Multi-stranded Nucleic Acid Research Articles

Mass Spectroscopic Study of G-Quadruplex.

Mass spectrometry (MS) is an analytical tool complimentary for being sensitive, accurate, and versatile in its application, such as the identification of multistranded nucleic acid assemblies, including G-quadruplex. More specifically, electrospray ionization mass spectrometry (ESI-MS) has been successfully applied to probe various G-quadruplex formations and G-quadruplex-ligand interactions. The benefit of the ESI process is that the noncovalent interactions, which typically stabilize the multistranded motifs of G-quadruplex in solution, are preserved in the gas phase. Here we use ESI-MS to describe the structural characterization of G-quadruplex structures found in three G-rich sequences, as well as the ligand binding. Detailed structural information of G-quadruplexes and their ligand-bound complexes (such as the cation/ligand binding stoichiometry, and the number of strands and G-quartets) can be obtained from a single spectrum using this ESI-MS-based method.

Novel Alternative and Multistranded DNA Microarrays: Z‐DNA, Triplex DNA and Quadruplex DNA

At the ends of chromosomes are segments of DNA called telomeres, which permit cells to replicate, protect DNA, and repair DNA. Multistranded and alternative DNA structures play important roles in gene expression. Novel alternative, multistranded, and helical transitional nucleic acid microarrays have been constructed, viz., Z‐DNA, triplex DNA and quadruplex DNA. These microarrays allow for characterization of the structure and function of Z‐DNA, triplex DNA and quadruplex DNA under different conditions. They allow for identification of drugs that bind to multistranded nucleic acids and for characterization of genomic elements that contain triplex DNA and telomeres. The novel microarrays will allow for a new approach towards studying gene expression and drug discovery. The new microarrays go beyond the limitations of conventional DNA microarrays, which focus on the primary structure of nucleic acids (i.e., base pairs) and ignore DNA secondary structure. These microarrays can be used for aging, cancer and infectious disease (e.g., tuberculosis, malaria and AIDS) research. The microarrays can also be employed for enhancing or inhibiting of gene expression. With these next generation DNA microarrays, scientists will have access to all of the uncharted structures that control gene expression, and aid in developing new classes of drugs for disease. Supported by a 2010 NYIT ISRC grant.

Novel Multistranded, Alternative, Plasmid and Helical Transitional Dna and Rna Microarrays: Implications for Therapeutics

Novel multistranded and alternative DNA, RNA and plasmid microarrays (transitional structural nucleic acid microarrays) have been developed that allows for the immobilization of intact, nondenatured, double-stranded DNA, double-stranded RNA, and alternative and multistranded nucleic acids. It also allows for the study of transitional changes that occur in the structure of DNA and RNA. Alternative types of DNA, RNA and multistranded nucleic acids are immobilized by a variety of different surface chemistries (i.e., noncovalent or covalent) onto a novel substrate surface. This technology represents the next generation of microarrays, which will aid in the characterization of nucleic acid structure and function, and accelerate the discovery of drugs that bind to nucleic acids. In addition, we demonstrate four novel techniques that are the first practical applications of the microarray, that is, transitional structural chemogenomics, transitional structural chemoproteomics, transitional structural pharmacogenomics and transitional structural pharmacoproteomics. These novel nucleic acid microarrays, together with pharmacogenomics, can be used to improve the study of DNA and RNA structure, gene expression, drug development and treatment of various diseases.

The Werner Syndrome Protein Binds Replication Fork and Holliday Junction DNAs as an Oligomer

Werner syndrome is an inherited disease displaying a premature aging phenotype. The gene mutated in Werner syndrome encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Both WRN helicase and exonuclease preferentially utilize DNA substrates containing alternate secondary structures. By virtue of its ability to resolve such DNA structures, WRN is postulated to prevent the stalling and collapse of replication forks that encounter damaged DNA. Using electron microscopy, we visualized the binding of full-length WRN to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both wild-type WRN and a helicase-defective mutant bind with exceptionally high specificity (>1000-fold) to DNA secondary structures at the replication fork and at Holliday junctions. Little or no binding is observed elsewhere on the DNA molecules. Calculations of the molecular weight of full-length WRN revealed that, in solution, WRN exists predominantly as a dimer. However, WRN bound to DNA is larger; the mass is consistent with that of a tetramer.

