Coupling aptamer biosensors to signal amplification
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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.