Unusual Nucleic Acid Structures Research Articles

Last year at Marienbad: Unusual nucleic acid structures

Last year at Marienbad: Unusual nucleic acid structures

Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures

Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1, encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS is unknown, nor is it understood why some, but not all expanded motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, while (AAAAG)n•(CTTTT)n is the most common non-pathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic form unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Non-pathogenic repeats did not form G-quadruplexes. Triple-stranded structures formed by the pathogenic motifs, but not by the non-pathogenic motifs. G- and C-richness of the pathogenic strands, (AAGGG)n•(CCCTT)n, favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, each biophysically stabilized by increased hydrogen-bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the non-pathogenic motif (AAAAG)n•(CTTTT)n. The quadruplex ligand, TMPyP4 binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.

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GENE THERAPY Reactions ShareShare onFacebookTwitterWechatLinked InRedditEmail C&EN, 2023, 101 (18), p 3June 5, 2023Cite this:C&EN 101, 18, 3(Credit: C&EN)Figure1of2(Credit: C&EN)Figure1of2Letters to the editorPeptide nucleic acids targeting noncanonical DNA and RNAAs a molecular biologist, I always enjoy reading articles concerning DNA and RNA. Your excellent article in the April 3, 2023, issue of C&EN (page 18) titled “The Promise of Peptide Nucleic Acids” discussed an important topic relating to gene therapy—that is, antisense therapeutics and gene- editing tools. Peptide nucleic acids (PNAs) can open a double-stranded (ds) DNA molecule and inhibit its gene expression. PNAs bind to DNA and RNA with high specificity and selectivity, resulting in PNA-DNA and PNA-RNA hybrid structures. Therefore, they have exciting potential as diagnostic tools and therapeutic agents.However, only briefly discussed in the article is the possibility of developing PNAs to target nucleic acid secondary structures such as RNA hairpins. More focus needs to be placed on all the other nucleic acid secondary structures, going beyond canonical right-handed ds-B-DNA, right-handed ds-A-RNA, and single-stranded (such as messenger RNA) nucleic acids based on nucleotide sequences—that is, the hybridization of base pairs.Researchers need to consider targeting exotic, noncanonical nucleic acids—that is, alternative and multistranded DNA and RNA structures. Local transitions in the structure of canonical ds-B-DNA are functionally significant biological molecules, regulating many cellular processes, such as transcription and translation. These exotic nucleic acid structures have also been connected to genetic instability in the presence or deficiency of DNA damage, with or without DNA replication. DNA and RNA possess dynamic structural variability resulting in a variety of unusual nucleic acid structures, such as cruciform DNA, DNA-RNA hybrids, slipped-strand DNA, and parallel-stranded DNA.Expanding the therapeutic capabilities of PNAs beyond canonical B-DNA would expand PNAs’ use in therapeutics. The expansion should include alternative ds-DNA structures such as left-handed ds-Z-DNA and left-handed ds-Z-RNA. It should also include multistranded DNA structures such as triplex DNA (intermolecular and intramolecular, like H-DNA), G4-quadruplex DNA, and i-motif DNA.If possible, targeting noncanonical nucleic acid structures with PNAs would increase the chance of developing novel therapeutics that can target all genomic DNA and RNA structures in genes and telomeres. We are witnessing the transition of gene medicine from an experimental technology into a real-world, practical approach for developing nucleic acid–based therapeutics, such as used for gene silencing or gene editing (like CRISPR) of pathological conditions.Using PNAs as therapeutic tools for targeting both canonical and noncanonical nucleic acids will allow for their full potential and optimal expansion of gene therapy.Claude GagnaBronxville, New YorkRecognizing nitrogen-fixing bacteria’s potential to reduce fertilizer useIn the article titled “Growing More Food with Less Chemicals,” which appeared in the May 8, 2023, issue of C&EN (page 28), there was a glaring omission.What was left out was the research and development of genetically modified nitrogen-fixing bacteria that can be applied to seed, foliage, or soil to reduce fertilizer use.Herbert B. ScherMoraga, California

An ELISA-based platform for rapid identification of structure-dependent nucleic acid–protein interactions detects novel DNA triplex interactors

Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)–mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure–protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide–induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair.

G-quadruplexes in helminth parasites.

