Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H

To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Mapping tRNA structure in solution using double-strand-specific ribonuclease V1 from cobra venom.

A method for mapping all base-paired stems in both elongation and initiator tRNAs is described using double-stranded-specific ribonuclease V1 from the venom of the cobra Naja naja oxiana. 32p-end-labeled RNA is first partially digested with double-strand-specific V1 nuclease under near physiological conditions, and the resultant fragments are than electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel run in 90% formamide. After autoradiography, the base-paired nucleotides are definitively located by comparing V1 generated bands with fragments of known length produced by both Neurospora endonuclease and base-specific ribonucleases. Using the substrates yeast tRNAPhe an E, coli tRNAfMet of known three-dimensional structure, we find V1 nuclease to cleave entirely within every base-paired stem. Our studies also reveal that nuclease V1 will digest paired nucleotides not hydrogen-bonded by standard Watson-Crick base-pairing. In yeast tRNAPhe cleavage of both wobble base-pairs and nucleotides involved in tertiary base-base hydrogen bonding is demonstrated.     

Secondary structure formation precedes tertiary structure in the refolding of ribonuclease A.

The kinetics of refolding of ribonuclease A were monitored by circular dichroism (CD), tyrosine fluorescence and absorbance in the -40 to -10 degrees C range using a methanol cryosolvent. The native-like far-ultraviolet CD signal returned in the dead-time of the mixing, whereas the native absorbance and fluorescence signals returned in a multiphasic process at rates several orders of magnitude more slowly. Thus the secondary structure was formed much more rapidly than the tertiary structure. In addition, the absorbance signal showed evidence of an early intermediate in which one, or more, tyrosine residues was in a transiently more polar environment. A total of four kinetic phases were observed by absorbance in refolding, the slowest two of which had energies of activation consistent with proline isomerization. A refolding scheme involving initial hydrophobic collapse, concurrent with secondary structure formation, followed by much slower rearrangement to the native tertiary structure is proposed.     

Intracellular mRNA cleavage by 3' tRNase under the direction of 2'-O-methyl RNA heptamers

Mammalian tRNA 3′ processing endoribonuclease (3′-tRNase) can cleave any RNA at any site under the direction of small guide RNA (sgRNA) in vitro. sgRNAs can be as short as heptamers, which are much smaller than small interfering RNAs of ~21 nt. Together with such flexibility in substrate recognition, the ubiquity and the constitutive expression of 3′-tRNase have suggested that this enzyme can be utilized for specific cleavage of cellular RNAs by introducing appropriate sgRNAs into living cells. Here we demonstrated that the expression of chloramphenicol acetyltransferase can be downregulated by an appropriate sgRNA which is introduced into Madin–Darby canine kidney epithelial cells as an expression plasmid or a synthetic 2′-O-methyl RNA. We also showed that 2′-O-methyl RNA heptamers can attack luciferase mRNAs with a high specificity and induce 3′-tRNase-mediated knock-down of the mRNAs in 293 cells. Furthermore, the MTT cell viability assay suggested that an RNA heptamer can downregulate the endogenous Bcl-2 mRNA in Sarcoma 180 cells. This novel sgRNA/3′-tRNase strategy for destroying specific cellular RNAs may be utilized for therapeutic applications.     

Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.     

Probing ribosome structure using short oligodeoxyribonucleotides: the question of resolution

The structure of ribosomal RNA in situ can be probed using short, complementary DNA oligomers. As these oligomers bind to exposed, single-stranded regions of rRNA, the stability of the hybridized complex can be assayed. Differences in binding stability between cDNA probes of similar length and composition may be indicative of the presence of competing structure, such as base-paired rRNA regions, tRNA interactions or protein interactions. In this study the degree to which such interactions can be distinguished is studied. It is found that by using suitable controls, interactions between rRNA and tRNA or rRNA can be discriminated to a resolution of one or two bases. This resolution promises to be important in delineating the higher-order structure of the rRNA.     

An efficient process for synthesis of 2'-O-methyl and 3'-O-methyl guanosine from 2-aminoadenosine using diazomethane and the catalyst stannous chloride

An improved strategy for the selective synthesis of 2'-O-methyl and 3'-O-methyl guanosine from 2-aminoadenosine is reported by using the catalyst stannous chloride. The regioselectivity of the 2' and 3'-O-alkylation was achieved by optimizing the addition, timing, and concentration of the catalysts and diazomethane during the methylation reaction. An efficient and selective alkylation at 2'-OH of 2-aminoadenosine was achieved by mixing a stoichiometric amount of stannous chloride at room temperature in DME The reaction mixture was stirred at 50 degrees C for 1 min and immediately followed by addition of diazomethane. The resulting 2'-O-methyl 2-aminoadenosine was treated with the enzyme adenosine deaminase, which resulted in an efficient conversion to the desired 2'-O-methylguanosine (98% yield). The product was isolated by crystallization. In contrast, the methylation at 3'-OH of 2-aminoadenosine was achieved by mixing a stoichiometric amount of stannous chloride in DMF and stirring at 50 degrees C for 15 min, followed by addition of diazomethane. The resulting mixture containing 3'-O-methyl-2-aminoadenosine in 90% yield and 2'-O-methyl-2-aminoadenosine in 10% yield was treated with the enzyme adenosine deaminase, which preferentially deaminated only 3'-O-methyl-2-aminoadenosine, resulting in the production of 3'-O-methylguanosine in 88% yield. Due to the extremely low solubility 3'-O-methylguanosine, the compound precipitated and was isolated by centrifugation. This synthetic route obviates the chromatographic purification. Selective monomethylation is achieved by using the unprotected ribonucleoside. As a result, the method described herein represents a significant improvement over the current synthetic approach by providing superior product yield and economy, a much more facile purification of 2',3'-O-methylated isomers, and eliminating the need for protected ribonucleosides reagents.