Intracellular build-up RNAi with single-strand circular RNAs as siRNA precursors.

We herein report a new approach for RNA interference, so-called "build-up RNAi" approach, where single-strand circular RNAs with a photocleavable unit or disulfide moiety were used as siRNA precursors. The advantages of using these circular RNA formats for RNAi were presented in aspects of immunogenicity and cellular uptake.

Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA.

Achiral, acyclic nucleic acids: synthesis and biophysical studies of a possible prebiotic polymer.

The search for prebiotic, nucleic acid precursors is, at its best, a speculative undertaking. Given the complex structure of RNA, it is not very likely that RNA was the first information system in the universe and thus finding possible precursor/s i.e. pre-RNA remains an open challenge. We, in this paper, have tried to construct nucleic acid polymers with a simple acyclic, achiral backbone. Such a linear, achiral backbone may have been formed from simple monomers that may have existed in the "prebiotic soup". We have shown that such polymers are capable of identifying the complementary "other self" and thus forming a potential system for information storage and transmission. This study thus involves investigation of nucleic acid analogues with a modified backbone that are likely to have formed in the prebiotic setting.

Multiple microRNAs derived from chemically synthesized precursors regulate thrombospondin 1 expression.

Thrombospondin 1 (THBS1) is a secreted protein with a variety of biological functions, including a potent anti-angiogenic activity and activation of latent transforming growth factor beta (TGF-ß). In many human cancers it is expressed at low levels, although mutations in the THBS1 gene have been rarely reported. Instead, the loss of THBS1 expression has been proposed to be due to transcriptional and post-transcriptional deregulations. In a systematic screen of predicted microRNA (miRNA) binding sites in the THBS1 3' untranslated region (UTR) we employed chemically synthesized pre-miRNAs-a new class of pre-miRNA mimics-to show that several miRNAs (let-7a, miR-18a, miR-29b, miR-194, and miR-221) can modulate THBS1 expression at the post-transcriptional level. Sequence-specific downregulation of THBS1 by let-7a, miR-18a or by a small interfering RNA induced TGF-ß1 and SMAD4 transcript levels. Ectopic expression of latent TGF-ß1 reduced THBS1 protein expression and was associated with increased expression of let-7a, let-7-b, and miR-18a in cells. These data suggest an inverse correlation of THBS1 and latent TGF-ß1 expression levels possibly involving miRNAs.

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α.

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5' hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-ß signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

Systematic screens of proteins binding to synthetic microRNA precursors.

We describe a new, broadly applicable methodology for screening in parallel interactions of RNA-binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre-miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts. Detection of bound RBPs is achieved by protein or tag-specific antibodies allowing crude cell lysates to be used as a source of RBP. We selected 70 pre-miRNAs with phylogenetically conserved loop regions and 25 precursors of other well-characterized miRNAs for chemical synthesis in 3'-biotinylated form. An equivalent set in unmodified form served as inhibitors in affinity determinations. By testing three RBPs known to regulate miRNA biogenesis on this set of pre-miRNAs, we demonstrate that Lin28 and hnRNP A1 from cell lysates or as recombinant protein domains recognize preferentially precursors of the let-7 family, and that KSRP binds strongly to pre-miR-1-2.

A route to enantiopure RNA precursors from nearly racemic starting materials.

The single-handedness of biological molecules is critical for molecular recognition and replication processes and would seem to be a prerequisite for the origin of life. A drawback of recently reported synthetic routes to RNA is the requirement for enantioenriched reactants, which fails to address the puzzle of how the single chirality of biological molecules arose. Here, we report the synthesis of highly enantioenriched RNA precursor molecules from racemic starting materials, with the molecular asymmetry derived solely from a small initial imbalance of the amino-acid enantiomers present in the reaction mixture. Acting as spectators to the main reaction chemistry, the amino acids orchestrate a sequence of physical and chemical amplification processes. The emergence of molecules of single chirality from complex, multi-component mixtures supports the robustness of this synthesis process under potential prebiotic conditions and provides a plausible explanation for the single-handedness of biological molecules before the emergence of self-replicating informational polymers.

Using synthetic precursor and inhibitor miRNAs to understand miRNA function.

