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

Abstract

Folding pathways of large RNAs are poorly understood. We have addressed this question by hybridizing in vitro transcripts, which varied in size, to an array of antisense oligonucleotides. All transcripts included a common sequence and all but one shared the same start-point; the other had a small deletion of the 59 end. Minimal free energy calculations predicted quite different folds for these transcripts. However, hybridization to the array showed predominant features that were shared by transcripts of all lengths, though 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 results support theories that RNA folding creates local stable states that are trapped early in the transcription or folding process. As the transcript elongates, interactions are added between regions that are transcribed early and those transcribed late. The method here described helps in identifying regions in the transcripts that take part in long-range interactions.

Structural analysis of hepatitis C RNA genome using DNA microarrays

“…Experiments to measure the effect of different single-stranded nucleic acid structures, under high salt conditions, on the rate of duplex formation on solid supports, have shown that RNA or cDNA structural motifs may impede hybridization between probe and target and, therefore, bias the results obtained in gene expression arrays (49)(50)(51)(52). Here, we have reversed the argument and, by taking advantage of the solid support hybridization techniques, we have aimed at evaluating the structural degree of long RNA molecules with potential application to HCV diagnostics and therapeutics.…”

“…This makes some sequences to remain accessible to further longrange interactions, while others show restricted accessibility to distant regions. As elongation of the RNA chain proceeds, more structures are added to the transcript which could mask the accessible sites previously obtained (51). Therefore, one should be cautious when interpreting accessible positions, since long-range interactions may involve not only the ends of an RNA transcript but also its internal regions, otherwise revealed by the loss of hybridization signals that results from extending the transcript length.…”

Plasminogen activator inhibitor 1: Mechanisms of its synergistic regulation by growth factors

“…This procedure was successful in identifying accessible regions on the mRNAs encoding β-globin, RhoA and luciferase (Zhang, Mao et al 2003). Microarrays were developed to examine the structure of an mRNA over a range of about 100 nt (Milner, Mir et al 1997;Sohail, Akhtar et al 1999;Sohail and Southern 2000). These arrays are produced using an innovative approach to We analyzed the accessible sequences on Lcn2 mRNA by both tiled microarray and computational methods to identify optimal AS-ODNs.…”

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

“…We have described a method to identify RNA sites that are favorable for hybridization with random oligonucleotide libraries and extension by reverse transcriptase, and have called them extendible sites+ The first question to ask is what is the relationship between extendible sites and RNA accessible sites described in the literature+ Accessible sites are loosely defined as those regions on an RNA molecule that can form stable complexes with complementary oligonucleotides+ However, conditions under which this stability is achieved as well as the length of the oligonucleotides used for analysis can significantly vary in different methods+ In one of the first methods (Hogenauer, 1970;Lewis & Doty, 1970;Uhlenbeck et al+, 1970;Uhlenbeck, 1972), RNA accessible regions were identified by using equilibrium dialysis to measure the association constants for the hybridization of 3-and 4-mer oligoribonucleotides to complementary regions of tRNA+ The tri-and tetranucleotides used in these studies are possibly the shortest oligonucleotides that can be employed for RNA probing, because 3-4 nt is the minimal size of a stable duplex corresponding to a nucleation complex (Craig et al+, 1971;Porschke & Eigen, 1971)+ Therefore, very short oligonucleotides bind to RNA regions that are not only in a single-stranded conformation, but are also flexible enough to adopt a duplex conformation upon binding (Uhlenbeck et al+, 1970)+ On the other side of the length spectrum, hybridization with arrays of oligonucleotides (Milner et al+, 1997;Mir & Southern, 1999;Sohail et al+, 1999) generally employs much longer probes+ Although the method can analyze oligonucleotides ranging from 1 to 21 nt, optimal binding is usually achieved with 12-to 17-mers+ Because accessible regions of such length do not likely appear frequently in RNA structures, the assumption is that long oligonucleotides initiate binding at short regions that are sterically accessible for nucleation, and the heteroduplex subsequently propagates into more structured RNA regions+ Kinetic restrictions on the perturbations of RNA secondary structure at the oligonucleotide binding site may explain the long hybridization times (3-18 h) used in this method+…”