Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.

Published

Journal Article

We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3'-OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3'-OH of an RNA-DNA hybrid. We found that the 3'-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format.

Full Text

Duke Authors

Cited Authors

  • Tjong, V; Yu, H; Hucknall, A; Chilkoti, A

Published Date

  • January 2013

Published In

Volume / Issue

  • 85 / 1

Start / End Page

  • 426 - 433

PubMed ID

  • 23194025

Pubmed Central ID

  • 23194025

Electronic International Standard Serial Number (EISSN)

  • 1520-6882

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/ac303132j

Language

  • eng