| Abstract
| - The interactions between two key macromolecular species, nucleic acids and proteins, control many importantbiological processes. There have been limited effectivemethodologies to study these interactions in real time. Inthis work, we have applied a newly developed molecularbeacon (MB) DNA probe for the analysis of an enzyme,lactate dehydrogenase (LDH), and for the investigation ofits properties of binding with single-stranded DNA. Molecular beacons are single-stranded oligonucleotide probesdesigned to report the presence of specific complementarynucleic acids by fluorescence detection. The interactionbetween LDH and MB has resulted in a significantfluorescence signal enhancement, which is used for theelucidation of MB/LDH binding properties. The processesof binding between MB and different isoenzymes of LDHhave been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the bindingconstant is 1.9 × 10-7 M-1. We have also studied salteffects, binding sites, temperature effects, pH effects, andthe binding specificities for different isoenzymes. Ourresults demonstrate that MB can be effectively used forsensitive protein quantitation and for efficient protein−DNA interaction studies. MB has a signal transductionmechanism built within the molecule and can thus beused for the development of rapid protein assays and forreal-time measurements.
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