RNA conformational sampling. I. Single-nucleotide loop closure

We consider the loop-closure problem for nucleic acids and describe an efficient numerical algorithm for closing single-nucleotide loops in nucleic acids. Using six new internal coordinates to represent the nucleotide conformation, which we call the R-representation, the original closure problem with six free torsion angles in each nucleotide can be reduced to one with only four degrees of freedom. Simple numerical techniques have been used to solve the resulting loop-closure equations, and a test of the closure algorithm on a set of RNAs consisting of more than 7000 nucleotides was able to regenerate the native torsion angles in every nucleotide in the test set without exception. We show how the conformational probability density transforms when the original torsion angle representation is mapped onto the new R-representation. We also present statistical evidence showing that the delta and nu(2) torsion angles are coupled, and how this coupling affects the conformation probability density in the R-representation. In addition to the backbone, the same loop-closure algorithm can also be applied to close the ribose ring. The algorithm is freely available at http://tyrosine.use.edu/closure.     

High-performance affinity chromatography of messenger RNA.

A 50-mer of thymidylic acid, (dT)50, was coupled to silica inside prepacked columns using the N-hydroxysuccinimide chemistry. The resulting (dT)50-silica columns were used to resolve oligomers of adenylic acid, (dA)19-24, and to separate poly(A) mRNA (messenger RNA) from Saccharomyces. Oligomers which differed in length by a single nucleotide base were readily resolved. Using either (dT)50- or (dT)18-silica, poly(A) mRNA could be purified in as little as 8 min. The poly(A) mRNA isolated appeared to be full length and could be used directly for T4 RNA ligase and RNAse A and T1 enzymatic reactions. The (dT)50-silica column was used to fractionate total poly(A) mRNA by tail length. While the separation was primarily due to poly(A) tail length, most fractions appeared to contain multiple tail lengths. Whether this represents an intrinsic feature of the RNA or a limitation of the method is discussed. These studies show that polynucleotides in the kilobase size range can be separated rapidly and with good resolution on DNA-silica.     

siRNA. A guide for RNA silencing

RNAi is routinely used to eliminate gene activity for experimental purposes. However, the precise molecular mechanism of RNAi is unknown. Recent papers partially illuminate this mechanism in human cells, advancing the potential application of RNAi toward the treatment of human disease.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

U.V. INDUCED CONFORMATIONAL CHANGES IN TRANSFER RNA

Abstract— The quantum yields for the u.v. inactivation of the amino acid acceptor function of E. coli transfer RNA (for val, phe and lys) and for the loss of its conformation, as a function of exposure, have been determined following irradiation at 280, 265 and 254 nm. Our results suggest that u.v. damage produces a change in the conformation of transfer RNA which in turn inactivates it, and that the anticodon is not the u.v. sensitive site. Calculations indicate that a small number of photoproducts inactivate the transfer RNA.     

Branched RNA nanostructures for RNA interference

Branched RNAs with three- or four-way junctions were designed by assembling single-stranded RNA for RNA interference. Human Dicer transformed branched RNAs into about 20 base pairs of double-stranded RNA, which is a standard siRNA species. Our tetramer design provides a potent silencing effect over a period of 5 days.     

Inhibited neoplastic phenotype by the c-Ha-ras antisense RNA.

A 2.0 kb fragment DNA plasmid which expresses antisense to the upstream first exon of c-Ha-ras oncogene was transfected into Ha-ras transformed cell lines, GCM-3T3 and REF-4.3. The transfection leads to the inhibition of malignant behaviour, shown by decreasing of growth speed, colony forming ability on soft agar, tumorigenicity in nude mice and increasing of differentiation degree. In GCM-3T3 cells the lung metastasis frequency became much less (from 60% to 12.5%) after the transfection and the expression of ras oncogene product, and p21 protein was obviously decreased in the transfected cells. This work has first shown the inhibitory effect of antisense RNA on neoplastic behaviour in China.     

Electron affinities of the DNA and RNA bases.

Adiabatic electron affinities (AEAs) for the DNA and RNA bases are predicted by using a range of density functionals with a double-zeta plus polarization plus diffuse (DZP++) basis set in an effort to bracket the true EAs. Although the AEAs exhibit moderate fluctuations with respect to the choice of functional, systematic trends show that the covalent uracil (U) and thymine (T) anions are bound by 0.05-0.25 eV while the adenine (A) anion is clearly unbound. The computed AEAs for cytosine (C) and guanine (G) oscillate between small positive and negative values for the three most reliable functional combinations (BP86, B3LYP, and BLYP), and it remains unclear if either covalent anion is bound. AEAs with B3LYP/TZ2P++ single points are 0.19 (U), 0.16 (T), 0.07 (G), -0.02 (C), and -0.17 eV (A). Favorable comparisons are made to experimental estimates extrapolated from photoelectron spectra data for the complexes of the nucleobases with water. However, experimental values scaled from liquid-phase reduction potentials are shown to overestimate the AEAs by as much as 1.5 eV. Because the uracil and thymine covalent EAs are in energy ranges near those of their dipole-bound counterparts, preparation and precise experimental measurement of the thermodynamically stable covalent anions may prove challenging.     

