Coupling aptamer biosensors to signal amplification.

摘要

Nucleic acids amplification methods can be extremely useful for the identification and quantitation of nucleic acid analytes, but are more difficult to adapt to the detection of non-nucleic acid targets. To facilitate the development of nucleic acid amplification for small molecule and protein analytes, we have developed the use of aptazyme and conformation-switching aptamers to generate amplification signals upon interaction with their cognate analytes.; We have developed chip-based rolling circle amplification (RCA) for the detection of ATP utilizing a DNA aptazyme that could catalyze the ligation and circularization of a single-stranded DNA substrate upon ATP recognition. The method has demonstrated that aptazyme-coupled chip-based RCA could sensitively detect ATP and the reproducible signals can be easily read and acquired within a few minutes.; In addition to the design of aptazyme mediated ligation for the detection of small molecules, we have been interested in the adaptation of structure-switching aptamers to generate analyte-dependent ligations. We have developed a novel type of conformationswitching aptamer that can be circularized by T4 DNA ligase upon interaction with its protein target, PDGF. Using this structure-switching aptamer real-time RCA can be used to quantitate PDGF down to low-nanomolar range, even against a background of cellular lysate. Our results also demonstrate that real-time RCA has advantages over chip-based RCA.; Furthermore, we have coupled conformation-switching aptamers with binding to an antisense oligonucleotide in a way that leads to ligation and the formation of a novel amplicon for real-time PCR. We have explored different strategies from four-piece to two-piece ligations. Our results show that the three-piece has sensitivity and simplicity over the four-piece ligation. However, both four-piece and three-piece ligations require ligation time as long as 8 hours, which is not practical for clinical diagnostics. Therefore, we have simplified the detection into a two-piece ligation, where the antisense sequence is attached to the aptamer and upon binding to protein analyte (PDGF or thrombin) the displaced antisense sequence is ligated to a substrate oligonucleotide. By real-time amplification (PCR) of the ligated product we find that the conformation-switching aptamers can sensitively and specifically detect thrombin or PDGF at picomolar level against a background of cellular lysate. The principal advantage of this method is that it can potentially be applied to a wide variety of analytes, thereby allowing the development of numerous amplificable aptamer biosensors.