Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
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- 作者：Rachel E. Armstrong ; Geoffrey F. Strouse
- 刊名：Bioconjugate Chemistry
- 出版年：2014
- 出版时间：October 15, 2014
- 年：2014
- 卷：25
- 期：10
- 页码：1769-1776
- 全文大小：373K
- ISSN：1520-4812
文摘
Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM 鈥?203 渭M) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the 螖G values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd鈥檚 for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.