Canonical Duplex Research Articles

Native Mass Spectrometric Insights into the Formation and Stability of DNA Triplexes.

Deoxyribonucleic acid is a genetic biomacromolecule that contains the inherited information required to build and maintain a living organism. While the canonical duplex DNA structure is rigorously characterized, the structure and function of higher order DNA structures such as DNA triplexes are comparatively poorly understood. Previous literature has shown that these triplexes can form under physiological conditions, and native mass spectrometry offers a useful platform to study their formation and stability. However, experimental conditions including buffer salt concentration, pH, and instrumentation parameters such as ion mode can confound analysis by impacting the amount of triplex observed by mass spectrometry. Using model 30mer Y-type triplex sequences, we demonstrate the influence a range of experimental variables have on the detection of DNA triplex structures, informing suitable conditions for the study. When carefully considered conditions are used, mass spectrometry offers a powerful complementary tool for the analysis of higher order DNA assemblies.

Probing the binding and interaction of curcumin-tryptophan conjugate to G-quadruplex motif via spectroscopic and molecular docking approach

A potential ligand with the ability to interact with the structure of G-quadruplexes has been promising strategy for develop anticancer drug. Curcumin, a natural antioxidant is known to induce apoptosis in the tumour cells and was previously found as G-quadruplex binder. We investigated how the conjugation of tryptophan with curcumin affects its ability as G-quadruplex DNA binder. We have reported the differential interaction of Curcumin-tryptophan (CT) with a parallel G-quadruplex(GQ) formed by the nuclease hypersensitivity element (NHEIII) Pu27 present of the protooncogene and human telomeric G-quadruplex sequence. Taken into account, ligand CT has a stronger binding affinity for the G-quadruplex over the canonical duplex DNA. We have used biophysical approach such as uv-visible, fluorescence, circular dichroism, differential scanning calorimetry and molecular docking experiment to show that curcumin tryptophan interacts with the G-quadruplex. The results indicate that CT has a binding affinity of 106 M−1 for G-quadruplexes. Thermodynamic studies show that the ligand CT destabilizes the G-quadruplexes (6–8 °C) due to aromatic core stacking between the tetrad. The anti-proliferative experiment of the synthesized ligand CT in vitro on several cancer cell lines such as HeLa cells and MCF-7 cell lines shows that it is more effective against MCF-7. Thus, present studies report Curcumin-tryptophan as a potential G-quadruplex binder that destabilizes the quadruplex structure .

Inverted strand polarity yields thermodynamically stable G-quadruplexes and prevents duplex formation within extended DNA.

DNA G-quadruplexes (G4) formed in guanine-rich sequences play a key role in genome function and maintenance, interacting with multiple proteins. However, structural and functional studies of G4s within duplex DNA have been challenging because of the transient nature of G4s and thermodynamic preference of G-rich DNA to form duplexes with their complementary strand rather than G4s. To overcome these challenges, we have incorporated native nucleotides in G-rich sequences using commercially available inverted 3'-O-DMT-5'-O-phosphoramidites of native nucleosides, to give 3'-3' and 5'-5' linkages in the centre of the G-tract. Using circular dichroism and 1H nuclear magnetic resonance spectroscopies and native gel electrophoresis, we demonstrate that these polarity-inverted DNA sequences containing four telomeric repeats form G4s of parallel topology with one lateral or diagonal loop across the face of the quadruplex and two propeller loops across the edges of the quadruplex. These G4s were stable even in the presence of complementary C-rich DNA. As an example, G4 assemblies of inverted polarity were shown to bind to the hinge region of Heterochromatin Protein 1α (HP1α), a known G4-interacting domain. As such, internal polarity inversions in DNA provide a useful tool to control G4 topology while also disrupting the formation of other secondary structures, particularly the canonical duplex.

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.

Sensitivity of the RNA Structure to Ion Conditions as Probed by Molecular Dynamics Simulations of Common Canonical RNA Duplexes.

