Novel DNA probes for detection and quantification of protein molecules

Abstract

Molecular Beacons (MBs), oligonucleotide probes that possess an inherent signal transduction mechanism and operate on the principle of "detection without separation", are becoming widely used in detection and quantification of biologically significant molecules both in vivo and in vitro. Until recently, MBs were used for oligonucleotide detection, primarily in PCR and solution studies. Here the use of these novel probes for both oligonucleotide and protein detection in complex solution will be discussed. These probes can be used as substrate for enzyme catalyzed cleavage reactions and allow for direct kinetic analysis of such enzymatic activity. Also, we demonstrate the use of these probes for detecting binding events by protein molecules such as SSB (single-stranded binding protein from E. coli) that bind but do not cleave these single stranded flourescent DNA probes. To further demonstrate the applicability of these probes toward protein detection, we show that MBs can be used to discriminate between isozymes of proteins such as lactate dehydrogenase. We will also briefly discuss the development of molecular aptamer beacon for specific protein recognition in real-time and in homogeneous solutions.