Hybridase cleavage of RNA. I. Oligonucleotide probes for regiospecific RNA cleavage]

New oligonucleotide probes for regiospecific cleavage of RNA molecules by hybridase (RNase H) are suggested. RNase H from E. coli is shown to site-specifically split eight phosphodiester bonds in RNA in the heteroduplex, formed by 5S rRNA and d(ACCACCGCGCT). The partial substitution of deoxycytidines in position 5, 6, 8, 10 of the probe by 2'-O-methylcytidines leads to unique (regiospecific) RNA cleavage between U25 and C26.     

Hybridase cleavage of RNA. II. Automatic synthesis of mixed oligonucleotide probes

Several oligo(ribodeoxyribo)nucleotides to be used as probes in the RNase H-induced hydrolyses of 5S ribosomal RNA E. coli were synthesized on the Victoria-4M automatic gene synthesizer by the phosphoramidite approach, which allows for introducing ribonucleotides into any position of the oligomer. 2'-Hydroxy function was protected by tert-butyldimethylsilyl group whose hydrophobicity simplified isolation of the oligonucleotides by reverse-phased HPLC.     

Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2'-fluoronucleoside and arabinofuranosylcytosine]

A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.     

Hybridase cleavage of RNA. III. Use of UV-spectroscopy for studying the kinetic properties of E. coli RNAase properties]

A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.     

Site-directed cleavage of RNA.

Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region. Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem. The method is proved to be generally applicable to RNA containing secondary structures.     

Complementary role of two fragments of domain V of 23 S ribosomal RNA in protein folding.

We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298).     

Site specific enzymatic cleavage of RNA.

The hybridization of a DNA oligonucleotide a specific tetramer or longer) will direct a cleavage by RNase H (EC 3.1.4.34) to a specific site in RNA. The resulting fragments can then be labeled at their 5' or 3' ends, purified, and sequenced directly. This procedure is demonstrated with two RNA molecules of known sequence: 5.8S rRNA from yeast (158 nucleotides) and satellite tobacco necrosis virus (STNV) RNA (1240 nucleotides).     

Enzymatic cleavage of RNA by RNA

The catalytic activity of E. coli RNase P, an enzyme essential for tRNA biosynthesis in vivo, resides in the RNA subunit of the enzyme. This RNA, which has all the properties of a classical enzyme, can cleave precursor tRNAs in vitro in the total absence of proteins.     

Enzymatic cleavage of RNA by RNA

The discovery and characterization of the catalytic RNA subunit of the enzyme ribonuclease P ofEscherichia coli is described.Nobel lecture given on December 8, 1989, by Professor Sidney Altman, and published in LES PRIX NOBEL 1989, printed in Sweden by Norstedts Tryckeri, Stockholm, Sweden, 1990, republished here with the permission of the Nobel Foundation, the copyright holder.     

Escherichia coli topoisomerase III-catalyzed cleavage of RNA.

Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture. Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium. The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III. The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed. Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.     

Complementary RNA in the chicken sarcoma cells and Rous sarcoma virus]

The subcellular localization in chicken Rous sarcoma of nucleotide sequence, complementary to Rous sarcoma virus RNA was examined by RNA/RNA molecular hybridization. The preparations of radioiodinated virion RNA were annealed with RNAs from different fractions (nuclei, mitochondria, free and membrane-bound polyribosomes) isolated from chicken Rous sarcoma. Formation of RNA-ase resistant hybrids between the viral 125I-RNA and RNA from the mitochondria and membrane-bound polyribosomes was revealed. The latter were characterized by a higher relative redundancy of nucleotide sequences complementary to virion RNA than that in the former, by factor 446. The role of complementary ribonucleotide sequences is discussed.     

Use of specific endonuclease cleavage in RNA sequencing.

Nonradioactive RNA fragments may be sequenced by incorporation of (3H)-label into 3'-terminal positions, controlled digestion with specific ribonucleases, and separation according to size of the digestion products on polyethyleneimine- (PEI-) cellulose thin layers. This combination of techniques allows one to measure accurately distances of specific cleavage sites from the labeled terminal positions. The cleavage specificities of RNases T1, U2, and A are utilized to identify the positions of G, A, and pyrimidine residues respectively. C and U may be distinguished by mobility differences on PEI-cellulose thin layers at ph 2.6. The procedure is simple, rapid, and highly sensitive; as little as 0.5 - 1 microgram of a RNA of the size of tRNA will be needed to sequence all fragments in a complete RNase digest.     

Complementary transcribed T4 RNA. Association with the polysomes.

Pulse labelled bacteriophage T₄ RNA isolated from polysomes from either early or late infected cells were found to contain complementary RNA. The fraction of complementary late RNA was higher in the heaviest late polysomes. The RNA not associated with polysomes appeared to contain insignificant amounts of complementary RNA. The significance of these findings are discussed.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Specific cleavage of ribosomal RNA caused by alpha sarcin.

Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment. The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes. The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits. The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA. The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis. Alpha sarcin also causes the specific cleavage of the 23S RNA of the E. coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.