On chip quadruplex priming amplification for quantitative isothermal diagnostics.

Nucleic acid testing is a common technique for medical diagnostics. For example, it is used to detect HIV treatment failure by monitoring viral load levels. Quadruplex Priming Amplification (QPA) is an isothermal nucleic acid amplification technique that requires little power and few chemical reagents per assay, all features that make QPA well suited for point-of-care (POC) diagnostics. The QPA assay can be further optimized by integrating it with microfluidic devices that can automate and combine multiple reaction steps and reduce the quantity and cost of reagents per test. In this study, a real-time, exponential QPA reaction is demonstrated for the first time in a microfluidic chip, where the reaction was not inhibited and supported performance levels comparable to a commercially-available, non-microfluidics setup.

Quadruplex priming amplification for the detection of mRNA from surrogate patient samples.

Simple and rapid methods for detecting mRNA biomarkers from patient samples are valuable in settings with limited access to laboratory resources. In this report, we describe the development and evaluation of a self-contained assay to extract and quantify mRNA biomarkers from complex samples using a novel nucleic acid-based molecular sensor called quadruplex priming amplification (QPA). QPA is a simple and robust isothermal nucleic acid amplification method that exploits the stability of the G-quadruplex nucleotide structure to drive spontaneous strand melting from a specific DNA template sequence. Quantification of mRNA was enabled by integrating QPA with a magnetic bead-based extraction method using an mRNA-QPA interface reagent. The assay was found to maintain >90% of the maximum signal over a 4 °C range of operational temperatures (64-68 °C). QPA had a dynamic range spanning four orders of magnitude, with a limit of detection of ~20 pM template molecules using a highly controlled heating and optical system and a limit of detection of ~250 pM using a less optimal water bath and plate reader. These results demonstrate that this integrated approach has potential as a simple and effective mRNA biomarker extraction and detection assay for use in limited resource settings.

Folding of the thrombin aptamer into a G-quadruplex with Sr(2+): stability, heat, and hydration.

It has been shown that the DNA aptamer d(G(2)T(2)G(2)TGTG(2)T(2)G(2)) adopts an intramolecular G-quadruplex structure in the presence of K+. Its affinity for trombin has been associated with the inhibition of thrombin-catalyzed fibrin clot formation. In this work, we used a combination of spectroscopy, calorimetry, density, and ultrasound techniques to determine the spectral characteristics, thermodynamics, and hydration effects for the formation of G-quadruplexes with a variety of monovalent and divalent metal ions. The formation of cation-aptamer complexes is relatively fast and highly reproducible. The comparison of their CD spectra and melting profiles as a function of strand concentration shows that K+, Rb+, NH(4)+, Sr(2+), and Ba(2+) form intramolecular cation-aptamer complexes with transition temperatures above 25 degrees C. However, the cations Li+, Na+, Cs+, Mg(2+), and Ca(2+) form weaker complexes at very low temperatures. This is consistent with the observation that metal ions with ionic radii in the range 1.3-1.5 A fit well within the two G-quartets of the complex, while the other cations cannot. The comparison of thermodynamic unfolding profiles of the Sr(2+)-aptamer and K+ -aptamer complexes shows that the Sr(2+)-aptamer complex is more stable, by approximately 18 degrees C, and unfolds with a lower endothermic heat of 8.3 kcal/mol. This is in excellent agreement with the exothermic heats of -16.8 kcal/mol and -25.7 kcal/mol for the binding of Sr(2+) and K+ to the aptamer, respectively. Furthermore, volume and compressibility parameters of cation binding show hydration effects resulting mainly from two contributions: the dehydration of both cation and guanine atomic groups and water uptake upon the folding of a single-strand into a G- quadruplex structure.

Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration.

We report on the physicochemical effects resulting from incorporating a 5-(3-aminopropyl) side chain onto a 2'-deoxyuridine (dU) residue in a short DNA hairpin. A combination of spectroscopy, calorimetry, density and ultrasound techniques were used to investigate both the helix-coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC) [N = dU, deoxythymidine (dT) or 5-(3-aminopropyl)-2'-deoxyuridine (dU*)], and the interaction of each hairpin with Mg(2+). All three molecules undergo two-state transitions with melting temperatures (T(M)) independent of strand concentration that indicates their intramolecular hairpin formation. The unfolding of each hairpin takes place with similar T(M) values of 64-66 degrees C and similar thermodynamic profiles. The unfavorable unfolding free energies of 6.4-6.9 kcal/mol result from the typical compensation of unfavorable enthalpies, 36-39 kcal/mol, and favorable entropies of approximately 110 cal/mol. Furthermore, the stability of each hairpin increases as the salt concentration increases, the T(M)-dependence on salt yielded slopes of 2.3-2.9 degrees C, which correspond to counterion releases of 0.53 (dU and dT) and 0.44 (dU*) moles of Na(+) per mole of hairpin. Absolute volumetric and compressibility measurements reveal that all three hairpins have similar hydration levels. The electrostatic interaction of Mg(2+) with each hairpin yielded binding affinities in the order: dU > dT > dU*, and a similar release of 2-4 electrostricted water molecules. The main result is that the incorporation of the cationic 3-aminopropyl side chain in the major groove of the hairpin stem neutralizes some local negative charges yielding a hairpin molecule with lower charge density.

