Modified bases in RNA reduce secondary structure and enhance hybridization.
Secondary structure in RNA targets is a significant barrier to short DNA probes. However, when such targets are the end product of an in vitro amplification scheme, it is possible to carry out transcription in the presence of nucleoside triphosphate analogues that reduce secondary structure of the RNA without impairing subsequent hybridization. Here we show that nucleoside triphosphates of 2-aminoadenine (nA) and 2-thiouracil (sU) are taken up by T7 RNA polymerase and that the resulting RNA possesses reduced secondary structure and improved accessibility to DNA probes. The hybridization properties of short RNA transcripts were studied using a new gel mobility shift assay from which melting temperatures were determined. RNA hairpins that contained nA and sU were able to hybridize to DNA probes under conditions where the unmodified hairpins did not. DNA-RNA hybrids that contained nA and sU in the RNA strand exhibited enhanced specificity, increased stability, and greater equality of base pairing strength than the same hybrids without modifications. Substitution of guanine (G) with inosine (I) further reduced secondary structure, but RNA with this base hybridized nonselectively. The high stability of nA-T and A-sU base pairs in DNA-RNA hybrids, combined with the destabilizing effect of the nA-sU couple in RNA targets, accounts for the improved hybridization properties. These results suggest that incorporation of nA and sU during in vitro transcription is a promising strategy for enhancing the performance of oligomeric DNA probes with an RNA target.
Gamper
Howard B
HB
Division of Hematology/Oncology, University of Pennsylvania School of Medicine, BRB II/III Room 713, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. hbgamper@mail.med.upenn.edu
Gewirtz
Alan
A
Edwards
Jillian
J
Hou
Ya-Ming
YM
D015075:2-Aminopurine / Q000031:analogs & derivatives* / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology; D001482:Base Composition; D020137:Base Pair Mismatch; D020029:Base Pairing; D015342:DNA Probes / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability; D006358:Hot Temperature; D007288:Inosine / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D013889:Thiouracil / Q000378:metabolism; D014764:Viral Proteins
D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral; D014764:Viral Proteins; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D013889:Thiouracil; D007288:Inosine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases