Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals.

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human beta-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

Inhibition of pre-mRNA splicing by synthetic branched nucleic acids.

The cellular transformation of a precursor mRNA (pre-mRNA) into its mature or functional form proceeds by way of a splicing reaction, in which the exons are ligated to form the mature linear RNA and the introns are excised as branched or lariat RNAs. We have prepared a series of branched compounds (bRNA and bDNA), and studied the effects of such molecules on the efficiency of mammalian pre-mRNA splicing in vitro. Y-shaped RNAs containing an unnatural L-2'-deoxycytidine unit (L-dC) at the 3' termini are highly stabilized against exonuclease hydrolysis in HeLa nuclear extracts, and are potent inhibitors of the splicing pathway. A bRNA containing internal 2'-O-methyl ribopyrimidine units and L-dC at the 3' ends was at least twice as potent as the most potent of the bRNAs containing no 2' modifications, with an IC50 of approximately 5 micro M. Inhibitory activity was maintained in a branched molecule containing an arabino-adenosine branchpoint which, unlike the native bRNAs, resisted cleavage by the lariat- debranching enzyme. The data obtained suggest that binding and sequestering of a branch recognition factor by the branched nucleic acids is an early event, which occurs prior to the first chemical step of splicing. Probably, an early recognition element preferentially binds to the synthetic branched molecules over the native pre-mRNA. As such, synthetic bRNAs may prove to be invaluable tools for the purification and identification of the putative branchpoint recognition factor.

Synthesis of lariat-DNA via the chemical ligation of a dumbbell complex.

[reaction: see text] An efficient synthesis of a medium-sized DNA lariat through the chemical ligation of a Y-shaped dumbbell precursor is described. The methodology requires only commercially available phosphoramidites and reagents and affords regioisomerically pure lariat molecules. Characterization of the lariat by T(m) analysis reveals that the molecule displays markedly enhanced thermal stability and unimolecular association-dissociation kinetics, consistent with DNA dumbbell behavior.

Branchpoint sugar stereochemistry determines the hydrolytic susceptibility of branched RNA fragments by the yeast debranching enzyme (YDBR).

A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.

Solid-phase synthesis of branched oligonucleotides.

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single-stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2',5'- and 3',5'-phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre-mRNA splicing. The protocols described herein allow for the facile solid-phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion-exchange HPLC and PAGE are also illustrated.

Template-mediated synthesis of lariat RNA and DNA.

Nucleic acid "lariats" have been of great interest to the biological community since their discovery two decades ago as splicing intermediates in the biosynthesis of messenger RNA (lariat RNA introns). We report here the first synthesis of lariat DNA and RNA via template-mediated chemical ligation of Y-shaped oligonucleotides. The method allows for the synthesis of lariat DNA of any base composition as well as the more biologically relevant lariat RNA. Typically, branched precursors and complementary linear templates ("splints") were dissolved in an equimolar ratio at a total concentration of 10(-4) M, and ligation was promoted by addition of cyanogen bromide in a pH 7.6 buffer. The template-directed cyclization was very efficient, since the amount of circularized lariat product observed in all cases was in the 40-60% range. The lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition confirmed by MALDI-TOF mass spectrometry. Thermal denaturation and circular dichroism studies of lariat:RNA and lariat:DNA duplexes were fully supportive of the isolated "lasso" structures. Further characterization was conducted by enzymatic degradation with spleen phosphodiesterase (a 3'-exonuclease) and the RNA lariat debranching enzyme, a specific 2',5'-phosphodiesterase.

Selective inhibition of HIV-1 reverse transcriptase (HIV-1 RT) RNase H by small RNA hairpins and dumbbells.

We present here the design of a novel class of RNA inhibitors of the RNase H domain of HIV-1 RT, a ribonuclease activity that is essential for viral replication in vivo. Specifically, we show that small RNA hairpins and dumbbells can selectively inhibit the RNase H activity of HIV-1 RT without affecting other cellular RNases H (e.g., E. coli and human RNase H). These results suggest that the inhibitors do not interact with the nucleic acid binding site of RT RNase H, as this region should be well conserved among the various enzymes. The most potent inhibitors displayed IC50 values in the 3-8 microM range. Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by the hairpin and dumbbell aptamers, a property not previously observed for any nucleic acid aptamer directed against RT RNase H. The results described here suggest a noncompetitive binding mechanism, as outlined in the differential inhibitory characteristics of each of the nucleic acid aptamers against the bacterial, human, and viral RNase H homologues.