Nucleic Acid Substrates Research Articles

Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination

The AID/APOBEC polynucleotide cytidine deaminases have historically been classified as either DNA mutators or RNA editors based on their first identified nucleic acid substrate preference. DNA mutators can generate functional diversity at antibody genes but also cause genomic instability in cancer. RNA editors can generate informational diversity in the transcriptome of innate immune cells, and of cancer cells. Members of both classes can act as antiviral restriction factors. Recent structural work has illuminated differences and similarities between AID/APOBEC enzymes that can catalyse DNA mutation, RNA editing or both, suggesting that the strict functional classification of members of this family should be reconsidered. As many of these enzymes have been employed for targeted genome (or transcriptome) editing, a more holistic understanding will help improve the design of therapeutically relevant programmable base editors.

Binding and compaction function of structural domains of SARS-CoV-2 nucleocapsid (N) protein on a single stranded nucleic acid substrate

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes for four major structural proteins. The nucleocapsid (N) protein is highly conserved among coronaviruses and is the only structural protein that binds the viral RNA packaged in the virion. One function of the N protein is the formation of a helical ribonucleoprotein (RNP) structure, which organizes and compacts the viral RNA inside the virion This process requires both nucleic-acid (NA) binding and interprotein interactions, which have been associated with the two structured N-terminal and C-terminal domains (NTD and CTD) within the N protein.

Monitoring helicase-catalyzed unwinding of multiple duplexes simultaneously.

Helicases catalyze the unwinding of duplex nucleic acids to aid a variety of cellular processes. Although helicases unwind duplex DNA in the same direction that they translocate on single-stranded DNA, forked duplexes provide opportunities to monitor unwinding by helicase monomers bound to each arm of the fork. The activity of the helicase bound to the displaced strand can be discerned alongside the helicase bound to the translocase strand using a forked substrate with accessible duplexes on both strands labeled with different fluorophores. In order to quantify the effect of protein-protein interactions on the activity of multiple monomers of the Bacteroides fragilis Pif1 helicase bound to separate strands of a forked DNA junction, an ensemble gel-based assay for monitoring simultaneous duplex unwinding was developed (Su et al., 2019). Here, the use of that assay is described for measuring the total product formation and rate constants of product formation of multiple duplexes on a single nucleic acid substrate. Use of this assay may aid characterization of protein-protein interactions between multiple helicase monomers at forked nucleic acid junctions and can assist with the characterization of helicase action on the displaced strand of forked duplexes.

Enhanced Triplex Hybridization of DNA and RNA via Syndiotactic Side Chain Presentation in Minimal bPNAs.

General design principles for recognition at noncanonical interfaces of DNA and RNA remain elusive. Triplex hybridization of bifacial peptide nucleic acids (bPNAs) with oligo-T/U DNAs and RNAs is a robust recognition platform that can be used to define structure-function relationships in synthetic triplex formation. To this end, a set of minimal (mw < 1 kD) bPNA variants was synthesized to probe the impact of amino acid secondary structural propensity, stereochemistry, and backbone cyclization on hybridization with short, unstructured T-rich DNA and U-rich RNAs. Thermodynamic parameters extracted from optical melting analyses of bPNA variant hybrids indicated that there are two bPNA backbone modifications that significantly improve hybridization: alternating (d, l) configuration in open-chain dipeptides and homochiral dipeptide cyclization to diketopiperazine. Further, binding to DNA is preferred over RNA for all bPNA variants. Thymine-uracil substitutions in DNA substrates revealed that the methyl group of thymine accounts for 71% of ΔΔGDNA-RNA for open-chain bPNAs but only 40% of ΔΔGDNA-RNA for diketopiperazine bPNA, suggesting a greater sensitivity to RNA conformation and more optimized stacking in the cyclic bPNA. Together, these data reveal pressure points for tuning triplex hybridization at the chiral centers of bPNA, backbone conformation, stacking effects at the base triple, and the nucleic acid substrate itself. A structural blueprint for enhancing bPNA targeting of both DNA and RNA substrates includes syndiotactic base presentation (as found in homochiral diketopiperazines and d, l peptides), expansion of base stacking, and further investigation of bPNA backbone preorganization.

Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes.

We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100-1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.

