Nucleic acid aptamers possess many useful features as affinity reagents, including facile chemical synthesis, reversible folding, thermal stability and low cost, making them a powerful alternative to antibodies and other protein-based reagents. However, over the past two decades, aptamers have suffered from the fact that 1) the conventional method of aptamer generation (SELEX) is lengthy, labor intensive and often does not yield aptamers with sufficient affinity (<1 nM) and specificity;2) there is no "standard protocol" that can be generally applied to most protein targets to generate aptamers;and 3) the characterization steps to measure the affinity and specificity of candidate aptamers are lengthy and resource-intensive, because each aptamer must be measured individually. We believe that these challenges arise from deficiencies in the conventional methodology of performing the selection, which has not changed significantly since its initial description 20 years ago. We also believe that these problems can be solved, by systematically taking fundamentally different approaches towards the three central stages of the process - selection, analysis and characterization of the aptamers. We propose here the development of such a system. We will combine three distinctly novel technologies -microfluidic selection, next-generation aptamer sequencing, and SPR Imaging - to develop the Quantitative Parallel Aptamer Selection System (QPASS) platform. The QPASS platform will generate specific aptamers with sub-nanomolar affinities (Kd) for a wide range of protein targets within 3 rounds of selection, identify a pool of the best candidates by next generation DNA sequencing and bioinformatic analysis, and home in on the optimal aptamer sequence by the parallel synthesis and measurement of the affinities of thousands of aptamer candidates. Individually, each component represents a significant technological advance. Combined this integrated approach offers an opportunity to revolutionize the process of aptamer generation.

Public Health Relevance

Affinity reagents represent a cornerstone of modern biotechnology, because they can bind specifically to their target molecules with high affinities. However, current methods of affinity reagent generation is lengthy, expensive and often irreproducible. Here we propose a novel technology platform capable of rapidly generating high performance affinity reagents by combining three distinctly novel technologies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54DK093467-02
Application #
8335434
Study Section
Special Emphasis Panel (ZRG1-BST-M (51))
Program Officer
Sechi, Salvatore
Project Start
2011-09-25
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
2
Fiscal Year
2012
Total Cost
$1,062,807
Indirect Cost
$165,221
Name
University of California Santa Barbara
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
094878394
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
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