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

Pathogenic mutations reveal a role of RECQ4 in mitochondrial RNA:DNA hybrid formation and resolution

The synthesis of mitochondrial DNA (mtDNA) is a complex process that involves the formation and resolution of unusual nucleic acid structures, such as RNA:DNA hybrids. However, little is known about the enzymes that regulate these processes. RECQ4 is a DNA replication factor important for mtDNA maintenance, and here, we unveil a role of human RECQ4 in regulating the formation and resolution of mitochondrial RNA:DNA hybrids. Mitochondrial membrane protein p32 can block mtDNA synthesis by restricting RECQ4 mitochondrial localization via protein–protein interaction. We found that the interaction with p32 was disrupted not only by the previously reported cancer-associated RECQ4 mutation, del(A420-A463), but also by a clinical mutation of the adjacent residue, P466L. Surprisingly, although P466L mutant was present in the mitochondria at greater levels, unlike del(A420-A463) mutant, it failed to enhance mtDNA synthesis due to the accumulation of RNA:DNA hybrids throughout the mtDNA. Biochemical analysis revealed that P466L mutation enhanced RECQ4 annealing activity to generate RNA:DNA hybrids at the same time reduced its unwinding activity to resolve this structure. Hence, P466L mutation led to a reduced efficiency in completing mtDNA synthesis due to unresolved RNA:DNA hybrids across mtDNA.

Interaction with single-stranded DNA-binding protein localizes ribonuclease HI to DNA replication forks and facilitates R-loop removal.

DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R-loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R-loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R-loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction between Escherichia coli RNase HI and the single-stranded DNA-binding protein (SSB) in this process. Interaction with SSB localizes RNase HI foci to DNA replication sites. Mutation of rnhA to encode an RNase HI variant that cannot interact with SSB but that maintains enzymatic activity (rnhAK60E) eliminates RNase HI foci. The mutation also produces a media-dependent slow-growth phenotype and an activated DNA damage response in cells lacking Rep helicase, which is an enzyme that disrupts stalled transcription complexes. RNA polymerase variants that are thought to increase or decrease R-loop accumulation enhance or suppress, respectively, the growth phenotype of rnhAK60E rep::kan strains. These results identify a cellular role for the RNase HI/SSB interaction in helping to clear R-loops that block DNA replication.

Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair.

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

Orienting tetramolecular G-quadruplex formation: the quest for the elusive RNA antiparallel quadruplex.

DNA and RNA G-quadruplexes (G4) are unusual nucleic acid structures involved in a number of key biological processes. RNA G-quadruplexes are less studied although recent evidence demonstrates that they are biologically relevant. Compared to DNA quadruplexes, RNA G4 are generally more stable and less polymorphic. Duplexes and quadruplexes may be combined to obtain pure tetrameric species. Here, we investigated whether classical antiparallel duplexes can drive the formation of antiparallel tetramolecular quadruplexes. This concept was first successfully applied to DNA G4. In contrast, RNA G4 were found to be much more unwilling to adopt the forced antiparallel orientation, highlighting that the reason RNA adopts a different structure must not be sought in the loops but in the G-stem structure itself. RNA antiparallel G4 formation is likely to be restricted to a very small set of peculiar sequences, in which other structural features overcome the formidable intrinsic barrier preventing its formation.

Investigation of mRNA quadruplex formation in Escherichia coli

The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

Predictable suppression of gene expression by 5′-UTR-based RNA quadruplexes

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure–function relationship of synthetic quadruplex sequences in 5′-UTRs in multiple mammalian cell-lines. We establish a universal ‘translational suppressor’ effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of ‘GGG’-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

Design, Synthesis, and Evaluation of Novel Biarylpyrimidines: A New Class of Ligand for Unusual Nucleic Acid Structures

Biarylpyrimidines are characterized as selective ligands for higher-order nucleic acid structures. A concise and efficient synthesis has been devised incorporating Suzuki biaryl cross-coupling of dihalopyrimidines. Two ligand series are described based on the parent thioether 4,6-bis[4-[[2-(dimethylamino)ethyl]mercapto]phenyl]pyrimidine (1a) and amide 4,6-bis(4[(2-(dimethylamino)ethyl)carboxamido]phenyl)pyrimidine (2a) compounds. In UV thermal denaturation studies with the poly(dA) x [poly(dT)]2 triplex structure, thioethers showed stabilization of the triplex form (Delta Tm < or = 20 degrees C). In contrast, amides showed duplex stabilization (Delta Tm < or = 15 degrees C) and either negligible stabilization or specific destabilization (Delta Tm = -5 degrees C) of the triplex structure. Full spectra of nucleic acid binding preferences were determined by competition dialysis. The strongest interacting thioether bound preferentially to the poly(dA) x [poly(dT)]2 triplex, K(app) = 1.6 x 10(5) M(-1) (40 x K(app) for CT DNA duplex). In contrast, the strongest binding amide selected the (T2G20T2)4 quadruplex structure, K(app) = 0.31 x 10(5) M(-1) (6.5 x K(app) for CT DNA duplex).

