Self-dissociative primers for nucleic acid amplification and detection based on DNA quadruplexes with intrinsic fluorescence.

We have developed a new method, quadruplex priming amplification, to greatly simplify nucleic acid amplification and real-time quantification assays. The method relies on specifically designed guanine-rich primers, which after polymerase elongation are capable of spontaneous dissociation from target sites and forming DNA quadruplex. The quadruplex is characterized by significantly more favorable thermodynamics than the corresponding DNA duplexes. As a result, target sequences are accessible for the next round of priming and DNA amplification proceeds under isothermal conditions with improved product yield. In addition, the quadruplex formation is accompanied by an increase in intrinsic fluorescence of the primers, allowing simple and accurate detection of product DNA.

A real-time assay for monitoring nucleic acid cleavage by quadruplex formation.

Direct and straightforward methods to follow nucleic acid cleavage are needed. A spectrophotometric quadruplex formation assay (QFA) was developed, which allows real-time monitoring of site-specific cleavage of nucleic acids. QFA was applied to study both protein and nucleic acid restriction enzymes, and was demonstrated to accurately determine Michaelis-Menten parameters for the cleavage reaction catalyzed by EcoRI. QFA can be used to study the mechanisms of protein-nucleic acid recognition. QFA is also a useful tool for dissecting individual nicking rates of a double-stranded cleavage.

The HIV-1 central DNA flap region contains a "flapping" third strand.

Due to the discontinuous nature of HIV-1 plus-strand DNA synthesis, a 99-nt plus-strand overhang termed the "central DNA flap" is present near the center of the proviral DNA prior to integration. The flap appears to have stabilizing and/or protective effects on viral DNA, which has been hypothesized to be due to a specific conformation adopted by the three-stranded region. The 5' end of the flap sequence is very purine rich and has the potential to adopt different secondary structures (e.g., duplex, triplex or quadruplex). In the present work, circular dichroism spectroscopy and thermal unfolding techniques were used to characterize an 89-nt long DNA sequence designed to mimic the three-stranded region at the 5' end of HIV-1 proviral DNA. The effect of addition of the HIV-1 nucleocapsid protein (NC) on the nucleic acid structure was also examined. Although, guanine-rich short oligonucleotides derived from the DNA flap demonstrated CD spectra characteristic to parallel quadruplexes, this analysis reveals that the extended 89-nt construct folds into a canonical duplex with a "flapping" third strand both in the absence and presence of NC.

Unfolding of DNA quadruplexes induced by HIV-1 nucleocapsid protein.

The human immunodeficiency virus type 1 nucleocapsid protein (NC) is a nucleic acid chaperone that catalyzes the rearrangement of nucleic acids into their thermodynamically most stable structures. In the present study, a combination of optical and thermodynamic techniques were used to characterize the influence of NC on the secondary structure, thermal stability and energetics of monomolecular DNA quadruplexes formed by the sequence d(GGTTGGTGTGGTTGG) in the presence of K+ or Sr2+. Circular dichroism studies demonstrate that NC effectively unfolds the quadruplexes. Studies carried out with NC variants suggest that destabilization is mediated by the zinc fingers of NC. Calorimetric studies reveal that NC destabilization is enthalpic in origin, probably owing to unstacking of the G-quartets upon protein binding. In contrast, parallel studies performed on a related DNA duplex reveal that under conditions where NC readily destabilizes and unfolds the quadruplexes, its effect on the DNA duplex is much less pronounced. The differences in NC's ability to destabilize quadruplex versus duplex is in accordance with the higher DeltaG of melting for the latter, and with the inverse correlation between nucleic acid stability and the destabilizing activity of NC.

Optical absorption assay for strand-exchange reactions in unlabeled nucleic acids.

The nucleic acid exchange reaction is a common feature for genetic recombination, DNA replication and transcription. Due to the fact that in the strand-exchange reactions the reactant and product molecules have similar or identical nucleotide sequences, the reaction is undetectable. As a rule, the nucleic acids with radioactive or fluorescence labels are used in such studies. Besides the fact that the labels can perturb the reaction and pose a health risk to the investigators, the assays usually involve extra experimental steps: quenching the reaction, separation, visualization and quantification of the products. Here, we describe a straightforward, direct and precise method to study strand-exchange reaction of unlabeled nucleic acids by real-time measurements of optical absorption. The method takes advantage of the property of some guanine-rich oligonucleotides to adopt monomolecular quadruplex conformation in the presence of certain cations. The conformation is characterized by significant absorption in long-wavelength range of the ultraviolet region where usually other secondary structures are transparent. The 'signal' oligonucleotide is incorporated into reactant duplex by annealing with target sequence. Adding the replacement sequence initiates the release of the 'signal' oligonucleotide into solution, which is accompanied by ultraviolet absorption in long-wavelength range.

Mg2+-induced triplex formation of an equimolar mixture of poly(rA) and poly(rU).

