically defined nucleosome arrays containing site-specifically positioned 5-formylcytosine (5fC) residues. We show that the extent of array compaction, as well as nucleosome positioning, dramatically influence the ability of TDG to excise 5fC from DNA, indicating that the chromatin structure is likely a key determinant of whether 5fC is removed from the genome or retained as an epigenetic mark. Furthermore, the H2A.Z/H3.3 double-variant nucleosome and the pioneering transcription factor forkhead box A1 (FOXA1), both of which are implicated in shaping the chromatin landscape during demethylation of tissue-specific enhancers, differentially regulate TDG activity on chromatin. Together, this work provides the first direct evidence that the higher order chromatin structure regulates active DNA demethylation through TDG and provides novel insights into the mechanism of 5fC turnover at enhancers.

Watson-Crick base pairing rules provide a powerful approach for engineering DNA-based nanodevices with programmable and predictable behaviors. In particular, DNA strand displacement reactions have enabled the development of an impressive repertoire of molecular devices with complex functionalities. By relying on DNA to function, dynamic strand displacement devices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation in living systems has been a slow process due to several persistent challenges, including nuclease degradation. To circumvent these issues, researchers are increasingly turning to chemically modified nucleotides as a means to increase device performance and reliability within harsh biological environments. In this review, we summarize recent progress toward the integration of chemically modified nucleotides with DNA strand displacement reactions, highlighting key successes in the development of robust systems and devices that operate in living cells and in vivo. We discuss the advantages and disadvantages of commonly employed modifications as they pertain to DNA strand displacement, as well as considerations that must be taken into account when applying modified oligonucleotide to living cells. Finally, we explore how chemically modified nucleotides fit into the broader goal of bringing dynamic DNA nanotechnology into the cell, and the challenges that remain. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing.

Biology relies almost exclusively on homochiral building blocks to drive the processes of life. Yet cross-chiral interactions can occur between macromolecules of the opposite handedness, including a previously described polymerase ribozyme that catalyzes the template-directed synthesis of enantio-RNA. The present study sought to optimize and generalize this activity, employing in vitro evolution to select cross-chiral polymerases that use either mono- or trinucleotide substrates that are activated as the 5'-triphosphate. There was only modest improvement of the former activity, but dramatic improvement of the latter, which enables the trinucleotide polymerase to react 102-103-fold faster than its ancestor and to accept substrates with all possible sequence combinations. The evolved ribozyme can assemble long RNAs from a mixture of trinucleotide building blocks, including a two-fragment form of the ancestral polymerase ribozyme. Further improvement of this activity could enable the generalized cross-chiral replication of RNA, which would establish a new paradigm for the chemical basis of Darwinian evolution.

Development of biocompatible tools for intracellular imaging of RNA expression remains a central challenge. Herein, we report the use of heterochiral strand-displacement to sequence-specifically interface endogenous d-miRNAs with an l-RNA version of the fluorogenic aptamer Mango III, thereby generating a novel class of biocompatible miRNA sensors. Fluorescence activation of the sensor is achieved through the displacement of an achiral blocking strand from the l-Mango aptamer by the d-RNA target. In contrast to d-Mango, we show that the l-Mango sensor retains full functionality in serum, enabling a light-up fluorescence response to the target. Importantly, we employ a self-delivering version of the l-Mango sensor to image the expression of microRNA-155 in living cells, representing the first time l-oligonucleotides have been interfaced with a living system. Overall, this work provides a new paradigm for the development of biocompatible hybridization-based sensors for live-cell imaging of RNAs and greatly expands the utility of fluorogenic aptamers for cellular applications.