Simulation and modeling of nucleic acid structure, dynamics and interactions

In moving towards the simulation of larger nucleic acid assemblies over longer timescales that include more accurate representations of the environment, we are nearing the end of an era characterized by single nanosecond molecular dynamics simulation of nucleic acids. We are excited by the promise and predictability of the modeling methods, yet remain prudently cautious of sampling and force field limitations. Highlights include the accurate representation of subtle drug–DNA interactions, the detailed study of modified and unusual nucleic acid structures, insight into the influence of dynamics on the structure of DNA, and exploration of the interaction of solvent and ions with nucleic acids.

INCORPORATION OF 8-HISTAMINYL-DEOXYADENOSINE [8-(2-(4-IMIDAZOLYL)ETHYLAMINO)-2′-DEOXYRIBOADENOSINE] INTO OLIGODEOXYRIBONUCLEOTIDES BY SOLID PHASE PHOSPHORAMIDITE COUPLING

ABSTRACT The 3′phosphoramidite of 8-histaminyl deoxyadenosine has been prepared and successfully incorporated into a short oligodeoxyribonucleotide. The synthetic methodology leading to this preparation is given and the implications for developing new DNAzymes as well as probing unusual nucleic acid structures are discussed.

Zipper-like Watson-Crick base-pairs

A series of DNA heptadecamers containing the DNA analogues of RNA E-like 5′-d(GXA)/(AYG)-5′ motifs (X/Y is complementary T/A, A/T, C/G, or G/C pair) were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers reveal excellent resolution in NMR spectra and exhibit many unusual nuclear Overhauser effects (NOEs) that allow for good characterization of an unusual zipper-like conformation with zipper-like Watson-Crick base-pairs; the potential canonical X · Y H-bonding is not present, and the central X/Y pairs are transformed instead into inter-strand stacks that are bracketed by sheared G · A base-pairs. Such phenomenal structural change is brought about mainly through two backbone torsional angle adjustments, i.e. δ from C2′-endo to C3′-endo for the sugar puckers of unpaired residues and γ from gauche+ to trans for the following 3′-adenosine residues. Such motifs are analogous to the previously studied (GGA)2 motif presumably present in the human centromeric (TGGAA)n tandem repeat sequence. The novel zipper-like motifs are only 4–7 deg. C less stable than the (GGA)2 motif, suggesting that inter-strand base stacking plays an important role in stabilizing unusual nucleic acid structures. The discovery that canonical Watson-Crick G · C or A · T hydrogen-bonded pairs can be transformed into stacking pairs greatly increases the repertoire for unusual nucleic acid structural motifs.

NMR Studies of DNA Structures Containing Sheared Purine•Purine and Purine•Pyrimidine Base Pairs

Summary Sheared purine•purine or purine•pyrimidine base pairs are important motifs in nucleic acid structures. They can exist either as tandem base pairs or as a single base pair closing hairpin mini-loops. Presence of such stable motifs greatly increases the structural repertoire of nucleic acid molecules and may play important roles in the contemporary nucleic acid studies. Several RNA crystal structures containing adjacent cross-strand base stacking have been solved. In this review, I will discuss the NMR studies and structural features for DNA molecules containing sheared G•A, A•A, or A•C base pairs. Special attention will be focused on exchangeable proton data, which have turned out to be crucial to the determination of unusual nucleic acid structures. The special features of the partial HIV-1 RT 21-mer act as a good example for correlating the 31P chemical shifts to the ζ and α torsional angles of the backbone phosphate diesters.

NAMOT2--a redesigned nucleic acid modeling tool: construction of non-canonical DNA structures.

