A Chimeric DNA/RNA Antiparallel Quadruplex with Improved Stability.

Nucleic acid quadruplexes are proposed to play a role in the regulation of gene expression, are often present in aptamers selected for specific binding functions and have potential applications in medicine and biotechnology. Therefore, understanding their structure and thermodynamic properties and designing highly stable quadruplexes is desirable for a variety of applications. Here, we evaluate DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG) in the presence of either K+ or Sr2+ . TBA predominantly folds into a chair-type configuration containing two G-tetrads, with G residues in both syn and anti conformation. All chimeras with DNA→RNA substitutions (G→g) at G residues requiring the syn conformation demonstrated strong destabilization. In contrast, G→g substitutions at Gs with anti conformation increased stability without affecting the monomolecular chair-type topology. None of the DNA→RNA substitutions in loop positions affected the quadruplex topology; however, these substitutions varied widely in their stabilizing or destabilizing effects in an unpredictable manner. This analysis allowed us to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all-DNA construct. These results have implications for a variety of quadruplex-based applications including for the design of dynamic nanomachines.

Structure of Tetrahelical DNA Homopolymers Supports Quadruplex World Hypothesis.

We previously reported a tetrahelical monomolecular architecture of DNA, tmDNA, which employs G-quartets and an all-parallel GGGTGGGTGGGTGGG (G3T) quadruplex as the repeating unit. Based on thermodynamic and kinetic studies, we proposed that covalently joined (G3T) n units formed an uninterrupted programmable homopolymer; however, structural evidence for the tmDNA architecture was lacking. Here, we used NMR spectroscopy of wild-type and single-inosine-substituted constructs to characterize both monomolecular (G3T)2 and bimolecular quadruplex-Mg-coupled versions of tmDNA. The NMR results support an architecture consisting of uninterrupted stacked G-tetrads in both the monomolecular constructs and bimolecular assemblies. Taken together, these data support the formation of a stable programmable homopolymeric tmDNA architecture, which may have been a precursor to the modern-day Watson-Crick DNA duplex.

Isothermal amplification of long DNA segments by quadruplex priming amplification.

Amplification of long DNA segments with the highest possible specificity and lowest bias is one of the main goals of modern genomics. Quadruplex priming amplification (QPA) is a single-primer isothermal method, which employs the free energy of quadruplex structures as the driving force for DNA amplification without any extra reaction components. As a result, QPA represents one of the simplest isothermal assays and was previously shown to be suitable for amplification of relatively short DNA sequences. The current study reveals that single-primer QPA can be used for both exponential and linear amplification of relatively long DNA segments (>100 nt), and switching between these modes can be accomplished by simple re-design of the primer used. While exponential amplification resulted in production of some undesired higher molecular weight species, linear QPA demonstrated highly specific amplification of the target molecules without any side products.

Sr(2+) induces unusually stable d(GGGTGGGTGGGTGGG) quadruplex dimers.

Guanine-rich sequences are able to form quadruplexes consisting of G-quartet structural units. Quadruplexes play an important role in the regulation of gene expression and have therapeutic and biotechnological potential. The HIV-1 integrase inhibitor, (GGGT)4 , and its variants demonstrate unusually high thermal stability. This property has been exploited in the use of quadruplex formation to drive various endergonic reactions of nucleic acids such as isothermal DNA amplification. Quadruplex stability is mainly determined by cations, which specifically bind into the inner core of the structure. In the present work, we report a systematic study of a variant of the HIV-1 integrase inhibitor, GGGTGGGTGGGTGGG (G3T), in the presence of alkali and alkaline-earth cations. We show that Sr(2+) -G3T is characterized by the highest thermal stability and that quadruplex formation requires only one Sr(2+) ion that binds with low micromolar affinity. These concentrations are sufficient to drive robust isothermal quadruplex priming DNA amplification reaction. The Sr(2+) -quadruplexes are also able to form unusually stable dimers through end-to-end stacking. The multimerization can be induced by a combination of quadruplex forming cations (i.e., K(+) or Sr(2+) ) and non-specific Mg(2+) .

Stable Domain Assembly of a Monomolecular DNA Quadruplex: Implications for DNA-Based Nanoswitches.

In the presence of K(+) ions, the 5'-GGGTGGGTGGGTGGG-3' (G3T) sequence folds into a monomolecular quadruplex with unusually high thermal stability and unique optical properties. In this study we report that although single G3T molecules unfold and fold rapidly with overlapping melting and refolding curves, G3T multimers (G3T units covalently attached to each other) demonstrate highly reproducible hysteretic behavior. We demonstrate that this behavior necessitates full-length tandem G3T monomers directly conjugated to each other. Any modification of the tandem sequences eliminates the hysteresis. The experimentally measured kinetic parameters and equilibrium transition profiles suggest a highly specific two-state transition in which the folding and unfolding of the first G3T monomer is rate-limiting for both annealing and melting processes. The highly reproducible hysteretic behavior of G3T multimers has the potential to be used in the design of heat-stimulated DNA switches or transistors.

Exponential quadruplex priming amplification for DNA-based isothermal diagnostics.

Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point-of-care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self-dissociate from the primer-binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 10(10)-fold amplification of DNA signal in less than 40 min.

Quadruplex priming amplification combined with nicking enzyme for diagnostics.

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin.

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3-methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI-based QPA is a useful assay for DNA amplification and detection.

Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.

Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.

Effect of Zn(II) on the reduction and accumulation of Cr(VI) by Arthrobacter species.

Natural habitats are often characterized by the coexistence of Zn and Cr. This study assessed the potential of two Gram-positive, Cr(VI)-reducing, aerobic bacterial strains belonging to Arthrobacter genera, which were isolated from basalt samples taken from the most polluted region of the Republic of Georgia, to remediate Cr(VI) in environments in the presence of Zn(II). Our batch experiments revealed that the addition of Zn(II) to the tested bacterial cells significantly enhanced the accumulation of Cr. According to electron spin resonance (ESR) measurements, the presence of Zn(II) ions did not change the nature of Cr(V) and Cr(III) complexes generated during the microbial reduction of Cr(VI). The efficiency of Cr(VI) reduction also remained unchanged after the addition of 50 mg/l of Zn(II) to the bacterial cells. However, at high concentrations of Zn(II) (higher than 200 mg/l), the transformation of Cr(VI) to Cr(V) and Cr(III) complexes decreases significantly. In addition, it was shown that the accumulation pattern of Zn in the tested bacterial species in the presence of 100 mg/l of Cr(VI) fits the Langmuir-Freundlich model well. The two tested bacterial strains exhibited different characteristics of Zn accumulation.