A Multipurpose Smart Probe Detection System for Homologous MicroRNAs

MicroRNAs (miRNAs) are now being detected by various analytical methods, including nucleic acid hairpin probes. However, these probes can only recognize one individual miRNA target. This means, for a given number of different miRNA targets, the same number of probes are needed. This is due to the sequence-specificity requirement of nucleic acids interactions, and of course, probe-target hybridizations. This project will develop a new multipurpose smart probe (MSP) detection system for Let-7 family of miRNAs (let-7a, -7b & -7c), which are homologous with very similar sequences. This means that only one smart probe will be able to recognize all the members of this family despite minor differences in their sequence. So, rather than one smart probe for each of let 7a, let-7b and let-7c, (one smart probe detecting one miRNA), this new MSP system will be able to recognize all the three miRNAs. In regular nucleic acid probe-target hybridizations, the probe is designed to only specifically recognize the perfect target sequence but not imperfect or mismatch sequences. This means the probe is specifically meant to interact (hybridize) with the perfect target sequence, while it should have little or no interaction with mismatch sequences because of the inherent sequence-specificity of the probes. So, even little non-specific hybridization between probe and mismatch sequences are undesirable. In this project, we will exploit non-specific hybridization between the MSP and the mismatch (non-target) sequences, which is usually undesirable. The MSP will naturally hybridize with perfect target sequence via specific hybridization. To a large extent, it will also hybridize with non-target (mismatch) sequences via non-specific hybridization. This means that the MSP can recognize the sequences of let-7a, let-7b and let-7c with almost equal propensity. Our hypothesis is that by combining this inherent specific/non-specific hybridization propensity of the MSP with nucleic acid blockers, which are purposely designed to completely block out certain specific or non-specific hybridizations, we can obtain sequence-specific hybridization of the MSP with any miRNA member of the let-7 family. We will measure fluorescence signal intensities of MSP-target hybrids and correlate the intensities with binding or hybridization strength. We will confirm our experimental results and conclusions with gel-electrophoresis. So, this project will demonstrate for the first time, a MSP for the sequence-specific detection of multiple miRNA sequences. As a preliminary proof of our hypothesis, preliminary estimates of melting temperature of probe-target hybrids of the MSP with each of let-7a, let-7b and let-7c revealed that they all have about the same melting temperature, which suggests the MSP will bind almost equally well with all three miRNAs.