RNA molecules play a key role in countless biochemical processes. RNA interactions, which are of highly diverse nature, are determined by the fact that RNA is a highly negatively charged polyelectrolyte, which leads to intimate interactions with an ion atmosphere. Although RNA molecules are formally single-stranded, canonical (Watson-Crick) duplexes are key components of folded RNAs. A double-stranded (ds) RNA is also important for the design of RNA-based nanostructures and assemblies. Despite the fact that the description of canonical dsRNA is considered the least problematic part of RNA modeling, the imperfect shape and flexibility of dsRNA can lead to imbalances in the simulations of larger RNAs and RNA-containing assemblies. We present a comprehensive set of molecular dynamics (MD) simulations of four canonical A-RNA duplexes. Our focus was directed toward the characterization of the influence of varying ion concentrations and of the size of the solvation box. We compared several water models and four RNA force fields. The simulations showed that the A-RNA shape was most sensitive to the RNA force field, with some force fields leading to a reduced inclination of the A-RNA duplexes. The ions and water models played a minor role. The effect of the box size was negligible, and even boxes with a small fraction of the bulk solvent outside the RNA hydration sphere were sufficient for the simulation of the dsRNA.

Sequence-specific binding behavior of coralyne toward triplex DNA: An ultrafast time-resolved fluorescence spectroscopy study.

Triplex DNA structure has potential therapeutic application in inhibiting the expression of genes involved in cancer and other diseases. As a DNA-targeting antitumor and antibiotic drug, coralyne shows a remarkable binding propensity to triplex over canonical duplex and thus can modulate the stability of triplex structure, providing a prospective gene targeting strategy. Much less is known, however, about coralyne-binding interactions with triplex. By combining multiple steady-state spectroscopy with ultrafast fluorescence spectroscopy, we have investigated the binding behaviors of coralyne with typical triplexes. Upon binding with a G-containing triplex, the fluorescence of coralyne is markedly quenched owing to the photoinduced electron transfer (PET) of coralyne with the G base. Systematic studies show that the PET rates are sensitive to the binding configuration and local microenvironment, from which the coexisting binding modes of monomeric (full and partial) intercalation and aggregate stacking along the sugar-phosphate backbone are distinguished and their respective contributions are determined. It shows that coralyne has preferences for monomeric intercalation within CGG triplex and pure TAT triplex, whereas CGC+ triplex adopts mainly backbone binding of coralyne aggregates due to charge repulsion, revealing the sequence-specific binding selectivity. The triplex-DNA-induced aggregation of coralyne could be used as a probe for recognizing the water content in local DNA structures. The strong π-π stacking of intercalated coralyne monomer with base-triplets plays an important role in stabilizing the triplex structure. These results provide mechanistic insights for understanding the remarkable propensity of coralyne in selective binding to triplex DNA and shed light on the prospective applications of coralyne-triplex targeted anti-gene therapeutics.

Modification at the C2'-O-Position with 2-Methylbenzothiophene Induces Unique Structural Changes and Thermal Transitions on Duplexes of RNA and DNA.

