Unrestricted accessibility of short oligonucleotides to RNA.

The propensity of RNA to fold into higher-order structures poses a major barrier to the use of short probes (<15 nucleotides) by preventing their accessibility. Introduction of the pseudo-complementary bases 2-aminoadenine (nA) and 2-thiouracil (sU) and the destabilizing base 7-deazaguanine (cG) into RNA provides a partial solution to this problem. While complementary in hydrogen bonding groups, nA and sU cannot form a stable base pair due to steric hindrance, and are thus pseudo-complementary. Each, however, recognizes the regular T/U and A complements, allowing pairing with oligonucleotides. Short pseudo-complementary RNAs can be prepared by in vitro transcription. Relative to standard transcripts, the modified transcripts possess reduced secondary structure and increased accessibility to short (8-mer) probes in the locked nucleic acid (LNA) configuration. They also hybridize to complementary probes with increased specificity and thermostability. Practical application of this strategy to oligonucleotide-based hybridization assays will require engineering of RNA polymerase for more efficient utilization of pseudo-complementary nucleoside triphosphates.