Montmorillonite catalysis of RNA oligomer formation in aqueous solution. A model for the prebiotic formation of RNA

Oligomers of adenylic acid of up to the 11-mer in length are formed by the reaction of the phosphorimidazolide of adenosine (ImpA) in pH 8 aqueous solution at room temperature in the presence of Na(+)-montmorillonite. These oligomers are joined by phosphodiester bonds in which the 3',5'-linkage predominates over the 2',5'-linkage by a 2:1 ratio. Reaction of a 9:1 mixture of ImpA, A5'ppA results in the formation of oligomers with a 3:1 ratio of 3',5'- to 2',5'-linked phosphodiester bonds. A high proportion of these oligomers contain the A5'ppA grouping. A5'ppA reacts much more rapidly with ImpA than does 5'-ADP (ppA) or 5'-ATP (pppA). The exchangeable cation associated with the montmorillonite effects the observed catalysis with Li+, Na+, NH4+, and Ca2+ being the more effective while Mg2+ and Al3+ are almost ineffective catalysts. 2',5'-Linked oligomers, up to the tetramer in length, are formed using UO2(2+)-montmorillonite. The structure analysis of individual oligomer fractions was performed by selective enzymatic and KOH hydrolytic studies followed by HPLC analysis of the reaction products. It is concluded from the composition of the oligomers that the rate of addition ImpA to a 3'-terminus containing a 2',5'-linkage is slower than the addition to a nucleoside joined by a 3',5'-linked phosphodiester bond. The potential importance of mineral catalysis of the formation of RNA and other oligomers on primitive Earth is discussed.     

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 °C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg²⁺ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.     

Distribution of type I phytochrome (phyA) RNA1 and RNA2 in etiolated pea seedlings.

Four-day-old etiolated pea seedlings were divided into 11 parts along the axis, from which poly(A)+ RNA and DNA were extracted. Using a slot-blot hybridization assay, the abundance of pea type I phytochrome (phyA) poly(A)+ RNA was measured in each portion of the etiolated pea seedling. For the quantification of the phyA RNA, pea phyA RNA synthesized in vitro was used as an RNA standard. The hook region contained the highest abundance of phyA RNA (approximately 0.3 ng phyA RNA per microgram DNA) in the etiolated seedling. Two mRNAs of different length (the shorter designated as RNA1 and the longer RNA2) are produced in detectable amounts from single pea phyA in the etiolated seedling; the ratio of the abundance of phyA RNA1 to phyA RNA2 was determined in each of the 11 parts by a primer extension assay. The abundance of phyA RNA1 in the plumule and hook regions was 3-5-fold higher than that of RNA2, whereas the ratio of their abundance was approximately unity in other regions. A time course study of the abundance of both RNAs was carried out during the imbibition of seeds and indicated that the accumulation of phyA RNA1 occurred more rapidly in the cotyledons than that of RNA2 during the first day of imbibition, whereas the accumulation of phyA RNA2 increased rapidly during the second day and became as high as that of phyA RNA1 by the third day.     

RNA as multitude/RNA as one

In the configurations formed by RNA and its ions there are structural possibilities not yet realized; some are hinted at in new work on the binding of an amino acid analog.     

A comparison of RNA with DNA in template-directed synthesis.

Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.     

Photochemistry of tobacco mosaic virus RNA. Effect of HCN

Abstract— In attempting to sort out possible mechanisms of photoreactivation of tobacco mosaic virus RNA (TMV-RNA) inactivated by ultraviolet radiation (u.v.) in buffer of ionic strength 0.25, we have investigated the effect of HCN on the quantum yield for u.v. inactivation of TMV-RNA and on the percent photoreactivation of inactivated TMV-RNA. Some photo-products produced by irradiation of model substances, polyuridylic acid (poly U) and polycytidylic acid (poly C), in the presence of HCN have also been studied. The ratio of the quantum yield for inactivation of TMV-RNA in the presence of HCN to that in the absence of HCN is 1.5, under non-photoreactivating conditions. By comparison, the ratio of the initial rates of loss of uracil residues in poly U under comparable conditions is 1.6; by contrast, the rate of loss of cytosine residues in poly C is unaffected by HCN. This similarity of ratios between poly U and TMV-RNA suggests that two of the mechanisms of u.v. inactivation of TMV-RNA at high ionic strength are akin to known reactions of uracil residues in poly U, i.e. hydrate and dimer formation. The photohydration reaction in poly U, as measured by the heat reversal of hydrated residues to uracil residues, is almost abolished by HCN, and the rate of dimerization, as measured by the appearance of dimer containing oligonucleotides following enzymatic hydrolysis of irradiated poly U, is reduced to half by HCN. HCN does not affect the rate of hydration of cytosine residues in poly C. Since photoreactivation of RNA inactivated in presence of HCN is only 60 per cent of that in absence of HCN it is suggested that uracil dimers are somehow involved in photoreactivation of TMV-RNA inactivated at high ionic strength.