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.
Kankia
Besik I
BI
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. bkankia@umn.edu
D052157:Aptamers, Nucleotide; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D054856:G-Quadruplexes; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013816:Thermodynamics
D052157:Aptamers, Nucleotide; D009841:Oligonucleotides; C081676:thrombin aptamer; D004247:DNA