Lateral flow assay for visualized screening secondary structure of short DNA sequence

Short DNA sequences that own non-canonical structure play important role in gene replication, transcription, repair and re-combination processes, and some of them are related with neurodegenerative disease. Although certain DNA simulation software can predict whether a short DNA sequence has secondary structure, the predicted results are sometimes inconsistent with the real case. Due to the short fragment, their secondary structure is uneasy to be experimentally detected, which usually requires nuclear magnetic resonance (NMR) characterization. Here we propose a lateral flow assay for visualized screening secondary structure of short DNA sequence. The short DNA sequence to be investigated is grafted on the 5'-end of hairpin monomers as an extended overhang. If secondary structure is present in the extended overhang, the secondary structure will inhibit the hybridization chain reaction (HCR) of hairpin monomers triggered by initiator strands, so that initiator strands remain and can be detected by subsequent lateral flow assay. The initiator strands act as bridging strands to link DNA-modified gold nanoparticles and T-capture strands on the lateral flow strip, inducing color development of test line. Secondary structure of short DNA sequence is thus converted to test line coloration on the strip, which makes the determination visual, portable and easy-to-operate. The strategy provides a new way for determination secondary structure of short DNA sequence and is helpful for structure-function relationship study.