Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase

A rapid and simple method for determining accessible sites in RNA that is i ndependent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequenc e-randomized libraries of DNA oligonucleotides linked to a common tag seque nce at their 5'-end. Annealed oligonucleotides are extended with reverse tr anscriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific p rimer to determine which regions of the RNA molecules were RNA extendible s ites, that is, sites available for oligonucleotide binding and extension. W e then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA ta rgets, human activated res (ha-ras), human intercellular adhesion molecule- 1 (ICAM-1), rabbit beta -globin, and human interferon-gamma (IFN-gamma). Ou r results were concordant with those of other researchers who had used RNas e H cleavage or hybridization with arrays of oligonucleotides to identify a ccessible sites on some of these targets. Further, we found good correlatio n between sites when we compared the location of extendible sites identifie d by RT-ROL with hybridization sites of effective antisense oligonucleotide s on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the r elationship between RNA extendible sites and RNA accessibility.