Oligonucleotides can be chemically modified for a variety of applications that include their use as biomaterials, in therapeutics, or as tools to understand biochemical processes, among others. This work focuses on the functionalization of oligonucleotides of RNA and DNA (12- or 14-nucleotides long) with methylbenzothiophene (BT), at the C2′-O-position, which led to unique structural features. Circular dichroism (CD) analyses showed that positioning the BT units on one strand led to significant thermal destabilization, while duplexes where each strand contained 4-BT rings formed a distinct arrangement with cooperativity/interactions among the modifications (evidenced from the appearance of a band with positive ellipticity at 235 nm). Interestingly, the structural arrays displayed increased duplex stabilization (>10 °C higher than the canonical analogue) as a function of [Na+] with an unexpected structural rearrangement at temperatures above 50 °C. Density functional theory–polarizable continuum model (DFT-PCM) calculations were carried out, and the analyses were in agreement with induced structural changes as a function of salt content. A model was proposed where the hydrophobic surface allows for an internal nucleobase rearrangement into a more thermodynamically stable structure, before undergoing full denaturation, with increased heat. While this behavior is not common, B- to Z-form duplex transitions can occur and are dependent on parameters that were probed in this work, i.e., temperature, nature of modification, or ionic content. To take advantage of this phenomenon, we probed the ability of the modified duplexes to be recognized by Zα (an RNA binding protein that targets Z-form RNA) via electrophoretic analysis and CD. Interestingly, the protein did not bind to canonical duplexes of DNA or RNA; however, it recognized the modified duplexes, in a [monovalent/divalent salt] dependent manner. Overall, the findings describe methodology to attain unique structural motifs of modified duplexes of DNA or RNA, and control their behavior as a function of salt concentration. While their affinity to RNA binding proteins, and the corresponding mechanism of action, requires further exploration, the tunable properties can be of potential use to study this, and other, types of modifications. The novel arrays that formed, under the conditions described herein, provide a useful way to explore the structure and behavior of modified oligonucleotides, in general.

Porphyrin induced structural destabilization of a parallel DNA G-quadruplex in human MRP1 gene promoter.

Porphyrins are among the first ligands that have been tested for their quadruplex binding and stabilization potential. We report the differential interaction of the positional cationic porphyrin isomers TMPyP3 and TMPyP4 with a parallel G-quadruplex (GQ) formed by 33-mer (TP) regulatory sequence present in the promoter region of the human multidrug resistance protein 1 (MRP1) transporter gene. This GQ element encompasses the three evolutionary conserved SP1 transcription factor binding sites. Taking into account that SP1 binds to a non-canonical GQ motif with higher affinity than to a canonical duplex DNA consensus motif, it is suggestive that GQ distortion by cationic porphyrin will have important implications in the regulation of MRP1 expression. Herein, we employed biophysical analysis using circular dichroism, visible absorption, UV-thermal melting and steady-state fluorescence spectroscopy, reporting destabilization of MRP1 GQ by cationic porphyrins. Results suggest that TMPyP4 and TMPyP3 interact with GQ with a binding affinity of 106 to 107 M-1 . Thermodynamic analysis indicated a significant decrease in melting temperature of GQ (ΔTm of 15.5°C-23.5°C), in the presence of 2 times excess of porphyrins. This study provides the biophysical evidence indicating the destabilisation of a parallel DNA G-quadruplex by cationic porphyrins.

Gold(III) porphyrins: Synthesis and interaction with G-quadruplex DNA

G-quadruplex nucleic acids (G4s) are RNA and DNA secondary structures involved in the regulation of multiple key biological processes. They can be found in telomeres, oncogene promoters, RNAs, but also in viral genomes. Due to their unique structural features, very distinct from the canonical duplexes or single-strands, G4s represent promising pharmacological targets for small molecules, namely G4-ligands. Gold(III) penta-cationic porphyrins, as specific G4 ligands, are able to inhibit HIV-1 infectivity and their antiviral activity correlates with their affinity for G4s. Up to now, one of the best antiviral compounds is meso-5,10,15,20-tetrakis[4-(N-methyl-pyridinium-2-yl)phenyl]porphyrinato gold(III) (1). Starting from this compound, we report a structure/affinity relationship study of gold(III) cationic porphyrins to find out the best porphyrin candidate for functionalization, in order to study the antiviral mechanism of action of these gold(III) porphyrins.

Oxidative stress-mediated epigenetic regulation by G-quadruplexes.

