Detection Of Single Nucleotide Polymorphism By Link - Rolling Amplification Reaction Combined With RNA FRET Probe

Accurate detection of sequence-specific nucleic acids and analysis of their variations at single nucleotide resolution have great significance for genetic research, diagnosis and personalized medicine. Due to its simplicity, robustness, specificity and versatility, RCA has been explored extensively to develop sensitive detection methods for single nucleotide polymorphism (SNP). Although RCA has been widely applied in various fields, some challenges of the RCA system for practical applications remain to be overcome. Firstly, the detection of RCA products generally needs electrophoresis separation or hybridizing fluorescence-labeled oligonucleotides to RCA products attached to a glass slide. Moreover, the ligation of padlock probes needs single strand (ss) DNA as the templates. The preparation of ssDNA templates from genomic DNA requires sophisticated procedure with restriction enzymes and exonucleases.To address the challenges of the RCA system, in this paper, we develop a RNA-based fluorescence resonance energy transfer (FRET) probe for monitoring of RCA products. The RNA molecule (14 nucleotides) containing a complementary sequence to the RCA products is labeled with a fluorophore at one end and a quencher at the other end in which the fluorescence can be quenched. The hybridization between the RNA molecules and RCA products can activate the ribonuclease H (RNase H) to digest the RNA FRET probes. The digestion of RNA FRET probes can restore the fluorescence signal and form the cycles of hybridization and digestion resulting in great signal amplification. The enzymatic signal amplification can bring the detection limit down to femtomolar, about four orders of magnitude lower than that of conventional MBs. On the other hand, we employ thermostable ligase to ligate the padlock probes with thermal cycles, where the double stranded genomic DNA can be directly serve as the template to circularize the padlock probes. More importantly, the circularized padlock probes can be linearly or exponentially increased with the thermal cycles, resulting in greatly enhanced sensitivity. Integration of the ligation of thermal cycles and the signal amplification of RNA-based FRET probes, the DNA targets can be detected as low as 10 aM at single nucleotide resolution.We have invented a new RNA FRET probe, which can be simply designed for detection of DNA targets without requirement of intensive optimization. More importantly, the RNA FRET probe can form the mechanism of target-dependent signal amplification with the catalysis of RNase H, leading to great improvement of detection sensitivity. As RCA assay has been widely applied in various fields, such as detection of SNP, DNA methylation, messenger RNA, microRNA, proteins, small molecules, and even cancer cells, our proposed assay strategy extends the application of RCA and provides a new platfrom for homogeneous detection of SNP.