Duplex Motifs Research Articles

Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Chemical Information and Computational Modeling of Targeting Hybrid Nucleic Acid Structures of PIM1 Sequences by Synthetic Pyrrole-Imidazole Carboxamide Drugs.

DNA can adopt various distinct structural motifs, such as quadruplex, duplex, i-motifs, etc. which have multifarious applications in biomedical therapeutics. Quadruplex-duplex hybrids (QDHs) consist of the juxtaposed quadruplex and duplex motifs and are thermally stable and biologically relevant. Selective binding toward these secondary structures plays an important role in the evaluation of the structure-specific ligands. Herein, several small molecules containing anthraquinone conjugated oligopyrrole, oligoimidazole, and pyrrole-imidazole derivatives have been screened for the binding of the quadruplex-duplex nucleic acid hybrids formed in PIM1 sequences through docking and molecular dynamics (MD) simulation studies. The binding interaction of the anthraquinone polypyrrole ligands has also been checked by performing different biophysical experiments. PIM1, being a coactivator of the MYC oncogene, can be targeted by these small molecules to control MYC expression which is overexpressed in the majority of human cancer cells. Accordingly, these cancer cell-specific and blood-compatible anthraquinone conjugated oligopyrrole ligands can be employed for anticancer therapeutic applications. Thus, the structure-activity relationship (SAR) of the screened ligands manifested prudent structural information for designing PIM1 QDHs targeting small molecules.

Self-Assembly of a 3D DNA Crystal Structure with Rationally Designed Six-Fold Symmetry.

Programming self-assembled designer DNA crystals with various lattices and functions is one of the most important goals for nanofabrication using nucleic acids. The resulting porous materials possess atomic precision for several potential applications that rely on crystalline lattices and cavities. Herein, we present a rationally designed and self-assembled 3D DNA crystal lattice with hexagonal symmetry. In our design, two 21-base oligonucleotides are used to form a duplex motif that further assembles into a 3D array. The interactions between the strands are programmed using Watson-Crick base-pairing. The six-fold symmetry, as well as the chirality, is directed by the Holliday junctions formed between the duplex motifs. The rationally designed DNA crystal provides a new avenue that could create self-assembled macromolecular 3D crystalline lattices with atomic precision. In addition, the structure contains a highly organized array of well-defined cavities that are suitable for future applications with immobilized guests.

Self‐Assembly of a 3D DNA Crystal Structure with Rationally Designed Six‐Fold Symmetry

Programming self-assembled designer DNA crystals with various lattices and functions is one of the most important goals for nanofabrication using nucleic acids. The resulting porous materials possess atomic precision for several potential applications that rely on crystalline lattices and cavities. Herein, we present a rationally designed and self-assembled 3D DNA crystal lattice with hexagonal symmetry. In our design, two 21-base oligonucleotides are used to form a duplex motif that further assembles into a 3D array. The interactions between the strands are programmed using Watson–Crick base-pairing. The six-fold symmetry, as well as the chirality, is directed by the Holliday junctions formed between the duplex motifs. The rationally designed DNA crystal provides a new avenue that could create self-assembled macromolecular 3D crystalline lattices with atomic precision. In addition, the structure contains a highly organized array of well-defined cavities that are suitable for future applications with immobilized guests.

Thioflavin T as a fluorescence light-up probe for both parallel and antiparallel G-quadruplexes of 29-mer thrombin binding aptamer.

A wide range of pathologies have been targeted with bimodular aptamers that contain both G-quadruplex (G4) and duplex motifs, while the structures and functions are poorly understood. G4-selective fluorescent dyes have served as facile tools to probe G4s, but not for bimodular aptamers, yet. Here, taking the 29-mer thrombin binding aptamer (TBA29) as an example, we demonstrated that 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium (ThT) was the most effective dye compared to NMM and PPIX in recognizing TBA29. Binding studies indicate that ThT recognized TBA29 via distinct buffer-dependent mechanisms. Specifically, ThT induced the formation of a bimolecular parallel G4 in cation-deficient buffer, showing 341-fold fluorescent enhancement. The competitive binding of thrombin disrupted the complex, leading to the monotonic fluorescence decrease. A similar mechanism was previously reported for the interaction between ThT and the 15-mer thrombin binding aptamer (TBA15). However, TBA29 bound with ThT in a more favorable state than TBA15, showing hyperchromic effects and two times stronger fluorescence enhancement. Differently, ThT bound with antiparallel TBA29/TBA15 in an intercalating/groove binding mode in 100mM KCl, generating 181/28-fold fluorescence enhancement, respectively. These results revealed that ThT recognized both parallel and antiparallel G4s of TBA29 more efficiently than it recognized TBA15. The duplex structure of TBA29 may play an important role in its interaction with ThT. Our study broadens the application of ThT in screening G4 to bimodular aptamers and provides some insights into the structures of TBA29, along with the interaction between ThT and TBA29. Our study also is useful for the development of structure-switching-based biosensors using bimodular aptamers. Graphical abstract The buffer-dependent binding mechanisms of ThT with TBA29, and the competitive (top)/noncompetitive (bottom) binding of thrombin with TBA29-ThT complex.