Although the majority of gene function studies center themselves around protein-encoding RNAs, the study of non-protein-encoding RNAs is becoming more widespread because of the discovery of hundreds of small RNA termed micro (mi) RNA that have regulator functions within cells. Currently, over 470 human miRNA genes are predicted to exist and are annotated within the "miRBase" public miRNA database ( http://microrna.sanger.ac.uk/ ). There is no denying that short interfering (si) and short hairpin (sh) RNAs have revolutionized how scientists approach understanding gene function; however, si and shRNAs are not effective for analyzing the function of miRNAs given that miRNAs are typically short (17-24 bases). In turn, new sets of agents that allow for the expression of miRNA above endogenous levels and inhibition of miRNAs have become a valuable technology for the study of these small regulatory RNAs. In this chapter, we provide step-by-step methods on how to utilize synthetic precursor and antisense inhibitor molecules for understanding miRNA function.

Chemical synthesis of a very long RNA oligomer, a 110mer precursor-miRNA candidate, with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group.

A long RNA oligomer, a 110mer with the sequence of a precursor-miRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We confirmed the identity of the synthetic 110mer by MALDI-TOF mass spectrometry, as well as HPLC, electrophoretic methods, RNase-digestion experiments, and its in vitro gene-silencing activity. The chemical synthesis of RNA oligomers of more than 100 nucleotides, which has until now been extremely difficult, can be practically realized by the CEM method.

Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate.

A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-mumol scale corresponds to 1 mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100 nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.

Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions.

We provide direct evidence that pre-mRNA splicing alters mRNP protein composition. Using a novel in vitro cross-linking approach, we detected several proteins that associate with mRNA exon-exon junctions only as a consequence of splicing. Immunoprecipitation experiments suggested that these proteins are part of a tight complex around the junction. Two were identified as SRm160, a nuclear matrix-associated splicing coactivator, and hPrp8p, a core component of U5 snRNP and spliceosomes. Glycerol gradient fractionation showed that a subset of these proteins remain associated with mRNA after its release from the spliceosome. These results demonstrate that the spliceosome can leave behind signature proteins at exon-exon junctions. Such proteins could influence downstream metabolic events in vivo such as mRNA transport, translation, and nonsense-mediated decay.

Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage.

Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3'-OH and 5'-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3'-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3'- S -phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3'- S -phosphorothiolate-modified ptRNA carrying a 7 nt 5'-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5'-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn(2+)or Cd(2+). To suppress aberrant cleavage, we also constructed a 3'- S -phosphorothiolate-modified ptRNA with a 1 nt 5'-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3'- S -phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.

Synthetic substrate analogs for the RNA-editing adenosine deaminase ADAR-2.

We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2[prime]-carbon of the edited adenosine. Methylation of the 2[prime]-OH caused a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine were deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction.

SV40 T1-mRNA trans-splicing and translation requires that the in vitro synthesized cRNA is capped before microinjection.

The purpose of this investigation was to study the effect on cap structure for trans-splicing in mammalian cells. The early SV40 Bst/Bam pre-mRNA (cRNA) was synthesized in vitro in both capped (cap-Bst/Bam-cRNA) and non-capped (Bst/Bam-cRNA) versions and microinjected into the nuclei of TC7 cells. Trans-splicing was monitored by immunofluorescence staining (T1-antigen) and by RT-PCR analysis. Cap-Bst/Bam-cRNA was trans-spliced with high efficiency, but not the Bst/Bam-cRNA molecules. Northern blot analysis revealed that both the capped and uncapped cRNA molecules had similar stability in the microinjected cells. The coinjected m7G(5')ppp(5')G cap analog did not inhibit the trans-splicing reaction in vivo and did not prevent nuclear export of the mRNA.

A two-step mechanism for 5' and 3' splice-site pairing.

A fundamental question in the splicing of precursor messenger RNA is how the 5' and 3' splice sites are recognized and paired during the splicing reaction. It has been proposed that spliceosome assembly in metazoan pre-mRNAs can be initiated through interaction between the 3' splice site and specific sequence elements on the downstream exon (an exonic enhancer or a 5' splice site). Pairing of the intronic 5' and 3' splice sites occurs subsequently. We report here that 5' and 3' splice sites located on separate synthetic pre-mRNA substrates can be efficiently trans-spliced if the 3' trans-splicing substrate contains these downstream sequence elements. Moreover, selection of the trans 5' splice site can occur after the second pre-spliceosomal complex A has assembled on the 3' trans-splicing substrate. Thus our data demonstrate that 5' and 3' splice-site pairing in metazoans can occur in two distinct steps.

Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates.

The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo.

Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P.

Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.