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

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

Mg(2+)-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 Mg(2+) 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°C revealed two differ ent processes: (i) Mg(2+) binding to unfolded poly(rA)·poly(rU) up to [Mg(2+)]/[phosphate] = 0.25; and (ii) poly(rA)·2poly(rU) triplex formation at [Mg(2+)]/[phosphate] between 0.25 and 0.5. The enthalpies of these two different processes are favorable and similar to each other, ∼–1.6 kcal mol(–1) of base pairs. Volume and compressibility effects of the first process are positive, 8 cm(3) mol(–1) and 24 × 10(–4) cm(3) mol(–1) bar(–1), respectively, and correspond to the release of water molecules from the hydration shells of Mg(2+) and the polynucleotides. The triplex formation is also accompanied by a positive change in compressibility, 14 × 10(–4) cm(3) mol(–1) bar(–1), but only a small change in volume, 1 cm(3) 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 Mg(2+). 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