Modified Bases in RNA Reduce Secondary Structure and Enhance Hybridization

Abstract

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.

Unrestricted accessibility of short oligonucleotides to RNA

“…2; HP25) of 25 bp, high in G/C content, was tested as a model substrate for the accessibility of probes. This RNA, previously designed and studied by us (Gamper et al 2004), was prepared by in vitro transcription with T7 RNA polymerase, in the presence of the pseudocomplementary nA + sU and/or the destabilizing hX or cG nucleoside triphosphates (Table 1). These modified nucleoside triphosphates attenuated the transcription of radiolabeled hairpin under standard reaction conditions.…”

“…Transcription in the presence of all three NTP analogs reduced hairpin synthesis to only 30% of the control reaction, regardless of whether cGTP or hXTP was used. While these levels of transcription efficiency were adequate for this study, the preparation of pseudo-complementary transcripts longer than 200 nt was problematic (Gamper et al 2004). …”

“…If the hairpin became destabilized because of the presence of modified bases, the T m of the hairpin should decrease relative to that of the unmodified RNA. The T m was determined by a recently developed gel mobility shift assay (Gamper et al 2004), where the accessibility of a 25-mer RNA probe to the RNA hairpin was monitored by gel electrophoresis under nondenaturing conditions. Determination of the accessibility over a range of temperatures (10 C-90 C) yielded a binding curve from which the T m was calculated (Table 2).…”

“…An RNA transcript ( Fig. 2; SS32) that embodied one arm of the 25-bp-long hairpin described earlier (Gamper et al 2004) was used as a target. This RNA, in the absence of modification, contained a hairpin at one end that was inaccessible to 12-mer DNA probes at 10 C in the presence of 5 mM MgCl 2 and 25 mM NaCl (Gamper et al 2004).…”

“…We have recently shown (Gamper et al 2004) that accessibility of short (12-mer) DNA probes to an RNA target can be improved if the RNA is substituted with the pseudocomplementary bases nA (also known as 2,6-diaminopurine) and sU and the destabilizing base hX (hypoxanthine, inosine) (Fig. 1).…”