Many cancer-associated genes are regulated by guanine (G)-rich sequences that are capable of refolding from the canonical duplex structure to an intrastrand G-quadruplex. These same sequences are sensitive to oxidative damage that is repaired by the base excision repair glycosylases OGG1 and NEIL1–3. We describe studies indicating that oxidation of a guanosine base in a gene promoter G-quadruplex can lead to up- and downregulation of gene expression that is location dependent and involves the base excision repair pathway in which the first intermediate, an apurinic (AP) site, plays a key role mediated by AP endonuclease 1 (APE1/REF1). The nuclease activity of APE1 is paused at a G-quadruplex, while the REF1 capacity of this protein engages activating transcription factors such as HIF-1α, AP-1 and p53. The mechanism has been probed by in vitro biophysical studies, whole-genome approaches and reporter plasmids in cellulo. Replacement of promoter elements by a G-quadruplex sequence usually led to upregulation, but depending on the strand and precise location, examples of downregulation were also found. The impact of oxidative stress-mediated lesions in the G-rich sequence enhanced the effect, whether it was positive or negative.

2'-O-Methylation can increase the abundance and lifetime of alternative RNA conformational states.

2′-O-Methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3′-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of flexible RNAs containing bulges and internal loops. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of low-populated short-lived excited states by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi-value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.

2'-Fluorinated Hydantoins as Chemical Biology Tools for Base Excision Repair Glycosylases.

The guanine oxidation products, 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), are mutagenic and toxic base lesions that are removed by Fpg, Nei, and the Nei-like (NEIL) glycosylases as the first step in base excision repair (BER). The hydantoins are excellent substrates for the NEIL glycosylases in a variety of DNA contexts beyond canonical duplex DNA, implicating the potential impact of repair activity on a multitude of cellular processes. In order to prepare stable derivatives as chemical biology tools, oligonucleotides containing fluorine at the 2'-position of the sugar of 8-oxo-7,8-dihydro-2'-deoxyguanosine2'-F-OG) were synthesized in ribo and arabino configuration. Selective oxidation of 2'-F-OG within a DNA oligonucleotide provided the corresponding 2'-F-Gh or 2'-F-Sp containing DNA. The 2'-F-hydantoins in duplex DNA were found to be highly resistant to the glycosylase activity of Fpg and NEIL1 compared to the unmodified lesion substrates. Surprisingly, however, some glycosylase-mediated base removal from both the 2'-F-ribo- and 2'-F-arabinohydantoin duplex DNA was observed. Notably, the associated β-lyase strand scission reaction of the 2'-F-arabinohydantoins was inhibited such that the glycosylases were "stalled" at the Schiff-base intermediate. Fpg and NEIL1 showed high affinity for the 2'-F-Gh duplexes in both ribo and arabino configurations. However, binding affinity assessed using catalytically inactive variants of Fpg and NEIL1 indicated higher affinity for the 2'-F-riboGh-containing duplexes. The distinct features of glycosylase processing of 2'-F-ribohydantoins and 2'-F-arabinohydantoins illustrate their utility to reveal structural insight into damage recognition and excision by NEIL and related glycosylases and provide opportunities for delineating the impact of lesion formation and repair in cells.

8-Pyrenylvinyl Adenine Controls Reversible Duplex Formation between Serinol Nucleic Acid and RNA by [2 + 2] Photocycloaddition.

Photocontrol of duplex formation between the totally artificial serinol nucleic acid (SNA) and target RNA was made possible using a photoresponsive nucleobase 8-pyrenylvinyl adenine (PVA). PVA residues in SNA can be induced to undergo intrastrand [2 + 2] photocycloaddition by 455 nm light. Effective cycloreversion of the PVA photodimer results from irradiation with 340 nm light. These reactions occurred in high yield, rapidly, selectively, and reversibly. When the PVA-SNA/RNA duplex was irradiated with 455 nm light, almost complete dissociation of the duplex was attained, and 340 nm light restored duplex formation by cycloreversion. This is the first example of use of photocycloaddition and cycloreversion to photoregulate canonical duplex formation and dissociation reversibly at constant temperature. Thus, SNA bearing PVA residues have potential for use in photocontrollable biological tools targeting endogenous RNAs in cells as well as photodriven SNA machines.

Parallel Guanine Duplex and Cytosine Duplex DNA with Uninterrupted Spines of AgI-Mediated Base Pairs.

Hydrogen bonding between nucleobases produces diverse DNA structural motifs, including canonical duplexes, guanine (G) quadruplexes, and cytosine (C) i-motifs. Incorporating metal-mediated base pairs into nucleic acid structures can introduce new functionalities and enhanced stabilities. Here we demonstrate, using mass spectrometry (MS), ion mobility spectrometry (IMS), and fluorescence resonance energy transfer (FRET), that parallel-stranded structures consisting of up to 20 G-AgI-G contiguous base pairs are formed when natural DNA sequences are mixed with silver cations in aqueous solution. FRET indicates that duplexes formed by poly(cytosine) strands with 20 contiguous C-AgI-C base pairs are also parallel. Silver-mediated G-duplexes form preferentially over G-quadruplexes, and the ability of Ag+ to convert G-quadruplexes into silver-paired duplexes may provide a new route to manipulating these biologically relevant structures. IMS indicates that G-duplexes are linear and more rigid than B-DNA. DFT calculations were used to propose structures compatible with the IMS experiments. Such inexpensive, defect-free, and soluble DNA-based nanowires open new directions in the design of novel metal-mediated DNA nanotechnology.

Atomic structures of excited state A-T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations.

NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3' and C4' spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.

High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

Pseudorotaxane formation via the slippage process with chemically cyclized oligonucleotides.

Circular nucleic acids have been utilized for versatile applications by taking advantage of the unique characteristic of their circular structure. In our previous study, we found that the chemically-cyclized ODN (cyODN) with double-tailed parts formed a pseudorotaxane structure with the target via the slippage process. We now report the investigation of the slippage properties and the mechanism of the slippage process using six different cyODNs. Our results indicate that the formation efficiency significantly depend on the temperature, the ring size, the target length and the mismatched position of the target. The kinetic studies also showed that this pseudorotaxane formation would proceed via a non-threaded structure which hybridizes with the target at the double-tailed parts. In addition, the resulting pseudorotaxanes showed interesting characteristics unlike the canonical duplex such as the hysteresis loop in the Tm measurements and the kinetic stabilization by lengthening the target. This information will be fundamentally important for finding new functions of circular nucleic acids and elucidating the threading mechanism regarding other synthetic small molecules and biopolymers.

Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin.

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)3) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.

X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues.

Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5′-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

Unique Length-Dependent Biophysical Properties of Repetitive DNA.

Expansion of a trinucleotide repeat (TNR) sequence is the molecular signature of several neurological disorders. The formation of noncanonical structures by the TNR sequence is proposed to contribute to the expansion mechanism. Furthermore, it is known that the propensity for expansion increases with repeat length. In this work, we use calorimetry to describe the thermodynamic parameters (ΔH, TΔS, and ΔG) of the noncanonical stem-loop hairpins formed by the TNR sequences (CAG)n and (CTG)n, as well as the canonical (CAG)n/(CTG)n duplexes, for n = 6-14. Using a thermodynamic cycle, we calculated the same thermodynamic parameters describing the process of converting from noncanonical stem-loop hairpins to a canonical duplex. In addition to these thermodynamic analyses, we used spectroscopic techniques to determine the rate at which the noncanonical structures convert to duplex and the activation enthalpy ΔH(⧧) describing this process. We report that the thermodynamic parameters of unfolding the stem-loop (CTG)n and (CAG)n hairpins, along with the thermodynamic and kinetic properties of hairpin to duplex conversion, do not proportionally correspond to the increase in length, but rather show a unique pattern that depends on whether the sequence has an even or odd number of repeats.