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements.

The interaction of hexamminecobalt(III), Co(NH(3))(6)(3+), with 160 and 3000-8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH(3))(6)(3+) binding up to the molar ratio [Co(NH(3))(6)(3+)]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH(3))(6)(3+)]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH(3))(6)(3+)]/[P] = 0.3. In the case of 3000-8000 bp DNA only two processes were observed: (i) binding up to [Co(NH(3))(6)(3+)]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B-->Psi transition after [Co(NH(3))(6)(3+)]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH(3))(6)(3+) binding on volume and compressibility have been obtained. The binding of Co(NH(3))(6)(3+) to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH(3))(6)(3+): DeltaV = 9 cm(3) mol(-1) and Deltakappa = 33 x 10(-4) cm(3) mol(-1) bar(-1). The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH(3))(6)(3+) and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.

Interaction of alkaline-earth metal ions with calf thymus DNA. Volume and compressibility effects in diluted aqueous solutions.

The binding of Mg2+, Ca2+, Sr2+ and Ba2+ ions to calf thymus DNA in solutions has been investigated by ultrasonic and densimetric techniques. The obtained parameters, the apparent molar volume, phiV, and the apparent molar adiabatic compressibility, phiK(S), are very sensitive to hydration of investigated molecules. The interaction between the cations and DNA is accompanied by overlapping their hydration shells and consequently releasing the water molecules from hydration shells to bulk state. The change in the hydration is reflected in the measured parameters, phiV and phiK(S). The magnitude of these hydration changes is determined by the position of the cation relative to DNA atomic groups involved in the binding, and thus can characterize the structure of cation-DNA complexes. The values of the dehydration effects of the binding, deltaphiV and deltaphiK(S), correspond to two direct or higher number of indirect contacts between calf thymus DNA and the cations.

Hydration effects of Ni(2+) binding to synthetic polynucleotides with regularly alternating purine-pyrimidine sequences.

High precision ultrasonic and densimetric techniques have been used to study the interaction of Ni(2+)ions with right-handed poly[d(G-C)].poly[d(G-C)], poly-[d(A-C)].poly[d(G-T)] and poly[d(A-T)].poly[d(A-T)] in 5 mM CsCl, 0.2 mM HEPES, pH 7.5 at 20 degrees C. From these measurements the changes in the apparent molar volume and the apparent molar adiabatic compressibility due to the interaction have been obtained. The volume effects of the binding, calculated per mole of Ni(2+)ions, range from 11.7 to 23.9 cm(3)mol(-1)and the compressibility effects range from 19.3 x 10(-4)to 43.1 x 10(-4)cm(3)mol(-1)bar(-1). These data are interpreted in terms of dehydration of the polynucleotides and Ni(2+)ions, i.e. the release of water molecules from the hydration shells of the molecules. An increase in G+C content gives an increase in volume and compressibility effects, indicating a rise in the extent of dehydration. The dehydration effects of Ni(2+)binding to poly[d(G-C)].poly[d(G-C)] are approximately twice those of poly[d(A-T)].poly[d(A-T)]. The volume and compressibility effects of Ni(2+)-EDTA complex formation have also been measured and used as a model system for quantitative estimation. These values revealed that Ni(2+)ions can coordinate two atomic groups of poly[d(G-C)]. poly[d(G-C)], while in the case of the Ni(2+)-poly[d(A-T)]. poly[d(A-T)] complex volume and compressibility effects correspond to one direct or two indirect (through water) contacts.

Liposome-DNA interaction: microcalorimetric study.

The authors applied differential scanning calorimetry (DSC) for studying the thermodynamic characteristics of DNA-liposome interactions. At the first stage, the melting curves of the 'order-disorder' thermal transition for lipid component and of the 'helix-coil' transition for DNA were obtained. At the second stage, the phase behavior of the DNA-lipid mixture as a function of both components (lipid/DNA ratio) was obtained. The liposome-DNA interaction was investigated comparing the melting curves of the pure components and the mixture.