Magnesium ions strongly influence the structure and biochemical activity of RNA. The interaction of Mg2+ with an equimolar mixture of poly(rA) and poly(rU) has been investigated by UV spectroscopy, isothermal titration calorimetry, ultrasound velocimetry and densimetry. Measurements in dilute aqueous solutions at 20 degrees C revealed two differ ent processes: (i) Mg2+ binding to unfolded poly(rA)*poly(rU) up to [Mg2+]/[phosphate] = 0.25; and (ii) poly(rA)*2poly(rU) triplex formation at [Mg2+]/[phosphate] between 0.25 and 0.5. The enthalpies of these two different processes are favorable and similar to each other, approximately -1.6 kcal x mol(-1) of base pairs. Volume and compressibility effects of the first process are positive, 8 cm3 x mol(-1) and 24 x 10(-4) cm3 x mol(-1) x bar(-1), respectively, and correspond to the release of water molecules from the hydration shells of Mg2+ and the polynucleotides. The triplex formation is also accompanied by a positive change in compressibility, 14 x 10(-4) cm3 x mol(-1) x bar(-1), but only a small change in volume, 1 cm3 x mol(-1). A phase diagram has been constructed from the melting experiments of poly(rA)*poly(rU) at a constant K+ concentration, 140 mM, and various amounts of Mg2+. Three discrete regions were observed, corresponding to single-, double- and triple-stranded complexes. The phase boundary corresponding to the transition between double and triple helical conformations lies near physiological salt concentrations and temperature.

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA.

The introduction of cationic 5-(omega-aminoalkyl)-2'-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2'-deoxyuridine ('control') or 5-(3-aminopropyl)-2'-deoxyuridine ('modified'). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the 'modified' duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15 degrees C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the 'control' duplex releases water while the 'modified' duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15 degrees C yielded a marginal differential DeltaG degrees term, positive DeltaDeltaH(ITC)-Delta(TDeltaS) compensation, negative DeltaDeltaV and a net release of counterions. The opposite signs of the differential enthalpy-entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the 'modified' duplex.

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies.

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.

HIV-integrase aptamer folds into a parallel quadruplex: a thermodynamic study.

Short guanine-rich sequences have a tendency to form quadruplexes that are stabilized by G-quartets with specific cation coordination. Quadruplexes are part of telomeres at the ends of chromosomes and play an important role in the regulation of gene expression. In addition, there is a strong interest in the therapeutic and biotechnological potential of quadruplex oligonucleotides. The HIV-integrase aptamer, d(GGGT)(4), demonstrated unusually favorable van't Hoff thermodynamics, and based on NMR studies the aptamer was proposed to fold into an antiparallel structure. Here we probed an apparent discrepancy between the NMR structure and the quadruplex topology suggested by circular dichroism (CD). Systematic thermodynamic analyses of d(GGGT)(4) and variants containing sequence modifications or missing specific nucleotides are consistent with a parallel quadruplex fold. CD studies carried out over a wide concentration range did not support a possible structural transition upon increasing strand concentration. Taken together, both optical and thermodynamic studies performed here strongly support a parallel fold for the d(GGGT)(4) aptamer.

Inner-sphere complexes of divalent cations with single-stranded poly(rA) and poly(rU).

A combination of ultrasound velocimetry, density, and UV spectroscopy has been employed to study the hydration effects of binding of Mn(2+) and alkaline-earth cations to poly(rA) and poly(rU) single strands. The hydration effects, obtained from volume and compressibility measurements, are positive due to overlapping the hydration shells of interacting molecules and consequently releasing the water molecules to bulk state. The volume effects of the binding to poly(rA), calculated per mole of cations, range from 30.6 to 40.6 cm(3) mol(-1) and the compressibility effects range from 59.2 x 10(-4) to 73.6 x 10(-4) cm(3) mol(-1) bar(-1). The volume and compressibility effects for poly(rU) are approximately 17 cm(3) mol(-1) and approximately 50 x 10(-4) cm(3) mol(-1) bar(-1), respectively. The comparative analysis of the dehydration effects suggests that the divalent cations bind to the polynucleotides in inner-sphere manner. In the case of poly(rU) the dehydration effects correspond to two direct coordination, probably between adjacent phosphate groups. The optical study did not reveal any effects of cation on the secondary structure or aggregation of poly(rU). In the case of single-helical poly(rA) binding is more specific: dehydration effects correspond to three to five direct contacts and must involve atomic groups of adenines, and the divalent cations stabilize and aggregate the polynucleotide.

DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics, hydration, sequence, and ionic effects.

The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG]] or cis-[Pt(NH(3))(2) [d(ApG]] cross-links, and their corresponding unmodified duplexes. The platinated duplexes are less stable and unfold with lower T(M)s (and Delta G degrees s) in enthalpy-driven reactions, which indicates a loss of favorable base-pair stacking interactions. The folding thermodynamics and hydration effects for the first set of decamers containing the d(GpG) cross-link was investigated by a combination of titration calorimetry, density, and ultrasound techniques. The hydration parameters showed an uptake of structural water by the platinated duplex and a release of electrostricted water by the control duplex. Relative to the unmodified duplex, the folding of the platinated duplex at 20 degrees C yielded a positive Delta Delta G degrees term [and positive Delta Delta H-Delta(T Delta S) compensation] and a negative differential volume change. The opposite signs of the Delta Delta G degrees and Delta Delta V terms confirmed its uptake of structural water. Further, solvent-accessible surface areas calculations for a similar pair of dodecamer duplexes indicated that the modified duplex has a 503 oeA(2) higher polar and nonpolar surface area that is exposed to the solvent. Therefore, the incorporation of a platinum adduct in duplex DNA disrupts favorable base-pair stacking interactions, yielding a greater exposure of aromatic bases to the solvent, which in turn immobilizes structural water. The overall results correlate nicely with the results reported in the available structural data of nuclear magnetic resonance solution studies.