Alexandria Digital Research Library

Development of novel technologies for discovery of high performance affinity reagents

Author:
Wang, Jinpeng
Degree Grantor:
University of California, Santa Barbara. Mechanical Engineering
Degree Supervisor:
H. Tom Soh
Place of Publication:
[Santa Barbara, Calif.]
Publisher:
University of California, Santa Barbara
Creation Date:
2013
Issued Date:
2013
Topics:
Engineering, Biomedical and Engineering, Materials Science
Keywords:
Aptamer
Directed evolution
Affinity reagents
Phage display
SELEX
High throughput screening
Genres:
Dissertations, Academic and Online resources
Dissertation:
Ph.D.--University of California, Santa Barbara, 2013
Description:

Comprehensive understanding of the proteome holds the promise for revolutionizing medicine. In order to functionally explore, understand and exploit the proteome, the availability of a comprehensive, well-characterized collection of renewable affinity reagents that specifically bind to the target proteins is an absolute necessity. Unfortunately, monoclonal antibodies cover only a small fraction of the proteome, despite decades of development. Thus, a quantum leap in affinity reagents discovery is urgently needed. Toward meeting this demand, we present two novel technologies that can generate high quality synthetic affinity reagents (nucleic acid aptamers and polypeptides) in a rapid and economic manner, towards accelerated exploration of the human proteome. First, we will present a high-throughput aptamer screening technology that reproducibly yields high quality aptamers.

Our method (termed Particle Display) transforms libraries of solution-phase aptamers into "aptamer particles", each displaying many copies of a single sequence on its surface. We then use fluorescence-activated cell sorting (FACS) to individually measure the relative affinities and specificities of 10 8 aptamer particles and sort them in a high-throughput manner. We used particle display to obtain high-affinity DNA aptamers for various protein targets, including proteins for which previous aptamer selection attempts have repeatedly failed. Second, we will expand the particle display technology to discover aptamer pairs that bind to distinct binding sites of the same protein target. We identified multiple pairs that bind to PAI-1 at different binding sites. Preliminary results showed that these aptamer pairs can be directly deployed in sandwich format assays and can reach pM range limit of detection in the presence of diluted serum.

Finally, we present the microfluidic phage selection (MiPS) system, which is the first microfluidic system for selecting phage-displayed peptides against surface markers from live mammalian cells. Compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well-known NRP-1-binding RPARPAR peptide.

Physical Description:
1 online resource (104 pages)
Format:
Text
Collection(s):
UCSB electronic theses and dissertations
ARK:
ark:/48907/f3v122w4
ISBN:
9781303732058
Catalog System Number:
990041153600203776
Rights:
Inc.icon only.dark In Copyright
Copyright Holder:
Jinpeng Wang
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