Watson-Crick Bonds Research Articles

Random words in free groups, non-crossing matchings and RNA secondary structures

Consider a random word $$X^n=(X_1,\ldots ,X_n)$$ in an alphabet consisting of 4 letters, with the letters viewed either as A, U, G and C (i.e., nucleotides in an RNA sequence) or $$\alpha $$ , $$\bar{\alpha }$$ , $$\beta $$ and $$\bar{\beta }$$ (i.e., generators of the free group $$\langle \alpha ,\beta \rangle $$ and their inverses). We show that the expected fraction $$\rho (n)$$ of unpaired bases in an optimal RNA secondary structure (with only Watson-Crick bonds and no pseudo-knots) converges to a constant $$\lambda _2$$ with $$0<\lambda _2<1$$ as $$n\rightarrow \infty $$ . Thus, a positive proportion of the bases of a random RNA string do not form hydrogen bonds. We do not know the exact value of $$\lambda _2$$ , but we derive upper and lower bounds for it. In terms of free groups, $$\rho (n)$$ is the ratio of the length of the shortest word representing X in the generating set consisting of conjugates of generators and their inverses to the word length of X with respect to the standard generators and their inverses. Thus for a typical word the word length in the (infinite) generating set consisting of the conjugates of standard generators grows linearly with the word length in the standard generators. In fact, we show that a similar result holds for all non-abelian finitely generated free groups $$\langle \alpha _1,\dots ,\alpha _k\rangle $$ , $$k\ge 2$$ .

Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology.

Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.

Real-Time Monitoring of the Multistate Conformational Dynamics of Polypurine Reverse Hoogsteen Hairpin To Capture Their Triplex-Affinity for Gene Silencing by smFRET Microspectroscopy.

Single-molecule Förster resonance energy transfer microspectroscopic methods are employed for real-time monitoring and to gain deeper insights into the formation of the polypurine reverse Hoogsteen hairpin (PPRH) and its triplex-forming activity. The heterogeneity in the behavior of individual PPRHs has been documented, and it is seen that the degree of anharmonic plasticity of the antiparallel hairpin is stabilized by the formation of reverse Hoogsteen (RH) bonds. While being involved in the hairpin formation, they flip reversibly between the open and closed conformations, irrespective of the concentration of ions present in their microenvironment. However, the nature of the cation present in the buffer plays a crucial role in determining the structural stability. The Watson Crick (WC) bonds are found to be more dynamic in the triplex compared to that of the RH base pairs, indicating the involvement of progressive WC bonds during the triplex motif formation by the PPRH. The majority of the intact triplex DNA attained a semifolded relaxed state before progressing toward a tightly folded state, emphasizing the fact that the folding mechanism pursues an ambiguous path in the mode of acquiring the final step of the triple helix motif. Moreover, the presence of triplex-forming sequences in the regulatory regions of the genome further provides an intricate link between the experimental results and sequence occurrence.

Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited transcriptomes.

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.

COMBO-FISH: A Versatile Tool Beyond Standard FISH to Study Chromatin Organization by Fluorescence Light Microscopy

Background: Fluorescence In Situ Hybridization (FISH) has become routine for bio-medical research and medical diagnosis, thereby offering a variety of probes and ready-to-use kits that fulfil requirements for many applications. However, conventional FISH relies on chemo- and/or thermal denaturation to improve target accessibility and use huge amounts of DNAs that needs to be bonded to the target site. COMBinatorial Oligo-nucleotide FISH (COMBO-FISH) offers possibilities to circumvent these shortcomings. Methods: COMBO-FISH uses either a set of oligo-nucleotide probes (15 – 25 mers) uniquely co-localizing at the target site or single oligo-stretches repetitively but exclusively binding to the target. These are designed by systematic sequence data base searches. COMBO-FISH probes form Hoogsteen or Watson-Crick bonds and protocols with or without thermal denaturation can be realized. The latter allows the combination of COMBO-FISH with immunostaining. Low amounts of probe DNA allow the best maintenance of native chromatin organization – a prerequisite for applying super-resolution single molecule localization microscopy (SMLM). Results: Specific labelling of the AMACR gene with an oligo-nucleotide probe set was applied in three different cell types. Hybridization efficiencies were determined by counting spots of high visibility and their radial positions were measured by confocal microscopy. The nuclear architecture revealed a non-random organization. Using uniquely, repetitively binding COMBO-FISH probes for centromere 9, Alu consensus and L1 sequences revealed a characteristic probe distribution in cell nuclei as being measured by SMLM. Comparison to theoretical data allowed determination of chromosome 9 radial positions without painting whole chromosomes. Three-colour staining by COMBO-FISH for Alu and L1 with immunostaining for heterochromatin is successfully demonstrated. Conclusions: COMBO-FISH is a powerful tool that circumvents shortcomings of standard FISH procedures using probes derived e.g., from BAC clones. The application for measurements of nuclear architecture by 3D confocal microscopy and of chromatin nano-architecture by SMLM using COMBO-FISH and immunostaining simultaneously has been demonstrated.