Improved Multi-Stranded Nucleic Acid Immobilization Technique: Enhanced Double-Stranded, Triple-Stranded and Four-Stranded DNA Microarray Platform

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Cell biology, chemogenomics and chemoproteomics – application to drug discovery

Cell biology has added immensely to the understanding of basic biologic concepts. However, scientists need to use cell biology more in the proteomic–genomic revolution. The authors have developed two novel techniques: transitional structural chemogenomics (TSCg) and transitional structural chemoproteomics (TSCp). TSCg is used to regulate gene expression by using ultrasensitive small-molecule drugs that target nucleic acids. By using chemicals to target transitional changes in the helical conformations of single-stranded (ss) and double-stranded (ds) DNA (e.g., B- to Z-DNA) and RNA (e.g., A- to Z-RNA), gene expression can be regulated (i.e., turning genes ‘on/off’ and variably controlling them). Alternative types of ds- and ssDNA and RNA (e.g., cruciform DNA) and other multistranded nucleic acids (e.g., triplex-DNA) are also targeted by this method. The authors’ second technique, TSCp, targets a protein before, during or after post-translational modifications, which alters the protein’s structure and function. These novel methods represent the next step in the evolution of chemical genomics and chemical proteomics. In addition, a novel multi-stranded (alternative) DNA, RNA and plasmid microarray has been developed that allows for the immobilization of intact, non-denatured dsDNA, alternative (i.e., exotic) and other multiple-stranded nucleic acids. This represents the next generation of nucleic acid microarrays, which will aid in the characterization of nucleic acids, studying the ageing process and improving the drug discovery process. The authors discuss how cell biology can be used to enhance genomics and proteomics. Cell biology will play a greater role during the postgenomic age and will help to enhance the omics/omes and drug discovery. It is the authors’ hope that these novel approaches can be used together with cellular biologic techniques to make major contributions towards understanding and manipulating different genomes.

Novel drug discovery and molecular biological methods, via DNA, RNA and protein changes using structure–function transitions: Transitional structural chemogenomics, transitional structural chemoproteomics and novel multi-stranded nucleic acid microarray

Nucleic acids and proteins are dynamic molecules that undergo structural changes which control gene expression. The authors have developed two novel techniques, viz., transitional structural chemogenomics and transitional structural chemoproteomics. Transitional structural chemogenomics is used to regulate gene expression, employing ultrasensitive small-molecule drugs targeted toward nucleic acids. Gene expression can be regulated by using chemicals to target transitional changes in the helical conformations of single-stranded (ss-) and double-stranded (ds-) DNA (e.g., B- to Z-DNA), and RNA (e.g., A- to Z-RNA). This method also targets alternative types of ds- and ss-DNA and RNA (e.g., cruciform DNA), and other multi-stranded nucleic acids (e.g., triplex-DNA). Our second technique, transitional structural chemoproteomics, targets a protein before, during or after post-translational modifications which alters its structure and function. Both a proteins' structured and unstructured regions are targeted. These two novel methods represent the next step in the evolution of chemical genomics and chemical proteomics. They allow for two approaches to regulate gene expression, viz., turning genes "on", "off" or variable control (e.g., dimmer switch). This article also discusses the confusion that exists between the term chemical genomics and other related subdisciplines, such as chemical proteomics. Additionally, we have developed a novel multi-stranded DNA, RNA and plasmid microarray which immobilizes intact nondenatured ds-DNA, alternative, and other multiple-stranded nucleic acids onto a substrate surface. This technique represents the next generation of nucleic acid microarrays, which will enhance the characterization of nucleic acids and the drug discovery process. These three novel techniques allow for a multifaceted approach that will greatly enhance the success of molecular biology, the "omics" and drug discovery. They represent the next era of gene expression tools.

Synthesis of fluorophore and quencher monomers for use in scorpion primers and nucleic acid structural probes.

We report the syntheses of monomers to incorporate fluorescein and methyl red within the sequence and at the termini of modified oligonucleotides. These monomers have been used in the solid-phase synthesis of fluorogenic oligonucleotides for genetic analysis and the study of multi-stranded nucleic acid structures.

Editorial: Fluorescence spectroscopy and nucleic acids

Fluorescence spectroscopy is an established tool for investigating the structure, folding, and conformational dynamics of proteins in solution. The widespread use of fluorescence techniques for the physicochemical characterization of proteins stems from the occurrence of the naturally fluorescent amino acids tryptophan and tyrosine in many proteins and the great sensitivity of the emission properties of these amino acids to perturbations in protein structure. Historically, the application of fluorescence spectroscopy to nucleic acids has been hindered by the lack of suitable intrinsic fluorophores (the exception are certain tRNAs that contain unusual fluorescent bases). However, with the advent of rapid and efficient methods for the solid phase synthesis and site-specific fluorescent labeling of DNA and RNA oligonucleotides, the power of fluorescence spectroscopy can now be brought to bear on nucleic acids. Indeed, the past decade has witnessed a tremendous growth in the number of studies in which fluorescence spectroscopic techniques have been used to characterize the structural properties and fundamental biophysical behaviors of nucleic acids and nucleic acid–protein complexes. Particular advantages of fluorescence-based methods include high detection sensitivity, fast time resolution, and ready adaptability to a wide range of solution conditions. Of the many spectroscopic phenomena that can be exploited to examine nucleic acids, fluorescence resonance energy transfer (FRET) lends itself to the broadest range of applications. This issue of Nucleic Acid Sciences outlines some of the methodologies employed and illustrates the rich variety of information that is forthcoming from FRET-based analyses of nucleic acids, highlighting studies that focus on structural characterization, thermodynamic properties, and kinetic processes. Since the efficiency of resonance energy transfer from a donor fluorophore to an acceptor reports on distances ranging from 10 to 100 Å, measurements of FRET can be used to define the global structure of nucleic acid complexes. This approach is especially powerful in the case of large or disordered complexes that are not amenable to structural analysis by x-ray crystallographic or NMR spectroscopic methods. Showing the broad applicability of this technique to structural questions, FRET has been used to elucidate the helical geometry of multistranded nucleic acid complexes (Singleton and Xiao), to explore the dynamic solution structure of branched DNA molecules (Klostermeier and Millar), to characterize the conformation of bent DNA (Parkhurst et al.), and to probe the global architecture of large macromolecule complexes (Heyduk and Niedziela-Majka). In addition to its utility in probing structural questions, FRET may be used to analyze nucleic acid complex stability and conformational transitions. Precise thermodynamic characterization of the impact of base lesions on DNA duplex stability is possible using sensitive FRET-based methods, giving insight into the biological consequences of DNA damage and the molecular basis of mutagenesis (Plum and Breslauer). As another example, the time-resolved FRET technique can be used to identify and quantify different tertiary structure conformers of RNA, which often play a role in regulating biological activity. This approach has been used to investigate the molecular forces that drive conformational transitions in the hairpin ribozyme (Klostermeier and Millar). Finally, FRET methods can also be harnessed to study the kinetics of nucleic acid conformational transitions, underscoring the great versatility of fluorescence techniques for biophysical characterization of nucleic acids. FRET and rapid kinetic methods have been used to study the dynamics of DNA bending by the transcription factor TBP in real time and to establish a detailed kinetic mechanism for the interaction of TBP with DNA (Parkhurst et al.). Similar methods have been used to monitor large-scale conformational changes during the catalytic cycle of the hairpin and hepatitis delta virus ribozymes (Walter et al.). The studies reviewed in this issue of Nucleic Acid Sciences illustrate the ability of fluorescence techniques to provide structural, thermodynamic, and kinetic information on a variety of biologically important DNA and RNA molecules. It is hoped that these examples will stimulate an even wider application of fluorescence techniques in the study of nucleic acids. While most of the examples presented here involve relatively short oligonucleotides, it is anticipated that the different fluorescent labeling strategies discussed herein will be combined to construct much larger DNA or RNA molecules. Thus, the fluorescence spectroscopic techniques described in this issue can be applied to a variety of dynamic macromolecular complexes, including the replisome, transcription complexes, and the ribosome. In addition, the fluorescence techniques can be extended to the single-molecule level, which should provide new insights into the conformational dynamics and biological function of nucleic acids (single-molecule fluorescence studies have been reviewed recently; T. J. Ha, Current Opinion in Structural Biology, 2001 Vol. 11, pp. 287–292).

