Mapping of accessible sites for oligonucleotide hybridization on hepatitis delta virus ribozymes

Abstract

Semi-random libraries of DNA 6mers and RNase H digestion were applied to search for sites accessible to hybridization on the genomic and antigenomic HDV ribozymes and their 3' truncated derivatives. An approach was proposed to correlate the cleavage sites and most likely sequences of oligomers, members of the oligonucleotide libraries, which were engaged in the formation of RNA-DNA hybrids. The predicted positions of oligomers hybridizing to the genomic ribozyme were compared with the fold of polynucleotide chain in the ribozyme crystal structure. The data exemplified the crucial role of target RNA structural features in the binding of antisense oligonucleotides. It turned out that cleavages were induced if the bound oligomer could adapt an ordered helical conformation even when it required partial penetration of an adjacent double-stranded region. The major features of RNA structure disfavoring hybridization and/or RNase H hydrolysis were sharp turns of the polynucleotide chain and breaks in stacking interactions of bases. Based on the predicted positions of oligomers hybridizing to the antigenomic ribozyme we chose and synthesized four antisense DNA 6mers which were shown to direct hydrolysis in the desired, earlier predicted regions of the molecule.

Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

“…For that purpose, the mRNA-122, 6-mers semirandom DNA libraries, and RNase H hydrolysis were used ( Figure 5(a)). This approach has been shown to be particularly useful in screening for target sites for antisense oligonucleotides in highly structured RNA molecules: region X of hepatitis C virus [34], and the genomic and antigenomic HDV ribozymes [35]. Most importantly, this approach was used for mapping the sites accessible to oligomer hybridization in the 5′-terminal region of human p53 mRNA that begins at P1 transcription initiation site [36].…”

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

“…The third group of methods relies on enzymatic detection of stable complexes formed between RNA and oligonucleotide probes (Campbell & Cech, 1995;Lieber & Strauss, 1995;Ho et al+, 1996;Birikh et al+, 1997;Matveeva et al+, 1997)+ In particular, the RNase H cleavage assay identifies RNA regions that form heteroduplexes with libraries of 6-to 11-mer random or semi-random oligonucleotides (Ho et al+, 1996;Wrzesinski et al+, 2000)+ The length of the oligonucleotides used in the RNase H method falls in the mid-range between the shorter and longer probes used in the methods discussed above+ Binding of mid-range probes may also perturb RNA secondary structure, although to a lesser extent than the longer probes (Wrzesinski et al+, 2000)+ In contrast to the conditions used for hybridization with arrays of oligonucleotides, the RNase H assay is performed under nearly physiological conditions and requires much shorter incubation times (from a few minutes to 1 h)+ The parameters used for the RT-ROL method are very close to those of the RNase H cleavage assay+ Indeed, the RT-ROL method analyzes the formation of 6-to 8-nt heteroduplexes, requires short hybridization times, and uses reaction conditions similar to that of RNase H assay+ Not surprisingly, therefore, the extendible sites mapped by RT-ROL correlate best with the accessible sites identified by the RNase H assay (Fig+ 5)+ On the other hand, the results of the RT-ROL and arrays of oligonucleotides methods correlated less well, as illustrated by the b-globin RNA example (Fig+ 6)+ The most obvious difference between the two methods is the lengths of oligonucleotide libraries used for hybridization with RNA+ Indeed, in the analysis of b-globin RNA, the optimal lengths of oligonucleotides used in array hybridization (Milner et al+, 1997) are 14-17 nt+ They are approximately twofold longer than the optimal lengths of the sequences used in RT-ROL+ The differences in oligonucleotide lengths and hybridization conditions that distinguish the RT-ROL and hybridization with oligonucleotide arrays methods would likely have a dramatic effect on the stability of the heteroduplexes detected by each method+ Heteroduplex stability can be estimated from the rate constant, k 1 , of the dissociative pathway as described (Reynaldo et al+, 2000)+ Under the hybridization conditions of the oligonucleotide arrays method (30 8C, 1 M NaCl), the lifetime, 1/k 1 , of a typical 14-mer heteroduplex (50% GϩC) is estimated to be .100 h, whereas under the conditions of the RT-ROL method (42 8C, 10 mM MgCl 2 ), the lifetime of an 8-mer duplex is expected to be approximately 10 ms+ This analysis suggests that the two methods rely upon fundamentally different mechanisms of accessible sites detection+ The oligonucleotide arrays method detects the formation of very stable heteroduplexes that require substantial and, likely, kinetically controlled rearrangements of local RNA structure, whereas the RT-ROL method, as well as the RNase H assay, analyzes short-lived, quickly hybridizing duplexes that probably cause only minor perturbations of RNA structure+ A combination of all three methods would be valuable to draw a complete picture of RNA accessibility+…”

“…The complexity and high cost of these experimental methods stimulated the development of theoretical methods for predicting accessible sites using RNA folding programs (Zuker, 1989;Stull et al+, 1992;Sczakiel et al+, 1993;Mathews et al+, 1999;Patzel et al+, 1999;Walton et al+, 1999)+ Although examples of the successful application of computational methods have been reported (Patzel et al+, 1999), the ability of RNA folding programs to predict accessible sites remains somewhat marginal due to poor prediction of RNA secondary structure augmented by the fact that the mechanisms of RNA "accessibility" are only beginning to emerge (Mir & Southern, 1999;Wrzesinski et al+, 2000)+ Here we describe a new experimental approach for mapping RNA accessible sites using reverse transcription with random oligonucleotide libraries (RT-ROL)+ This method uses oligonucleotides with random sequences for primer extension with reverse transcriptase to generate a series of cDNAs+ The cDNAs will initiate only at RNA sites that are favorable for hybridization and extension+ The extension products are PCR amplified and their lengths are determined by gel electrophoresis with a single nucleotide resolution to identify the sites where the extension occurred+ Because only the first step of this method involves RNA and the following analysis is performed on DNA molecules, no RNA labeling or RNA sequencing is required+ The products of a single RT reaction can be used multiple times with different sets of PCR primers to scan the full length of the RNA target molecule+…”

Rapid Determination of RNA Accessible Sites by Surface Plasmon Resonance Detection of Hybridization to DNA Arrays

“…The experimental methods8,9 tend to be time-consuming, labor-intensive, and due to their low-throughput nature are cost-prohibitive for screening a large number of RNA molecules. Computational methods have been developed to predict RNA folding patterns, providing a starting point to predict the accessible sites 10-16.…”