The folding of large RNAs studied by hybridization to arrays of complementary oligonucleotides

Folding pathways of large RNAs are poorly understood. We have addressed thi s question by hybridizing in vitro transcripts, which varied in size, to an array of antisense oligonucleotides. All transcripts included a common seq uence and all but one shared the same start-point; the other had a small de letion of the 5' end. Minimal free energy calculations predicted quite diff erent folds for these transcripts. However, hybridization to the array show ed predominant features that were shared by transcripts of all lengths, tho ugh some oligonucleotides that hybridized strongly to the short transcripts gave weak interaction with longer transcripts. A full-length RNA fragment that had been denatured by heating and allowed to cool slowly gave the same hybridization result as a shorter transcript. Taken together, these result s support theories that RNA folding creates local stable states that are tr apped early in the transcription or folding process. As the transcript elon gates, interactions are added between regions that are transcribed early an d those transcribed late. The method here described helps in identifying re gions in the transcripts that take part in long-range interactions.