Abstract
The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.
Highlights
Since its development over twenty years ago [1,2], the process of in vitro selection has led to the isolation of numerous functional nucleic acids, called aptamers, that bind a wide range of target molecules
It has been shown that small changes of sequence in loop regions of certain oligonucleotides has resulted in dramatic changes from one stable quadruplex topology to another [44]
In vitro selection has established itself as a powerful technique for the isolation of a wide range of functional nucleic acids
Summary
Since its development over twenty years ago [1,2], the process of in vitro selection has led to the isolation of numerous functional nucleic acids, called aptamers, that bind a wide range of target molecules. Much research has been carried out to convert aptamers into sensors by coupling their target recognition to diverse signalling platforms, generating many fluorescent, colorimetric, and electrochemical sensors (reviewed in [3,4]) Despite these advances, the process of in vitro selection that is used to select aptamers remains enigmatic and is in part responsible for hindering aptamers from reaching the mainstream. Any means of increasing the chances of isolating potent aptamers by improving an aspect of the current in vitro selection procedure would be of great value
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