title: Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.
abstract: 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.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708, United States.
authors.forename: Vinalia
authors.initials: V
authors.lastname: Tjong
authors.forename: Hua
authors.initials: H
authors.lastname: Yu
authors.forename: Angus
authors.initials: A
authors.lastname: Hucknall
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Chilkoti
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D005095:Exoribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D058105:Polymerization; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013499:Surface Properties
chemical_list: D002232:Carbocyanines; D005095:Exoribonucleases; D005456:Fluorescent Dyes; D012313:RNA; C089187:cyanine dye 3