The genomic DNA of eukaryotic cells exists in the form of chromatin, the structure of which controls the biochemical accessibility of the underlying DNA to effector proteins. In order to gain an in depth molecular understanding of how chromatin structure regulates DNA repair, detailed in vitro biochemical and biophysical studies are required. However, because of challenges associated with reconstituting nucleosome arrays containing site-specifically positioned DNA modifications, such studies have been limited to the use of mono- and dinucleosomes as model in vitro substrates, which are incapable of folding into native chromatin structures. To address this issue, we developed a straightforward and general approach for assembling chemically defined oligonucleosome arrays (i.e., designer chromatin) containing site-specifically modified DNA. Our method takes advantage of nicking endonucleases to excise short fragments of unmodified DNA, which are subsequently replaced with synthetic oligonucleotides containing the desired modification. Using this approach, we prepared several oligonucleosome substrates containing precisely positioned 2'-deoxyuridine (dU) residues and examined the efficiency of base excision repair (BER) within several distinct chromatin architectures. We show that, depending on the translational position of the lesion, the combined catalytic activities of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) can be either inhibited by as much as 20-fold or accelerated by more than 5-fold within compact chromatin (i.e., the 30 nm fiber) relative to naked DNA. Moreover, we demonstrate that digestion of dU by UDG/APE1 proceeds much more rapidly in mononucleosomes than in compacted nucleosome arrays, thereby providing the first direct evidence that internucleosome interactions play an important role in regulating BER within higher-order chromatin structures. Overall, this work highlights the value of performing detailed biochemical studies on precisely modified chromatin substrates in vitro and provides a robust platform for investigating DNA modifications in chromatin biology.

The Polycomb Repressive Complex 2 (PRC2) interacts promiscuously with G-quadruplex (G4) RNA structures. Herein, we tested the limit of this promiscuity by exploring the interaction of PRC2 with G4 RNAs comprised of l-ribonucleic acids (l-RNA), the enantiomer of naturally occurring d-RNA. Remarkably, we find that PRC2 binds similarly to both d- and l-G4 RNAs, suggesting that these interactions are independent of stereochemistry. Moreover, we show that d- and l-RNAs bind to the same site on PRC2, enabling l-G4 RNAs to outcompete native substrates for binding. This work challenges the prevailing assumption that l-oligonucleotides are "invisible" to native biology and provides a unique opportunity to develop a novel class of PRC2 inhibitors based on nuclease-resistant l-RNA.

The absence of a straightforward strategy to interface native d-DNA with its enantiomer l-DNA-oligonucleotides of opposite chirality are incapable of forming contiguous Watson-Crick base pairs with each other-has enforced a "homochiral" paradigm over the field of dynamic DNA nanotechnology. As a result, chirality, a key intrinsic property of nucleic acids, is often overlooked as a design element for engineering of DNA-based devices, potentially limiting the types of behaviors that can be achieved using these systems. Here we introduce a toehold-mediated strand-displacement methodology for transferring information between orthogonal DNA enantiomers via an achiral intermediary, opening the door for "heterochiral" DNA nanotechnology having fully interfaced d-DNA and l-DNA components. Using this approach, we demonstrate several heterochiral DNA circuits having novel capabilities, including autonomous chiral inversion of DNA sequence information and chirality-based computing. In addition, we show that heterochiral circuits can directly interface endogenous RNAs (e.g., microRNAs) with bioorthogonal l-DNA, suggesting applications in bioengineering and nanomedicine. Overall, this work establishes chirality as a design parameter for engineering of dynamic DNA nanotechnology, thereby expanding the types of architectures and behaviors that can be realized using DNA.

To facilitate isolation of l-aptamers with novel RNA-binding properties, we employed a cationic nucleotide, 5-aminoallyluridine, during the mirror image in vitro selection process. Through this effort, we identified a modified l-RNA aptamer (MlRA) capable of binding oncogenic precursor microRNA 19a (pre-miR-19a) with exceptional affinity, and we showed that cationic modification is absolutely critical for binding. Furthermore, formation of the MlRA-pre-miR-19a complex inhibited Dicer-mediated cleavage of the pre-miR, thus blocking formation of the mature functional microRNA. The MlRA reported here not only represents the first l-aptamer to be evolved by using modified nucleotides but also the first modified aptamer (of any type) to be selected against a structured RNA target. Our results demonstrate that functionalized l-aptamers, which are intrinsically nuclease-resistant, provide an attractive approach for developing robust RNA-binding reagents.

