Functional RNA microarrays for high-throughput screening of antiprotein aptamers.
High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.
Collett
James R
JR
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Cho
Eun Jeong
EJ
Lee
Jennifer F
JF
Levy
Matthew
M
Hood
Allysia J
AJ
Wan
Christine
C
Ellington
Andrew D
AD
D001483:Base Sequence; D015374:Biosensing Techniques; D019921:Biotinylation; D046228:Microarray Analysis / Q000379:methods*; D009113:Muramidase / Q000276:immunology; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000302:isolation & purification*; D014158:Transcription, Genetic
D016601:RNA-Binding Proteins; D012313:RNA; D009113:Muramidase