Polypurine Reverse Hoogsteen Hairpins as a Gene Silencing Tool for Cancer.

Polypurine reverse Hoogsteen (PPRH) molecules are DNA hairpins formed by two polypurine strands running in an antiparallel orientation and containing no nucleotide modifications. The two strands, linked by a pentathymidine loop, are bound through intramolecular reverse Hoogsteen bonds. Then, PPRHs can bind by Watson-Crick bonds to their corresponding polypyrimidine target in the dsDNA provoking a displacement of the polypurine strand of the duplex. We described the effect and mechanisms of action of PPRHs in cells using PPRHs designed against the template and coding strands of the dhfr gene. The proof of principle of PPRHs as a therapeutic tool was established using a PPRH against survivin in a xenograft prostate cancer tumor model. To improve the PPRHs effect, the influence of the length was studied obtaining a higher efficiency with longer molecules. To decrease the possible offtarget effect, when a purine interruption is found in the pyrimidine target, the PPRH sequence should contain both strands of the complementary base opposite to the interruption. Furthermore, the stability of PPRHs is higher than that of siRNAs, as evidenced by the longer halflife of the former in different types of serum and in PC3 cells. PPRHs do not induce the levels of the transcription factors nor the proinflammatory cytokines involved in the Toll-like Receptor pathway and they do not trigger the formation of the inflammasome complex. PPRHs can be used as therapeutic tools to target genes related to cancer progression, resistance to drugs or immunotherapy approaches.

Polypurine reverse-Hoogsteen (PPRH) oligonucleotides can form triplexes with their target sequences even under conditions where they fold into G-quadruplexes

Polypurine reverse-Hoogsteen (PPRH) oligonucleotides are non-modified DNA molecules composed of two mirror-symmetrical polypurine stretches linked by a five-thymidine loop. They can fold into reverse-Hoogsteen hairpins and bind to their polypyrimidine target sequence by Watson-Crick bonds forming a three-stranded structure. They have been successfully used to knockdown gene expression and to repair single-point mutations in cells. In this work, we provide an in vitro characterization (UV and fluorescence spectroscopy, gel electrophoresis and nuclease assays) of the structure and stability of two repair-PPRH oligonucleotides and of the complexes they form with their single-stranded targets. We show that one PPRH oligonucleotide forms a hairpin, while the other folds, in potassium, into a guanine-quadruplex (G4). However, the hairpin-prone oligonucleotide does not form a triplex with its single-stranded target, while the G4-prone oligonucleotide converts from a G4 into a reverse-Hoogsteen hairpin forming a triplex with its target sequence. Our work proves, in particular, that folding of a PPRH oligonucleotide into a G4 does not necessarily impair sequence-specific DNA recognition by triplex formation. It also illustrates an original example of DNA structural conversion of a G4 into a reverse-Hoogsteen hairpin driven by triplex formation; this kind of conversion might occur at particular loci of genomic DNA.

The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo.

RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson–Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.

Effect of Polypurine Reverse Hoogsteen Hairpins on Relevant Cancer Target Genes in Different Human Cell Lines.

We studied the ability of polypurine reverse Hoogsteen hairpins (PPRHs) to silence a variety of relevant cancer-related genes in several human cell lines. PPRHs are hairpins formed by two antiparallel polypurine strands bound by intramolecular Hoogsteen bonds linked by a pentathymidine loop. These hairpins are able to bind to their target DNA sequence through Watson-Crick bonds producing specific silencing of gene expression. We designed PPRHs against the following genes: BCL2, TOP1, mTOR, MDM2, and MYC and tested them for mRNA levels, cytotoxicity, and apoptosis in prostate, pancreas, colon, and breast cancer cell lines. Even though all PPRHs were effective, the most remarkable results were obtained with those against BCL2 and mammalian target of rapamycin (mTOR) in decreasing cell survival and mRNA levels and increasing apoptosis in prostate, colon, and pancreatic cancer cells. In the case of TOP1, MDM2, and MYC, their corresponding PPRHs produced a strong effect in decreasing cell viability and mRNA levels and increasing apoptosis in breast cancer cells. Thus, we confirm that the PPRH technology is broadly useful to silence the expression of cancer-related genes as demonstrated using target genes involved in metabolism (DHFR), proliferation (mTOR), DNA topology (TOP1), lifespan and senescence (telomerase), apoptosis (survivin, BCL2), transcription factors (MYC), and proto-oncogenes (MDM2).

