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.
Kankia
Besik I
BI
Department of Chemistry, University of Minnesota, MN 55455, USA. bkankia@umn.edu
D052157:Aptamers, Nucleotide / Q000032:analysis; D002942:Circular Dichroism; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D021881:DNA, Catalytic / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D054856:G-Quadruplexes; D007700:Kinetics; D012313:RNA / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D052157:Aptamers, Nucleotide; D021881:DNA, Catalytic; C081676:thrombin aptamer; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI