Abstract
Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.
Highlights
The study of traditional biochemistry through kinetics is centered about the understanding of enzymes as catalysts with their kinetic parameters kcat and KM [1]
Agarose gels or polyacrylamide gel electrophoresis (PAGE) in native/denaturing conditions with either SDS or urea are commonly used in kinetic studies of nucleic acids and proteins [2,3]
The detection and quantification of the minimal amount of 18-nucleotide single-stranded DNA that cannot only be detected, but reliably quantified, was determined for various dyes and our measurements showed that SYBR Gold can efficiently detect and quantify as low as 1 picomole of a short DNA product, whereas the detection limit of approximately 0.2 pmol, provided by the manufacturer, cannot be precisely quantified [10]
Summary
The study of traditional biochemistry through kinetics is centered about the understanding of enzymes as catalysts with their kinetic parameters kcat and KM [1]. Agarose gels or polyacrylamide gel electrophoresis (PAGE) in native/denaturing conditions with either SDS or urea are commonly used in kinetic studies of nucleic acids and proteins [2,3]. Substrate-labeled nucleic acids that are separated in gels are revealed by autoradiography films, or by biomolecular imager screens. The advantage of using 32-P in DNA or RNA is that it does not change its chemical nature because it has the same size and charge as any other phosphorus in the nucleic acid backbone. Radiolabeled substrate kinetic assays are considered discontinuous: small aliquots are removed before being separated by gel electrophoresis. The detection of labeled molecules is very sensitive, the quality of the data depends
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