A General Platform for Aptamer Mediated Capture of Specific Targets

Abstract

<p> The purpose of this research is to develop a general method for capturing and separating specific targets. Nucleic acid aptamers are short sequences of single-stranded DNA or RNA which have the ability to bind to the small organic or inorganic molecules such as protein and metal ions with high binding affinity. In this study, a bioconjugate re-usable system was developed. It can reversibly load or unload DNA aptamers.</p> <p> To allow separation, a thermally responsive polymer (N-isopropylacrylamide, PNIPAM) is used. This polymer can undergo a reversible phase transition upon adding NaCl and/or increasing temperature. A short sequence of single stranded DNA (ssDNA) was coupled to PNIPAM. The ssDNA will experience a reversible phase transition because of the PNIPAM.</p> <p> The DNA sequence for an aptamer can be extended to contain a sequence that is complementary to that of the ssDNA coupled to PNIPAM. Adding this extended aptamer to the conjugate will result in spontaneous hybridization of the two strands of DNA. These strands can be separated using an agent (e.g. urea) that destroys hydrogen bonding. The conjugate can be recovered using a reversible inverse phase transition.</p> <p> The same PNIPAM-ssDNA conjugate can be used reversibly for coupling different aptamers. The aptamers did not lose their binding ability when coupled with PNIPAM-ssDNA conjugates. In the process of precipitation separation targets, the PNIPAM-ssDNA conjugate showed little loss with applied phase transition. Moreover, the coupling efficiency of the ssDNA to PNIPAM conjugates was determined. The binding ability of the ATP aptamers to ATP was also investigated.</p>