Despite recent evidence suggesting that histone lysine acetylation contributes to base excision repair (BER) in cells, their exact mechanistic role remains unclear. In order to examine the influence of histone acetylation on the initial steps of BER, we assembled nucleosome arrays consisting of homogeneously acetylated histone H3 (H3K18 and H3K27) and measured the repair of a site-specifically positioned 2′-deoxyuridine (dU) residue by uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1). We find that H3K18ac and H3K27ac differentially influence the combined activities of UDG/APE1 on compact chromatin, suggesting that acetylated lysine residues on the H3 tail domain play distinct roles in regulating the initial steps of BER. In addition, we show that the effects of H3 tail domain acetylation on UDG/APE1 activity are at the nucleosome level and do not influence higher-order chromatin folding. Overall, these results establish a novel regulatory role for histone H3 acetylation during the initiation of BER on chromatin.

Heterochiral DNA strand-displacement reactions enable sequence-specific interfacing of oligonucleotide enantiomers, making it possible to interface native d-nucleic acids with molecular circuits built using nuclease-resistant l-DNA. To date, all heterochiral reactions have relied on peptide nucleic acid (PNA), which places potential limits on the scope and utility of this approach. Herein, we now report heterochiral strand-displacement in the absence of PNA, instead utilizing chimeric d/l-DNA complexes to interface oligonucleotides of the opposite chirality. We show that these strand-displacement reactions can be easily integrated into multicomponent heterochiral circuits, are compatible with both DNA and RNA inputs, and can be engineered to function in serum-supplemented medium. We anticipate that these new reactions will lead to a wider application of heterochiral strand-displacement, especially in the design of biocompatible nucleic acid circuits that can reliably operate within living systems.

The programmability of DNA/RNA-based molecular circuits provides numerous opportunities in the field of synthetic biology. However, the stability of nucleic acids remains a major concern when performing complex computations in biological environments. Our solution to this problem is l-(deoxy)ribose nucleic acids (l-DNA/RNA), which are mirror images (i.e. enantiomers) of natural d-nucleotides. l-oligonucleotides have the same physical and chemical properties as their natural counterparts, yet they are completely invisible to the stereospecific environment of biology. We recently reported a novel strand-displacement methodology for transferring sequence information between oligonucleotide enantiomers (which are incapable of base pairing with each other), enabling bio-orthogonal l-DNA/RNA circuits to be easily interfaced with living systems. In this perspective, we summarize these so-called ‘heterochiral’ circuits, provide a viewpoint on their potential applications in synthetic biology, and discuss key problems that must be solved before achieving the ultimate goal of the engineering complex and reliable functionality.

In vitro selection was used to obtain L-RNA aptamers that bind the distal stem-loop of various precursor microRNAs (pre-miRs). These L-aptamers, termed “aptamiRs”, bind their corresponding pre-miR target through highly specific tertiary interactions rather than Watson–Crick pairing. Formation of a pre-miR–aptamiR complex inhibits Dicer-mediated processing of the pre-miR, which is required to form the mature functional microRNA. One of the aptamiRs, which was selected to bind oncogenic pre-miR-155, inhibits Dicer processing under simulated physiological conditions, with an IC50 of 87 nM. Given that L-RNAs are intrinsically resistant to nuclease degradation, these results suggest that aptamiRs might be pursued as a new class of miR inhibitors.

An L-RNA aptamer was developed that binds the natural D-form of the HIV-1 trans-activation responsive (TAR) RNA. The aptamer initially was obtained as a D-aptamer against L-TAR RNA through in vitro selection. Then the corresponding L-aptamer was prepared by chemical synthesis and used to bind the desired target. The Kd of the L-aptamer for D-TAR RNA is 100 nM. The aptamer binds D-TAR RNA exclusively at the six-nucleotide distal loop, but does so through tertiary interactions rather than simple Watson-Crick pairing. This complex is the first example of two nucleic acids molecules of opposing chirality that interact through a mode of binding other than primary structure. Binding of the L-aptamer to D-TAR RNA inhibits formation of the Tat-TAR ribonucleoprotein complex that is essential for TAR function. This suggests that L-aptamers, which are intrinsically resistant to degradation by ribonucleases, might be pursued as an alternative to anti-sense oligonucleotides to target structured RNAs of biological or therapeutic interest.