The Role of Aromaticity, Hybridization, Electrostatics, and Covalency in Resonance-Assisted Hydrogen Bonds of Adenine-Thymine (AT) Base Pairs and Their Mimics.

Hydrogen bonds play a crucial role in many biochemical processes and in supramolecular chemistry. In this study, we show quantum chemically that neither aromaticity nor other forms of π assistance are responsible for the enhanced stability of the hydrogen bonds in adenine–thymine (AT) DNA base pairs. This follows from extensive bonding analyses of AT and smaller analogs thereof, based on dispersion-corrected density functional theory (DFT). Removing the aromatic rings of either A or T has no effect on the Watson–Crick bond strength. Only when the smaller mimics become saturated, that is, when the hydrogen-bond acceptor and donor groups go from sp2 to sp3, does the stability of the resulting model complexes suddenly drop. Bonding analyses based on quantitative Kohn–Sham molecular orbital theory and corresponding energy decomposition analyses (EDA) show that the stronger hydrogen bonds in the unsaturated model complexes and in AT stem from stronger electrostatic interactions as well as enhanced donor–acceptor interactions in the σ-electron system, with the covalency being responsible for shortening the hydrogen bonds in these dimers.

Repair of single-point mutations by polypurine reverse Hoogsteen hairpins.

Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.

Two-phase stretching of molecular chains.

Although stretching of most polymer chains leads to rather featureless force-extension diagrams, some, notably DNA, exhibit nontrivial behavior with a distinct plateau region. Here, we propose a unified theory that connects force-extension characteristics of the polymer chain with the convexity properties of the extension energy profile of its individual monomer subunits. Namely, if the effective monomer deformation energy as a function of its extension has a nonconvex (concave up) region, the stretched polymer chain separates into two phases: the weakly and strongly stretched monomers. Simplified planar and 3D polymer models are used to illustrate the basic principles of the proposed model. Specifically, we show rigorously that, when the secondary structure of a polymer is mostly caused by weak noncovalent interactions, the stretching is two phase, and the force-stretching diagram has the characteristic plateau. We then use realistic coarse-grained models to confirm the main findings and make direct connection to the microscopic structure of the monomers. We show in detail how the two-phase scenario is realized in the α-helix and DNA double helix. The predicted plateau parameters are consistent with single-molecules experiments. Detailed analysis of DNA stretching shows that breaking of Watson-Crick bonds is not necessary for the existence of the plateau, although some of the bonds do break as the double helix extends at room temperature. The main strengths of the proposed theory are its generality and direct microscopic connection.

Coding Polypurine Hairpins Cause Target-Induced Cell Death in Breast Cancer Cells

Polypurine reverse-Hoogsteen hairpins (PPRHs) are double-stranded DNA molecules formed by two polypurine stretches linked by a pentathymidine loop, with intramolecular reverse-Hoogsteen bonds that allow a hairpin structure. PPRHs bind to polypyrimidine targets by Watson-Crick bonds maintaining simultaneously a hairpin structure due to intramolecular Hoogsteen bonds. Previously, we described the ability of Template-PPRHs to decrease mRNA levels because these PPRHs target the template DNA strand interfering with the transcription process. Now, we designed Coding-PPRHs, a new type of PPRHs that directly target the pre-mRNA. The dihydrofolate reductase (dhfr) gene was selected as a target in breast cancer therapy. These PPRHs caused a high degree of cytotoxicity and a decrease in DHFR mRNA and protein levels, but by a different mechanism of action than Template-PPRHs. Coding-PPRHs interfere with the splicing process by competing with U2 auxiliary factor 65 for binding to the polypyrimidine target sequence, leading to a lower amount of mature mRNA. These new PPRHs showed high specificity as no off-target effects were found. The application of these molecules as therapeutic tools was tested in breast cancer cells resistant to methotrexate, obtaining a noticeable cytotoxicity even though the dhfr locus was amplified. Coding-PPRHs can be considered as new molecules to decrease gene expression at the mRNA level and an alternative to other antisense molecules.

Polypurine Hairpins Directed against the Template Strand of DNA Knock Down the Expression of Mammalian Genes

We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.

A Computational Study of Nucleosomal DNA Flexibility

Molecular dynamics simulations of the nucleosome core particle and its isolated DNA free in solution are reported. The simulations are based on the implicit solvent methodology and provide insights into the nature of large-scale structural fluctuations and flexibility of the nucleosomal DNA. In addition to the kinked regions previously identified in the x-ray structure of the nucleosome, the simulations support the existence of a biochemically identified distorted region of the DNA. Comparison of computed relative free energies shows that formation of the kinks is associated with little, if any, energy cost relative to a smooth, ideal conformation of the DNA superhelix. Isolated nucleosomal DNA is found to be considerably more flexible than expected for a 147 bp stretch of DNA based on its canonical persistence length of 500 Å. Notably, the significant bending of the DNA observed in our simulations occurs without breaking of Watson-Crick bonds. The computed relative stability of bent conformations is sensitive to the ionic strength of the solution in the physiological range; the sensitivity suggests possible experiments that might provide further insights into the structural origins of the unusual flexibility of the DNA.

