Rapid, Amplification- and Label-Free, Ultrahigh-Confidence Counting of Single RNA and Other Biomarker Molecules in Patient Samples
MicroRNAs (miRNAs) and other non-coding RNAs (ncRNAs) have recently emerged as promising diagnostic biomarkers for virtually all pathologies, including cancer, heart disease, diabetes, Alzheimer’s, and viral infection. We have developed a novel technology platform for the specific and ultra-sensitive detection of virtually any biomarker of interest through a kinetic “fingerprinting” approach called Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS). The SiMREPS platform achieves the rapid, amplification-free counting of single label-free miRNA and ncRNA molecules, bypasses thermodynamic limits of specificity, virtually eliminates false positives, utilizes no enzymes and hence requires minimal pre-treatment of biological samples prior to biomarker detection and avoids the introduction of sampling bias. We have demonstrated the ultrahigh-confidence single-molecule detection of endogenous miRNAs in minimally treated patient samples such as blood serum and cell extracts in under 30 minutes, as well as the >500-fold discrimination against a single nucleotide polymorphism. In addition, SiMREPS can utilize its kinetic “fingerprint” to sensitively detect changes in the accessibility of a sensor RNA or DNA upon biomarker binding to detect virtually any type of secondary biomarker.