Using a new set of reduced coordinates developed for describing regular and unusual nucleic acid structures, we have revised our nucleic acid modeling tool NAMOT2. NAMOT2 is general in terms of modeling different nucleic acid structures. A set of modifiable libraries allows users to customize their modeling environment. With this set of libraries, NAMOT2 can be used to model non-canonical structures such as parallel-stranded, triple-stranded and quadruple-stranded nucleic acid molecules. For modeling irregular structures (junctions, hairpin loops, etc.), we introduce a structural recipe approach. The complete procedure using NAMOT2 to construct the structure of a specific molecule is treated as the recipe for that structural motif. The existing recipes can be modified to generate new recipes for different structural motifs. Several examples of nucleic acids with non-canonical structures were modeled using NAMOT2. These examples include a DNA-drug complex, a DNA cube, a six-arm junction and a curved DNA molecule.

DNA Sequence GCGAATGAGC Containing the Human Centromere Core Sequence GAAT Forms a Self-complementary Duplex with Sheared G·A Pairs in Solution

The DNA sequence dGCGAATGAGC has a well-resolved, two-dimensional nuclear Overhauser (NOESY) spectrum that is suitable for high quality solution structure determination by NMR methods; in solution this sequence from a stable self-complementary duplex containing sheared G·A base-pairs. A total of 220 distance constraints derived from time-dependent NOE measurements were collected and refined by repeated back-calculation of the NOESY spectra. Distance information from imino proton studies and from exclusive two-dimensional correlated spectroscopy (E. COSY) and/or linewidth analysis was included in the structure calculation using the program DSPACE 4·2, followed by restrained energy minimization with the program DISCOVER using the AMBER force field. The energies of the distance geometry (DG) structures decreased rapidly in the first few cycles and approached - 510 ± 3 keal after 1000 cycles of conjugate gradient minimization (about 540 keal lower than in the initial DG structures). All 15 final DG structures converged to a single family of closely related structures with pair-wise r.m.s.d. values of 0·96±0·34 Å which was further reduced by energy minimization to 0·70±0·35 Å. Rather unusual structural features of the duplex are revealed in the final structures. The results indicate that, in addition to normal sequences with standard base-pairing, unusual nucleic acid structures can also be determined in solution with quite high precision by NMR/distance geometry methods.

Chemical Probe for Glycosidic Conformation in Telomeric DNAs

Chemical modifications are powerful tools to probe nucleic acid structure and protein-nucleic acid interactions. Nucleic acids can be modified either randomly by a variety of chemical reagents or site-specifically with synthetic base analog substitutions. We report here the use of 8-bromodeoxyguanosine (8-Br-dG) as a modification that is directly sensitive to a dihedral angle. 8-Br-dG should prove useful as a chemical probe for the conformation about glycosidic bonds in unusual nucleic acid structures.

Formation of multiple complexes between actinomycin D and a DNA hairpin: structural characterization by multinuclear NMR.

The solution conformations of a DNA oligomer and its complexes with the anticancer drug actinomycin D (ActD) were characterized using homo- and heteronuclear NMR techniques. Previous high-resolution NMR investigations of ActD-DNA complexes employed symmetric double-stranded DNA oligomers, yielding two identical symmetry-related complexes. In order to understand the important effects that neighboring base pairs and/or unusual nucleic acid structures may have on ActD binding specificity and orientation, we chose to study the oligonucleotide d(TCGCGTTTTCGCGA), which adopts a hairpin structure in solution. NOE cross-peak intensities were used to generate distance constraints for molecular dynamics simulations and structure determinations of the free oligonucleotide and for both complexes. A total of 86 intermolecular NOEs were identified for each complex, 27 of which involve exchangeable protons. These intermolecular NOEs along with changes in the phosphorus chemical shifts were used to determine the drug binding site on the DNA. As expected, ActD intercalated exclusively at the single d(GC) step in the DNA hairpin. Interestingly, although the two complexes, which differ by the orientation with which the asymmetric drug chromophore intercalates the DNA, were not formed in equal concentrations, their conformations are very similar. The RMS difference of the DNA hairpin in the two complexes is only 1.10 A. The structures of the minor groove binding pentapeptide rings are not affected by any of the changes in the normal double-helical structure imposed by the hairpin loop. The total pairwise RMS difference over all atoms for the four peptides (two per complex) in the calculated structures is 0.72 A. Conversely, the structure of the hairpin loop is not appreciably changed upon binding--the RMS difference between the free DNA loop region and the loop region in the two complexes is 1.68 A and only 0.43 A between the two complexes. Our data also support a possible conformation of the d(T)4 loop that does not possess a thymine-thymine "wobble" base pair.