Aptamers are ssDNA or RNA that bind specific targets with high affinity and selectivity. Their combination of high affinity and selectivity make them excellent drug candidates or diagnostic agents. Aptamers are isolated using a process referred to as SELEX (Systematic Evolution of Ligands by Exponential enrichment). In this process a nucleic acid library containing a random sequence region is incubated with the target of interest. Sequences with affinity for the target are separated from non-binding sequences using filter, chromatography or panning separations. These binding sequences are collected, amplified and purified, generating a new pool for further rounds of enrichment. A significant fraction of sequences in the pool exhibit affinity for the target after 8-15 rounds of selection. Aptamers have been successfully selected for a wide range of targets including small molecules, peptides, proteins and even entire cells. Unfortunately the process for isolating aptamers remains long and complex. In the previous funding cycle we introduced capillary electrophoresis selections (CE-SELEX) which reduced the time required to isolated aptamers from weeks to days. In the current submission we propose to further leverage the advantages of microfluidic systems to further simplify and speed the process. We will also develop microfluidic protocols for selections that are not easily performed using existing techniques. In particular we propose to: 1) Develop an automated, flow through, microfluidic SELEX device. We will combine a micro-FFE separation with flow through PCR and DNA purification to develop a fully automated microfluidic device. We anticipate that this device will be able to perform a single round of selection in as little as 10 minutes. 2) Develop a CE-SELEX protocol for isolating aptamers with affinity for membrane proteins. To date the vast majority of aptamers have been selected to bind soluble proteins. This is unfortunate since ligands for membrane proteins are much more valuable as drug candidates and diagnostic agents. CE-SELEX is ideally suited to perform selections against membrane proteins constituted in micelles or liposomes. 3) Develop a new protocol for isolating aptamers with catalytic activity. Hundreds of aptamers that catalyze reactions have been isolated. Unfortunately, due to the selection process used for isolating these aptamers, almost all of these nucleic acids directly take part in the reaction, limiting their effectiveness. We propose using techniques developed to detect and study individual enzyme molecules as the basis for a new method that isolates aptamers based directly on their catalytic activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063533-08
Application #
7645099
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2002-07-01
Project End
2011-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
8
Fiscal Year
2009
Total Cost
$274,266
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Johnson, Alexander C; Bowser, Michael T (2017) Micro free flow electrophoresis. Lab Chip 18:27-40
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Soller, Kailey J; Yang, Jing; Veglia, Gianluigi et al. (2016) Reversal of Phospholamban Inhibition of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) Using Short, Protein-interacting RNAs and Oligonucleotide Analogs. J Biol Chem 291:21510-21518
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Soller, Kailey J; Verardi, Raffaello; Jing, Meng et al. (2015) Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides. Sci Rep 5:13000
Jing, Meng; Verardi, Raffaello; Veglia, Gianluigi et al. (2014) Development of a Sensitive Assay for SERCA Activity Using FRET Detection of ADP. Anal Methods 6:1468-1474
Sheng, Yixiao; Bowser, Michael T (2014) Isolating single stranded DNA using a microfluidic dialysis device. Analyst 139:215-24
Taylor, Thane H; Frost, Nicholas W; Bowser, Michael T et al. (2014) Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies. Anal Bioanal Chem 406:1683-91
Yang, Jing; Bowser, Michael T (2013) Capillary electrophoresis-SELEX selection of catalytic DNA aptamers for a small-molecule porphyrin target. Anal Chem 85:1525-30

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