Structures and Properties of Multi-stranded Nucleic Acids

Nucleic acids exist in a number of structural states that may comprise one to four strands. Common features involve base-stacking and pairing of complementary bases via H-bonded interactions, although a wide variety of pairing motifs are found in higher-order structures. Another property is the marked flexibility of nucleic acids reflected in their solution behaviour, thermodynamic stability and interactions with proteins and small ligand molecules. The electrostatic properties of a nucleic acid and its ability to pack into high-order structure(s) are largely determined by the anionic phosphodiester backbone. This feature is particularly evident for triple-helical assemblies, where there is close proximity between phosphates and intrinsic thermodynamic stability is strongly influenced by ionic strength. Structural stabilisation of parallel triplexes can also be modulated by the presence of positive charge, achieved through either N3-protonation of cytosine bases or the deliberate incorporation of c harged residues into the third strand. Salt effects are also pronounced in four-stranded structures such as the i-motif and G-tetraplex (quadruplex) assemblies indeed, different structures are produced for the latter depending on the nature of the associated cation. High-order nucleic acid structures are currently a focus of intense interest in antisense and antigene strategies toward novel chemotherapeutic agents. An understanding of both their structural details and solution properties is essential for a rational approach to DNA-targeted drug design. Keywords: Multi-stranded Nucleic Acids, Oligonucleotides, torsions, DNA duplexes, Antiparallel Triplexes, Tetrahymena

Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides.

One of the most difficult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme-substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 A resolution when exposed to a synchrotron X-ray source.

Divalent transition metal cations counteract potassium-induced quadruplex assembly of oligo(dG) sequences.

Nucleic acids containing tracts of contiguous guanines tend to self-associate into four-stranded (quadruplex) structures, based on reciprocal non-Watson-Crick (G*G*G*G) hydrogen bonds. The quadruplex structure is induced/stabilized by monovalent cations, particularly potassium. Using circular dichroism, we have determined that the induction/stabilization of quadruplex structure by K+is specifically counteracted by low concentrations of Mn2+(4-10 mM), Co2+(0.3-2 mM) or Ni2+(0.3-0.8 mM). G-Tract-containing single strands are also capable of sequence-specific non-Watson-Crick interaction with d(G. C)-tract-containing (target) sequences within double-stranded DNA. The assembly of these G*G.C-based triple helical structures is supported by magnesium, but is potently inhibited by potassium due to sequestration of the G-tract single strand into quadruplex structure. We have used DNase I protection assays to demonstrate that competition between quadruplex self-association and triplex assembly is altered in the presence of Mn2+, Co2+or Ni2+. By specifically counteracting the induction/stabilization of quadruplex structure by potassium, these divalent transition metal cations allow triplex formation in the presence of K+and shift the position of equilibrium so that a very high proportion of triplex target sites are bound. Thus, variation of the cation environment can differentially promote the assembly of multistranded nucleic acid structural alternatives.