Mismatch-Induced Conformational Distortions in Polymerase β Support an Induced-Fit Mechanism for Fidelity

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.

Structure of a High Fidelity DNA Polymerase Bound to a Benzo[a]pyrene Adduct That Blocks Replication

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Antisense drug discovery: can cell-free screens speed the process?

Many conditions must be satisfied for an antisense drug to function. It must colocalize with its target RNA at a sufficient concentration for a bimolecular reaction to occur, and it must have a structure that favors association with its target. In addition, if the antisense compound is to form Watson-Crick bonds with the target RNA, it must be complementary to sites that are amenable to binding. Unfortunately, the peculiarities that cause certain sites to be especially vulnerable to antisense compounds are undefined, as discussed previously (Branch, 1998). Because vulnerable target sites have no common properties allowing them to be identified by sequence analysis, most target sites and their antisense counterparts are found through a trial and error process in which oligomers--each complementary to a different site in the target RNA--are tested individually to find the one with the greatest specificity and lowest inhibitory concentration (IC50). However, testing antisense molecules one at a time can be a taxing process, and there is great interest in developing cell-free screening methods that can reduce the number of compounds that must be tested in cells and in whole animals. These cell-free screens are designed to generate short lists of target sites that include the ideal site--the site most vulnerable to antisense ablation in vivo. They are based on the unproven assumption that ideal sites have distinctive properties, such as susceptibility to RNase H-mediated cleavage, that allow them to be detected in cell-free assays. This is a review of data emerging from studies using RNase H-based screens and a summary of the challenges confronting these and any similar methods that use naked RNAs as surrogates for intracellular RNAs. It is not yet clear if cell-free screening methods will be effective.

The Fragile X Syndrome Single Strand d(CGG)n Nucleotide Repeats Readily Fold Back to Form Unimolecular Hairpin Structures

Expansion of a d(CGG)n run within the 5'-untranslated region of the X-linked human gene FMR1 blocks FMR1 transcription, delays its replication, and precipitates the fragile X syndrome. We showed previously that d(CGG)n tracts aggregate into interstrand tetrahelical complexes (Fry, M., and Loeb, L. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4950-4954). Here we show that these sequences also form under physiological conditions in in vitro unimolecular hairpin structures. Folding is demonstrated by temperature-dependent mobility of d(CGG)n oligomers in a nondenaturing polyacrylamide gel, by UV-hyperchromicity of thermally denaturing oligomers, and by UV cross-linking of compact forms of d(CGG)n runs interspersed by thymidine clusters. That the compact d(CGG)n structures are unimolecular is suggested by their zero-order kinetics of formation. Diethyl pyrocarbonate modification reveals a single, 4-5 residue-long central or epicentral unpaired loop in folded d(CGG)n oligomers. The position of this loop remains unchanged by insertion of thymidine clusters into 15- or 33-mer d(CGG) tracts as indicated by KMnO4 probing of unpaired thymidines. The presence of a single loop in folded d(CGG)n oligomers and the accessibility of every guanine to dimethyl sulfate modification suggest that they are hairpin and not tetraplex structures. Modeling indicates that different d(CGG)n hairpins are stabilized by guanine-guanine Hoogsteen hydrogen bonds or by Hoogsteen and Watson-Crick bonds. If formed in vivo, d(CGG)n hairpins could entail slippage and trinucleotide expansion during replication and could obstruct FMR1 gene transcription and replication.

RecA protein mediates homologous recognition via non-Watson-Crick bonds in base triplets.

E. coli RecA protein, the prototype of a class, forms a helical nucleoprotein filament on single-stranded DNA that recognizes homology in duplex DNA, and initiates the exchange of strands in homologous recombination. The discovery of this reaction some years ago posed a quandary on how a third strand recognizes homology in duplex DNA, whose Watson-Crick bonds face inward in a hydrophobic core of stacked bases. Recent studies have shown that RecA protein promotes homologous recognition via non-Watson-Crick bonds in base triplets. The intermediates in the RecA reaction differ distinctly from triplex DNA that forms non-enzymically. The biological significance of the novel set of DNA interactions by which RecA protein effects homologous recognition is indicated by the importance of this protein in recombination, and the widespread distribution of homologous proteins in prokaryotes and eukaryotes.