Computational identification of biologically functional non-hairpin GC-helices in human Argonaute mRNA

BackgroundPerfectly formed duplex elements in RNA occur within folding units, often as a part of hairpin motifs which can be reliably predicted by various RNA folding algorithms. Double helices with consecutive Watson-Crick base-pairing may also be formed between distant RNA segments thereby facilitating long-range interactions of long-chain RNA that may be biologically functional. Here we addressed the potential formation of RNA duplex motifs by long-range RNA-RNA interactions of distantly located matching sequence elements of a single long-chain RNA.ResultsWe generated a Python-based software tool that identifies consecutive RNA duplex elements at any given length and nucleotide content formed by distant sequences. The software tool, dubbed RNAslider, is built on the theoretical RNA structure prediction algorithm Mfold. Source code and sample data sets are available on demand. We found that a small ratio of human genes including the Argonaute (Ago)-like gene family encode mRNAs containing highly GC-rich non-hairpin duplex elements (GC-helix) of equal to or more than 8 base pairs in length and we provide experimental evidence for their biological significance.ConclusionGC-helices are observed preferentially within the 5′-region of mRNAs in an evolutionarily conserved fashion indicating their potential biological role. This view is supported experimentally by post-transcriptional regulation of gene expression of a fusion transcript containing 5′-sequences of human mRNAAgo2 harbouring GC-helices and down-stream coding sequences of Renilla luciferase.

Accumulation of Fluorophores into DNA Duplexes To Mimic the Properties of Quantum Dots

By using perylene and pyrene as fluorophores, we have designed various fluorophore assemblies that mimic inorganic quantum dots in showing a high emission intensity, a large Stokes' shift, and a modulated emission maximum. For this purpose, we utilized two kinds of duplex motifs with D-threoninols as scaffolds: cluster and interstrand-wedged motifs. In the cluster motif, fluorophores are introduced into both strands to produce tentative pseudo-"base-pairs", in which the dyes strongly interact with each other and form dimers, trimers, or hexamers. In the interstrand-wedged motif, a base-pair is inserted between the fluorophores to suppress their direct interaction. These two motifs were applied to accumulate dyes within a DNA duplex, depending on their emission properties. Since pyrene exhibits strong excimer emission, the emission at 500 nm of a pyrene cluster motif strongly increased as the number of accumulated dyes increased, whereas the interstrand-wedged motif quenched pyrene monomer emission. In contrast, assembled perylenes, which are mostly quenched by dimerization, showed intense monomer emission in the interstrand-wedged motif, whereas perylene cluster motifs strongly suppressed perylene emission. These two motifs were then applied to the heteroassembly of pyrenes and perylenes. Both a large Stokes' shift and a modulation of the emission maximum, which are also characteristics of inorganic quantum dots, were successfully realized using fluorescent resonance energy transfer (FRET) and exciplex formation. These fluorophore assemblies thus obtained could be enzymatically ligated to longer DNA, demonstrating that this technique has the potential to be a versatile labeling agent for biomolecules.

Melting Behavior of DNA Complexes With Joined Triple and Duplex Motifs

One focus of our laboratory is to understand how sequence, duplex and triplex stabilities, and solution conditions affect the melting behavior of complex DNA structures. We used a combination of UV and circular dichroism (CD) spectroscopies and differential scanning calorimetry (DSC) techniques to obtain a full thermodynamic description of the melting behavior of seven DNA complexes involving joined triplex and duplex motifs. Six of these complexes are formed intramolecularly while the seventh forms an intermolecular complex. The circular dichroism spectra at low temperatures indicated that all complexes maintained the “B” conformation. UV and DSC melting curves of each complex show biphasic or triphasic transitions. However, the transition temperatures, TMs, of the intramolecular complexes remained constant with increasing strand concentration, while the TM of the intermolecular complex did not. This confirms their molecularity. Deconvolution of the DSC thermograms allowed us to determine standard thermodynamic profiles for the transitions of each complex. For each transition, the favorable folding free energy terms result from the characteristic compensation of a favorable enthalpy and unfavorable entropy contribution. The magnitude of these thermodynamic parameters (and associated TMs) indicate that the overall folding of each complex depends on several factors: a) the extent of the favorable heat contributions (formation of base pair and base triplet stacks that are compensated with both the ordering of the oligonucleotide and the putative uptake of protons and ions; b) inclusion of the more stable C+GC base triplets; c) stabilizing the duplex stem of the complex; d) complex molecularity; and e) solution conditions, such as pHand salt concentration. Overall, the melting behavior of each complex corresponds to the initial disruption of the triplex motif (removal of the third strand) followed by the partial or full unfolding of the duplex stem.