The C4'-oxidized abasic site is produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor agents. Independent generation of this alkali-labile lesion at defined positions within nucleosome core particles reveals that the histone proteins increase strand scission between 130- and 550-fold. Strand scission proceeds via a Schiff base intermediate, but the DNA-protein cross-links are unstable. The oxidized abasic site is removed in its entirety from the DNA and transferred to the lysine-rich tail region of the proximal histone protein in the form of a lactam. The modification is distributed over several residues within the amino-terminal tail of the proximal histone. Transfer of DNA damage to histones could affect gene regulation.

The reactivity of apurinic/apyrimidinic (AP) sites at different locations within nucleosome core particles was examined. AP sites are greatly destabilized in nucleosome core particles compared to free DNA. Their reactivity varied ~5-fold with respect to the location within the nucleosome core particles but followed a common mechanism involving formation of a Schiff base between histone proteins and the lesion. The identity of the histone protein(s) involved in the reaction and the reactivity of the corresponding DNA-protein cross-links varied with the location of the abasic site, indicating that while the relative rate constants for individual steps varied in a complex manner, the overall mechanism remained the same. The source of the accelerated reactivity was probed using nucleosomes containing AP89 and histone H3 and H4 variants. Mutating the five lysine residues in the amino tail region of histone H4 to arginines reduced the rate constant for disappearance almost 15-fold. Replacing histidine 18 with an alanine reduced AP reactivity more than 3-fold. AP89 in a nucleosome core particle composed of the H4 variant containing both sets of mutations reacted only4-fold faster than it did in naked DNA. These experiments reveal that nucleosome-catalyzed reaction at AP89 is a general phenomenon and that the lysine rich histone tails, whose modification is integrally involved in epigenetics, are primarily responsible for this chemistry.

Summary A synthetic genetic system, based on cross-replicating RNA enzymes, provides a means to evaluate alternative genetic codes that relate heritable information to corresponding molecular function. A special implementation of encoded combinatorial chemistry was used to construct complex populations of cross-replicating RNA enzymes in accordance with a user-specified code that relates genotype and phenotype on a molecule-by-molecule basis. The replicating enzymes were made to undergo self-sustained Darwinian evolution, resulting in the emergence of the most advantageous variants. These included both highly active enzymes that sustained the population as a whole and poorly active enzymes that survived as parasites of the active molecules. This evolutionary outcome was a consequence of the information capacity and fidelity of the genetic code, suggesting how these parameters should be adjusted to implement codes tailored to particular applications.

The C4'-oxidized abasic site (C4-AP) forms two types of interstrand cross-links with the adjacent nucleotides in DNA. Previous experiments revealed that dG does not react with the lesion and that formation of one type of cross-link is catalyzed by the opposing dA. iso-Guanosine·dC and 2-aminopurine·dT base pairs were used to determine why dG does not cross-link with C4-AP despite its well known reactivity with other bis-electrophiles. 7-Deaza-2'-deoxyadenosine was used to probe the role of the nucleotide opposite C4-AP in the catalysis of interstrand cross-link formation.

Here, a cross-chiral RNA polymerase is developed—an RNA enzyme that can catalyse the templated polymerization of activated mononucleotides that are of the opposite handedness—shedding light on how RNA-based life could have emerged. It is widely assumed that homochirality is a requirement for life and that biological macromolecules must be of the same stereochemical 'handedness' to interact efficiently. Working with Leslie Orgel and others, Gerald Joyce extended this idea in 1984 to suggest that homochirality may also be essential for the origins of life, as templated polymerization of RNA occurs readily in a homochiral system but is impaired in racemic mixtures. Now Joyce and co-author Jonathan Sczepanski show that RNAs of opposing chirality can work together. They devised a D-RNA enzyme that catalyses the polymerization of L-RNA on a L-RNA template — and vice versa. The catalytic potency of this ribozyme is sufficient for it to synthesize its own enantiomer by joining 11 component oligonucleotides. The ribozyme is thought to interact with its substrates via tertiary contacts rather than Watson–Crick base pairing. This unexpected finding will add a new dimension to thoughts on how life could have emerged in an 'RNA world'. Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer1. This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes before the origin of RNA-based life3,4. Once an RNA enzyme arose that could catalyse the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating d- and l-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyse the templated polymerization of activated mononucleotides of the opposite handedness. Here we develop such a cross-chiral RNA polymerase, using in vitro evolution starting from a population of random-sequence RNAs. The d-RNA enzyme, consisting of 83 nucleotides, catalyses the joining of l-mono- or oligonucleotide substrates on a complementary l-RNA template, and similar behaviour occurs for the l-enzyme with d-substrates and a d-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme’s activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides.