Colorimetric and electrochemical detection of ligase through ligation reaction-induced streptavidin assembly

We propose a concept for ligase detection by conversion of aggregation-based homogeneous analysis into surface-tethered electrochemical assay through streptavidin (SA)-biotin interaction. Sortase A (SrtA) served as the model analyte and two biotinylated peptides (bio-LPETGG and GGGK-bio) were used as the substrates. SrtA-catalyzed ligation of the peptide substrates led to the generation of bio-LPETGGGK-bio. The ligation product (bio-LPETGGGK-bio) induced the aggregation and color change of SA-modified gold nanoparticles (AuNPs) through the SA-biotin interactions, which could be assayed by the colorimetric method. Furthermore, we found that the bio-LPETGGGK-bio could trigger the assembly of tetrameric SA proteins with the formation of the (SA-bio-LPETGGGK-bio)n assemblies through the same interactions. The above results were further confirmed by atomic force microscopy and fluorescent imaging. The insulated assemblies were in-situ fabricated at the SA-modified gold electrode, thus hindering the electron transfer of [Fe(CN)6]3−/4− and leading to an increase in the electron-transfer resistance. The capability of the method for the detection of SrtA both in vitro and Staphylococcus aureus (S. aureus) has been demonstrated. SrtA with a concentration down to 1 pmol/L has been determined by the electrochemical analysis, which is lower than that achieved by the colorimetric assay (50 pmol/L). By integrating the advantages of homogeneous reaction and heterogeneous detection, the strategy serves as an ideal means for the fabrication of various sensing platforms by adopting biotin-labeled and sequence-specific peptide or nucleic acid substrates.

Probing DNA-protein interactions using single-molecule diffusivity contrast

Single-molecule fluorescence investigations of protein-nucleic acid interactions require robust means to identify the binding state of individual substrate molecules in real time. Here, we show that diffusivity contrast, widely used in fluorescence correlation spectroscopy at the ensemble level and in single-particle tracking on individual (but slowly diffusing) species, can be used as a general readout to determine the binding state of single DNA molecules with unlabeled proteins in solution. We first describe the technical basis of drift-free single-molecule diffusivity measurements in an anti-Brownian electrokinetic trap. We then cross-validate our method with protein-induced fluorescence enhancement, a popular technique to detect protein binding on nucleic acid substrates with single-molecule sensitivity. We extend an existing hydrodynamic modeling framework to link measured diffusivity to particular DNA-protein structures and obtain good agreement between the measured and predicted diffusivity values. Finally, we show that combining diffusivity contrast with protein-induced fluorescence enhancement allows simultaneous mapping of binding stoichiometry and location on individual DNA-protein complexes, potentially enhancing single-molecule views of relevant biophysical processes.

Writing a review on trapping RNase substrate complexes for structural studies as an undergraduate researcher

We conducted a major survey of ribonuclease (RNase) substrate complex structures to identify strategies used to inhibit cleavage of the RNA. This review is intended to serve as a guide for structural biologists interested in discovering mechanisms and functions of ribonucleases through structural studies. We searched the Protein Data Bank for RNase structures which include both RNase and nucleic acid substrate and vetted the results for structures in which the substrate remained intact. Then we organized the structures by category based on the method used for inhibition. Each of the methods include their owns pros and cons and often it is advantageous to use a mix of different methods to achieve inhibition. In addition to surveying current strategies, we identify some ideas for future directions in the field based on what we have learned.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR–Cas systems. Acrs usually block the ability of CRISPR–Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR–Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR–Cas activity in vivo. We conclude that misdirecting the CRISPR–Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.

High-yield purification of exceptional-quality, single-molecule DNA substrates

Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential anti-fungal targets.

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2′-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2′-PO4 attacks NAD+ to form an RNA-2′-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential antifungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1Δ mutation and in vitro in NAD+-dependent conversion of a 2′-PO4 to a 2′-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD+, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2′-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2′-PO4 nucleotide with arabinose-2′-PO4 diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2′-phospho-ADP-ribosylated intermediate. Our results implicate the 2′-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.

Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

ALKBH1 promotes lung cancer by regulating m6A RNA demethylation

Lung cancer has surpassed breast cancer as the leading cause of cancer death in females in developed countries and the leading cause of cancer death in males. Despite extensive research on lung cancer, the pathogenesis of lung cancer is not fully understood. ALKBH1 is a 2-oxoglutarate and Fe (II)-dependent dioxygenase responsible for the demethylation of 6-methyladenine (m6A) in RNA and is essential to multiple cellular processes in human. Numerous recent studies suggest that ALKBH1 plays a role in tumorigenesis and tumor progression, but the role of ALKBH1 in lung cancer is largely unknown. In this study, we demonstrated that the expression levels of ALKBH1 in lung cancer tissues and cells were up regulated. The invasion and migration abilities of lung cancer cells were significantly suppressed in vitro upon the silencing of ALKBH1 while they were significantly promoted upon its overexpression. We next characterized the enzyme biochemically by analyzing the contribution of essential residues Y184, H231, D233, H287, R338, and R344 to its m6A demethylation activity. Lastly, our 3.1-Å crystal structure of mouse ALKBH1 revealed that the N-terminal domain of the protein forms close contacted with the core catalytic domain and might be responsible for the recognition of nucleic acid substrates. In summary, our combined cellular, biochemical, and structural results provide insight into the potential ALKBH1-based drug design for cancer therapies.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

Programmed Allelic Mutagenesis of a DNA Polymerase with Single Amino Acid Resolution.

Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

The linker region of LINEs modulates DNA cleavage and DNA polymerization.

Long interspersed elements (LINEs) replicate by target primed reverse transcription (TPRT). Insertion involves two half reactions. Each half reaction involves DNA cleavage followed by DNA synthesis. The linker region, located just beyond the reverse transcriptase in the LINE open reading frame, contains a set of predicted helices that may form an α-finger, followed by a gag-like zinc-knuckle. Point mutations of moderately conserved amino-acid residues in the presumptive α-finger severely impair the DNA endonuclease and reverse transcriptase activities of the integration reaction during both half reactions. Mutations in the gag-like zinc-knuckle also impair DNA cleavage and DNA synthesis in some instances. Mutations in core residues that presumably disrupt the protein structure of the presumptive α-finger and the gag-like zinc-knuckle lead to a promiscuous DNA endonuclease and protein-nucleic acid complexes that get stuck in the well during analysis. The linker region appears to function as a protein, DNA, and RNA conformational switching area. The linker is used to properly position nucleic acid substrates into the active sites of the reverse transcriptase and of the DNA endonuclease.

The T4 TerL Prohead Packaging Motor Does Not Drive DNA Translocation by a Proposed Dehydration Mechanism.

A “DNA crunching” linear motor mechanism that employs a grip-and-release transient spring like compression of B- to A-form DNA has been found in our previous studies. Our FRET measurements in vitro show a decrease in distance from TerL to portal during packaging; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem of translocating Y-DNA that conforms to B- and A- structure. In normal translocation into the prohead the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The TerL motor cannot package A-form dsRNA. Our work reported here shows that addition of helper B form DNA:DNA (D:D) 20mers allows increased packaging of heteroduplex A-form DNA:RNA 20mers (D:R), evidence for a B- to A-form spring motor pushing duplex nucleic acid. A-form DNA:RNA 25mers, 30mers, and 35mers alone are efficiently packaged into proheads by the TerL motor showing that a proposed hypothetical dehydration motor mechanism operating on duplex substrates does not provide the packaging motor force. Taken together with our previous studies showing TerL motor protein motion toward the portal during DNA packaging, our present studies of short D:D and D:R duplex nucleic acid substrates strongly supports our previous evidence that the protein motor pushes rather than pulls or dehydrates duplex substrates to provide the translocation into prohead packaging force.

Single-Molecule Studies of HIV-1 Gag Assembly

Gag is the key structural protein that mediates assembly of nascent HIV-1 virions around viral genomic RNA. Previous studies have established that Gag can spontaneously assemble around a variety of nucleic acid substrates into immature virus-like particles in vitro. Additionally, substrates which include the Psi signal, a small structural element found in the 5’ UTR of the HIV-1 genome, are selectively packaged over other substrates. In fact, the Psi signal is required for selective packaging of the genomic RNA over the large background of other RNAs in the cellular milieu. However, the mechanism of this assembly process and interactions with the Psi signal that lead to specificity are poorly understood. Here we have developed a single-molecule total internal reflection fluorescence microscopy assay to monitor the earliest steps of particle assembly under controlled conditions. Individual fluorescently-labeled Gag monomers are resolved as they spontaneously bind to and dissociate from immobilized RNA substrates derived from the genomic 5’ UTR. Statistical analysis of multiple single-molecule time trajectories allowed for the determination of the relative populations of various assembly intermediates along with rate constants for individual binding and dissociation steps. Experiments were performed under a variety of conditions including varied protein concentrations, mutations to Gag and the RNA substrates, and with proposed small-molecule cofactors present. Taken together, the results from these experiments elucidate details of molecular interactions involved in the initiation of particle assembly that may be opaque to other methodologies.

Substrate analogs that trap the 2′-phospho-ADP-ribosylated RNA intermediate of the Tpt1 (tRNA 2′-phosphotransferase) reaction pathway

The enzyme Tpt1 removes an internal RNA 2′-PO4 via a two-step reaction in which: (i) the 2′-PO4 attacks NAD+ to form an RNA-2′-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2′-phosphodiester yields 2′-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Because step 2 is much faster than step 1, the ADP-ribosylated RNA intermediate is virtually undetectable under normal circumstances. Here, by testing chemically modified nucleic acid substrates for activity with bacterial Tpt1 enzymes, we find that replacement of the ribose-2′-PO4 nucleotide with arabinose-2′-PO4 selectively slows step 2 of the reaction pathway and results in the transient accumulation of high levels of the reaction intermediate. We report that replacing the NMN ribose of NAD+ with 2′-fluoroarabinose (thereby eliminating the ribose O2″ nucleophile) results in durable trapping of RNA-2′-phospho-(ADP-fluoroarabinose) as a “dead-end” product of step 1. Tpt1 enzymes from diverse taxa differ in their capacity to use ara-2″F-NAD+ as a substrate.

Argonaute bypasses cellular obstacles without hindrance during target search

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.