Single-Round Patterned DNA Library Microarray Aptamer Lead Identification.

Jennifer A Martin,Jorge L Chávez,Joshua A Hagen,Yaroslav Chushak,Peter A Mirau,Nancy Kelley-Loughnane,Rajesh R Naik

doi:10.1155/2015/137489

Abstract

A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

Highlights

Aptamers are oligonucleotide molecular recognition elements selected through a synthetic iterative evolutionary process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) [1, 2]
A mutation of the streptavidin aptamer (SA) with an extra guanine base inserted at position 19 was used as a negative control due to its low binding observed with a variety of different targets in preliminary work [23]
This work illustrates the potential of DNA microarray technology for aptamer identification and highlights patterned libraries designed without prior binding sequence consideration as a viable solution to the limitations on microarray oligonucleotide surface density

Summary

Introduction

Aptamers are oligonucleotide molecular recognition elements selected through a synthetic iterative evolutionary process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment) [1, 2]. One example is the timescale of aptamer selection, which typically requires an average of 12 cycles and a minimum of 2–6 months, not including initial optimization processes, validation of aptamer candidates, or structural analysis [4, 5] This is typically a result of the low partitioning efficiency (the ability to separate binding sequences from nonbinders in a selection round) of conventional partitioning methods used in SELEX [6]. The sequences reported likely reflect the most abundant sequences present but may not report those that have artificially lower numbers due to factors such as PCR bias or cloning efficiency or due to low sampling of the entire sequence space (diversity) of the final pool [8]

Methods

Results

Conclusion