RNA aptamer microarrays for the specific detection of proteins and their potential use as molecular diagnostics for the treatment of HIV.

摘要

Aptamers are nucleic acid species with specific binding properties that are selected in vitro from large pools of random oligonucleotides. In the present work, I adapted microarray technology for the production of DNA oligonucleotide microarrays to print biotinylated RNA aptamer clones and pools onto streptavidin coated microarray slides in ways that preserved the specific protein binding functionalities of the aptamers. The resulting RNA aptamer microarrays could reliably detect fluorescently labeled HIV-1 reverse transcriptase (RT), hen egg white lysozyme, and other proteins in a dose-dependent manner, with linear signal responses that spanned up to 7 orders of magnitude of analyte concentration, and lower limits of detection in the pg/ml range. Aptamers on the microarray retained their specificity for target proteins in the presence of a 10,000 fold (w/w) excess of T-4 cell lysate protein. Aptamer microarray development was facilitated by the optimization of high-throughput and highly parallel methods for the enzymatic synthesis of 5' biotinylated RNA aptamer clones and pools. As an alternative, unmodified RNA aptamers were prepared for immobilization by appending an 18 nt generic linker sequence to their 3' ends that was hybridized to a 5' biotinylated LNA/DNA anchor oligonucleotide, thereby forming a duplex with a Tm of 80°C. Applications for RNA aptamer microarrays as molecular diagnostics for HIV-1 treatment were potentiated by detecting the specific capture of unlabeled RT on the microarrays using fluorescent antibody sandwich methods. Microarray tests of wild type and drug-resistant RT produced recombinantly in our laboratory indicated a nascent ability for RNA aptamers to distinguish between closely related RT phenotypes, thereby further demonstrating the potential utility of RNA aptamer-based diagnostics in the treatment of HIV.