Trinucleotide Rolling Circle Amplification: A Novel Method for the Detection of RNA and DNA

Most natural DNA and RNA are devoid of long trinucleotide (TN) sequences that lack one specific nucleotide (missing nucleotide (MN)). Here we developed a novel method that is based on rolling circle amplification (RCA), in which the TN-information of short TN stretches is sequence-specifically recognized, transferred, extended, amplified and detected by padlock probes that consist entirely of nucleotides complementary to the three nucleotides present in the target sequence (complementary TN-information). Upon specific head-to-tail annealing and ligation to the TN-target sequence, these padlock probes represent extended complementary TN versions of the target sequence that can be further amplified by trinucleotide rolling circle amplification (TN-RCA). Since during TN-RCA the MN (as dNTP) is not added, background amplification is minimized with endogenous RNA/DNA (which mostly would require all four dNTP). Therefore, various labelled dNTP can be added to the TN-RCA reaction that enables the separation, isolation and detection of the amplified single-stranded DNA (ssDNA). Here the TN-RCA method is exemplified with RNA/DNA from Zika virus and from human papilloma virus (HPV). TN-RCA is a novel isothermal amplification technique that can be used for sensitive sequence-specific detection and diagnosis of natural and synthetic DNA or RNA containing TN stretches with low background in short time.