The C2′-oxidized abasic lesion (C2-AP) is produced in DNA that is subjected to oxidative stress. The lesion disrupts replication and gives rise to mutations that are dependent upon the identity of the upstream nucleotide. Ape1 incises C2-AP, but the 5′-phosphorylated fragment is not a substrate for the lyase activity of DNA polymerase β (Pol β). Excision of the lesion is achieved by strand displacement synthesis in the presence of flap endonuclease (FEN1) during which C2-AP and the 3′-adjacent nucleotide are replaced. The oxidized abasic lesion is also a substrate for the bacterial UvrABC nucleotide excision repair system. These data suggest that the redundant nature of DNA repair systems provide a means for removing a lesion that resists excision by short patch base excision repair.

The C4'-oxidized abasic site (C4-AP) is a commonly formed DNA lesion, which generates two types of interstrand cross-links (ICLs). The kinetically favored cross-link consists of two full length strands and forms reversibly and exclusively with dA. Cross-link formation is attributed to condensation of C4-AP with the N6-amino group of dA. Formation of the thermodynamic ICL involves cleavage of the strand containing C4-AP on the 3'-side of the lesion. The ratios and yields of the ICLs are highly dependent upon the local sequence. Product analysis of enzyme-digested material reveals that the ICL with dA is a cyclic adduct. Formation of the thermodynamically favored cross-link is catalyzed by the surrounding DNA sequence and occurs favorably with dC and dA but not with dG or dT. Mechanistic studies indicate that beta-elimination from C4-AP is the rate-limiting step in the formation of the thermodynamic ICL and that the local DNA environment determines the rate constant for this reaction. The efficiency of ICL formation, the stability of the thermodynamic products, and their possible formation in cells (Regelus, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14032) suggest that these lesions will be deleterious to the biological system in which they are produced.

Duplex DNA containing an apurinic/apyrimidinic (AP) lesion undergoes cleavage significantly more rapidly in nucleosome core particles (NCPs) than it does when free. The mechanism of AP cleavage within NCPs was studied through independently generating lesions within them. AP mediated DNA cleavage within NCPs is initiated by DNA-protein cross-link (DPC(un)) formation followed by β-elimination to give DPCs containing cleaved DNA (DPC(cl)). Hydrolysis of DPC(cl) produces a DNA single strand break (SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate-determining step. Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are involved in both DPC formation and β-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at superhelical location 1.5, but other amino acids (e.g., lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA–protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA–protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA–protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.

The DNA interstrand cross-link (ICL) resulting from the C4'-oxidized abasic site (C4-AP) is a unique clustered lesion comprised of a cross-link adjacent to a nick. The ICL is a substrate for the UvrABC nucleotide excision repair system. The strand containing the nick is preferentially incised, but the nick influences the cleavage sites. Moreover, in approximately 15% of the molecules, the strand opposite the nick is incised, resulting in a more toxic double-strand break. This is the first example in which an interstrand cross-link is converted by nucleotide excision misrepair into a more deleterious double-strand break.

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress. A recent report suggests that this lesion forms interstrand cross-links. Using duplexes in which C4-AP is produced from a synthetic precursor, we show that the lesion produces interstrand cross-links in which both strands are in tact and cross-links in which the C4-AP containing strand is cleaved. The yields of these products are dependent upon the surrounding nucleotide sequence. When C4-AP is opposed by dA, cross-link formation occurs exclusively with an adjacent dA on the 5'-side. Moreover, formation of the lower molecular weight cross-link is promoted by an opposing adenine. When the opposing dA is replaced by dT, the activity of the adenine can be rescued by adding the free base. This is a rare example in which DNA promotes its own modification, an observation that is all the more important because of the biological significance of the product produced.