Structural features of a three-stranded DNA junction containing a C-C junctional bulge.

We have examined the stability of junctional base pairs in a three-way DNA junction with two unpaired cytidine residues at the branch point using two-dimensional nuclear Overhauser effect spectroscopy in H2O solution. Our data directly support the presence of two of the three junctional Watson-Crick base pairs, with indirect support for the third as well. These results complement the data presented in the preceding paper, where we examined the nonexchangeable proton resonance assignments of three-way DNA junctions from NOESY data in D2O solution. We have incorporated the NOE data from both sets of experiments, using this information as input for a combined distance geometry (DG) and simulated annealing (SA) protocol designed to derive three-dimensional structures of the junction molecule consistent with the NMR data. Although the data does not allow us to derive a unique solution for the structure of the molecule, certain conformational features are invariably present in our models. We demonstrate the existence of a preferred, pair-wise stacking arrangement between two of the three helices in the junction. Furthermore, the remaining duplex stem is situated so that it always forms an acute angle with just one of the arms from the quasi-continuous helix. The unpaired residues provide an extended backbone segment linking two of the helices together. The first unpaired base on the 5' end loops out from the interior of the molecule to reside along the minor groove of one helix. The second is located within the interior of the molecule, stacking below one of the junctional base pairs. Our findings suggest that junctional base pair stacking is an important determinant in the conformation of multistranded nucleic acid junctions. In three-way junctions, the presence of unpaired bases at the branch point provides a relief from covalent constraints that would otherwise prevent the simultaneous realization of both base pairing and base pair stacking within the branch point of the molecule.

Construction and use of models designed to demonstrate some topological properties of multistranded nucleic acid structure and its possible bearing on recombination

A description is given of the construction and use of a simple model designed to show the consequences of association between duplexes to form some kind of four strand structure in recombination. The model was first devised to help in consideration of the work of Nash et al. (1980). Conclusions drawn from it may however be of wider interest. The model was designed with a detailed molecular model in mind (McGavin, 1971a, 1979). It could however apply to any close juxtaposition of duplexes. A close juxtaposition of duplexes is a feature of several models for recombination, although this is not often referred to explicitly as four strand structure.

Review: ethidium fluorescence assays. Part 1. Physicochemical studies.

DNA and RNA can be assayed rapidly and very sensitively by exploiting the enhanced fluorescence of ethidium intercalated into duplex regions. By assaying at different pHs and introducing a heating/cooling cycle, a great many physicochemical aspects of DNA and RNA can be studied avoiding the use of radiolabels, and often giving information not otherwise readily obtainable. Studies are described on duplex DNA which involve measurement of extinction coefficients, cross-linking by chemicals, Cot curve analysis as well as estimation of drug-DNA binding constants. The assays can be adapted to investigate multi-stranded nucleic acid structures. The use of covalently closed circular DNA also allows rapid and extremely sensitive measurements of nicking caused by irradiation or drugs.

Kinetic analysis of the annealing period in the formation of the Poly (A)-2Poly (U) triple helix.

AbstractThe slow kinetics of annealing processes in multistranded nucleic acids is spectrophotometrically investigated using poly(A)·2poly(U) as a model system. The absorbance changes at specific wavelengths show that double‐helical (A·U) base pairs appear as transient intermediates. The annealing process is identified by the enlargement of triple‐helical sequences at the cost of (A·U) base pairs and unpaired (U) residues. A large time range in the reorganization of mismatched chain configurations is characterized by a logarithmic dependence on time. This observation is quantitatively described by a kinetic model developed by Jackson. In Jackson's model the rate‐limiting process in the slow annealing phase of maximizing triple‐helical sequences, is the removal of strand entanglements, knots, and hairpin loops by complete unwinding of those helical stretches which stabilize the mismatched configurations. The results of the present study are briefly discussed in terms of optimum conditions for hybridization experiments and for the preparation of polynucleotide complexes commonly used to produce interferons.