DNA Complexes Containing Joined Triplex and Duplex Motifs: Melting Behavior of Intramolecular and Bimolecular Complexes with Similar Sequences

Our laboratory is interested in predicting the thermal stability and melting behavior of nucleic acids from knowledge of their sequence. One focus is to understand how sequence, duplex and triplex stabilities, and solution conditions affect the melting behavior of complex DNA structures, such as intramolecular DNA complexes containing triplex and duplex motifs. Nucleic acid oligonucleotides (ODNs), as drugs, present an exquisite selectivity and affinity that can be used in antigene and antisense strategies for the control of gene expression. In this work, we try to answer the following question: How does the molecularity of a DNA complex affect its overall stability and melting behavior? We used a combination of temperature-dependent UV spectroscopy and calorimetric (DSC) techniques to investigate the melting behavior of DNA complexes with a similar helical stem sequence, TC(+)TC(+)TC(+)T/AGAGAGACGCG/CGCGTCTCTCT, but formed with different strand molecularity. We determined standard thermodynamic profiles, and the differential binding of protons and counterions accompanying their unfolding. The formation of a DNA complex is accompanied by a favorable free energy term resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions. As expected, acidic pH stabilized each complex by allowing protonation of the cytosines in the third strand; however, the percentage of protonation increases as the molecularity decreases. The results help in the design of oligonucleotide sequences as targeting reagents that could effectively react with DNA or RNA sequences involved in human diseases, thereby increasing the feasibility of using the antigene and antisense strategies, respectively, for therapeutic purposes.

Structural and Functional Characterization of Potent Antithrombotic Oligonucleotides Possessing Both Quadruplex and Duplex Motifs

We report the results of a selection for single-stranded DNA oligonucleotide ligands to the serine protease thrombin using recently developed methods. This selection yielded a family of DNA sequences that conform to a consensus structure comprised of a unimolecular quadruplex motif and complementary flanking sequences capable of forming an additional Watson-Crick duplex motif. This novel quadruplex/duplex structure was not reported in a previous selection for DNA molecules which bind to thrombin [Bock et al. (1992) Nature 355, 564-566]. All quadruplex/duplex molecules tested bound to thrombin with higher affinity than quadruplex structures lacking the duplex structure. However, binding affinities did not always correlate with inhibitory potency since some molecules with high affinity were not potent inhibitors in vitro. 1H NMR spectroscopy studies demonstrated that the complementarity of bases in the duplex portion of a selected sequence allows it to form multimolecular structures. Constraining these molecules to the unimolecular quadruplex/duplex structure by bridging the 5' and 3' ends of the duplex motif with either triethylene glycol or disulfide bonds improved their thrombin inhibitory activity. All bridged quadruplex/duplex molecules were more potent inhibitors than molecules with only a quadruplex motif. Bridging the ends of these structures not only increased thrombin inhibition but also improved resistance to nucleases in serum more than 40-fold over the unbridged quadruplex. In addition, we have found that both the length and sequence of the duplex motif are important for inhibition.

Structure-Activity Studies of the Binding of Modified Peptide Nucleic Acids (PNAs) to DNA

Peptide nucleic acid (PNA) oligomers where one of the repeating backbone units is extended with a methylene group to either N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine were prepared. Alternatively, the linker to the nucleobase was extended from methylenecarbonyl to ethylenecarbonyl. The thermal stability of the hybrids between these PNA oligomers and complementary DNA oligonucleotides was significantly lower than that of the corresponding complexes involving unmodified PNA. However, the sequence selectivity was retained. Thymidyl decamers with all N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine backbones were prepared and shown to be unable to hybridize to the complementary (dA)(10) oligonucleotides, whereas a PNA decamer containing only ethylenecarbonyl linkers between the nucleobases and the N-(2-aminoethyl)glycine backbone showed weak but sequence-specific affinity for complementary DNA. All hybrids involving homopyrimidine PNA oligomers exhibited (PNA)(2)/DNA stoichiometry, whereas mixed-sequence PNA oligomers formed PNA/DNA duplexes. The preferred binding direction between the modified PNA and DNA in the duplex motifs was antiparallel, as previously reported for complexes involving unmodified PNA.