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[Effect of chain length of dialdehyde oligonucleotides on the rate of their interaction with the three-dimensional matrix of polyacrylamide gel].

The kinetics of the reaction of periodate oxidized oligonucleotides with polyacrylhydrazide gel was studied. The rate of the reaction is proportional to (see article), where M is the molecular weight of the oligonucleotide for the permeable gels. The rate of the reaction with given oligonucleotide decrease as the number of crosslinks in the gel matrix increase; a dramatic decrease of the rate occurs when oligonucleotides become too large to penetrate into the matrix. The rate of the reactions with short oligonucleotides does not depend upon the viscosity of the medium. ESR method revealed a considerable decrease of the rotational mobility of oligonucleotides captured by the gel matrix. It has been shown also that an increase of the number of cross-links leads to a decrease of the rotational mobility of the gel chains.

Acrylamides↗

PubMed1977

Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.

Adenosine↗

PubMed1975

Sequence determination of 5'-terminal and 3'-terminal T1 oligonucleotides of 18-S ribosomal RNA of a mouse cell line (L 5178 Y).

The 5' and 3'-terminal oligonucleotides of 18-S ribosomal RNA of L 5178 Y (a mouse cell line) obtained after total T1 ribonuclease hydrolysis were isolated by a diagonal procedure. They were localized on the fingerprint of T1-ribonuclease-hydrolysed 18-S RNA. These two oligonucleotides were partially hydrolysed by snake venom and spleen phsophodiesterases and resulting products were fractionated bidimensionally. Their base compositions were determined by total hydrolysis with piperidine or snake venom phosphodiesterase. From these results the following sequences were deduced: pU-A-C-C-U-G for the 5'-terminal oligonucleotide and G-A-U-C-A-U-U-Aoh for the 3'-terminal oligonucleotide. Quantitative studies indicated that these sequences represent at least 70% for the 5' oligonucleotide and 85% for the 3' oligonucleotide of the terminal sequences of the 18-S RNA.

Animals↗

PubMed1979

Analysis of large specific T1 oligonucleotides of 17S and 25S ribosomal RNAs from Saccharomyces cerevisiae.

The primary structure of 17S and 25S ribosomal RNAs from Saccharomyces cerevisiae has been analysed by two-dimensional fractionation of T1 oligonucleotides. This method consists of an electrophoresis at pH 3.5 followed by a homochromatography on DEAE-cellulose plates. After the second dimension, the large T1 oligonucleotides were hydrolyzed by pancreatic RNAse, followed by alkaline hydrolysis of the pancreatic products. By fractionating a mixture of tritiated HeLa cell ribosomal RNAs and 32 P yeast cell ribosomal RNAs, two autoradiographs were obtained; one corresponding to the 32P labelled material and the other to the tritiated labelled material. By superposition of the two autoradiographs, the mobility of the various T1 oligonucleotides can be accurately compared and it is shown that yeast 17S rRNA and human 18S rRNA have in common 5 large oligonucleotides and that yeast 25S rRNA and human 28S rRNA have 4 identical oligonucleotides.

Autoradiography↗

PubMed1976

Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants.

The RNase-T1-resistant oligonucleotides of two Prague Rous sarcoma viruses with temperature-sensitive (ts) DNA polymerases (DNA nucleotidyltransferases), termed ts LA 337 and 335 of one leukosis virus, RAV-6, and 20 of their recombinant progeny have been mapped relative to the 3' poly (A) terminus of the viral RNA. The resulting oligonucleotide maps have been ocrrelated with markers of the four known viral genetic elements encoded in the RNA of 10,000 nucleotides. In accord with previous results recombinant RNAs contained (i) oligonucleotides characteristic of the src gene, coding for sarcoma formation, between the poly(A) end and 2000 nucleotides and (ii) olignucleotides characteristic of the env gene, coding for the envelope glycoprotein, between 2500 and 5000 nucleo tides from the poly(A) end. (iii) A cluster of four oligonucleotides that mapped between 6000 and 8000 nucleotides from the 3' poly(A) end of each RNA was shared by both parental viruses and all recombinants. Since all other map segments of our recombinants failed to segregate with the ts- or wild-type markers of the parental DNA polymerase gene (pol), it was concluded that the ts pol lesion maps in this RNA segment. (iv) The 5' segment of each recombinant RNA contained a cluster of four to five oligonucleotides whose parental origin correlated with an electrophoretic marker of one of the parental virion proteins, p27, a major product of the viral gag gene. The gene order 5'-gag-pol-env-src-poly(A) is consistent with our data.

Antigens, Viral↗

PubMed1975

Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide.

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).

Alkaline Phosphatase↗

PubMed1978

Selectivity of RNA chain initiation in vitro. 2. Correlation of 5'-triphosphate-labeled oligonucleotides on polyethyleniminecellulose thin-layer chromatography with RNA transcripts of bacteriophage lambda cb2 and T7.

Methods are described for the correlation of 5'-terminal oligonucleotides separated by two-dimensional polyethyleniminecellulose thin-layer chromatogrphy with specific RNA transcripts made in vitro from DNA of phages T7 and lambdacb2. The 5'-terminal oligonucleotides transcribed from DNA containing mutations and alterations which affect the RNA transcripts from specific promoters were compared with fingerprints of RNA from wild type DNA. Specific RNAs were purified on polyacrylamide gels and digested and their 5'-terminal oligonucleotides subjected to chromatography. Transcription of DNA fragments containing specific promoters was carried out and the 5'-oligonucleotide fingerprints of the RNA products compared with fingerprints of the RNA from the whole DNA. Using these methods and information known about T7 and lambdacb2 RNA 5'-terminal sequences, it was possible to identify many of the oligonucleotides separated on the polyethyleniminecellulose chromatography system.

Base Sequence↗

PubMed1978

Modified nucleotides in T1 RNase oligonucleotides of 18S ribosomal RNA of the Novikoff hepatoma.

The primary structure of 18S rRNA of the Novikoff hepatoma cells was investigated. Regardless of whether the primary sequence of 18S rRNA is finally determined by RNA sequencing methods or DNA sequencing methods, it is important to identify numbers and types of the modified nucleotides and accordingly the present study was designed to localize the modified regions in T1 RNase derived oligonucleotide. Modified nucleotides found in 66 different oligonucleotide sequences included 2 m62A, 1 m6A, 1 m7G, 1m1cap3psi, 7 Cm, 13 Am, 9 Gm, 11 Um, and 38 psi residues. A number of these modified nucleotides are now placed in defined sequences of T1 RNase oligonucleotides which are now being searched for in larger fragments derived from partial T1 RNase digests of 18S rRNA. Improved homochromatography fingerprinting (Choi et al. (1976) Cancer Res. 36, 4301) of T1 RNase derived oligonucleotides provided a distinctive pattern for 18S rRNA of Novikoff hepatoma ascites cells. The 116 spots obtained by homochromatography contain 176 oligonucleotide sequences.

Base Sequence↗

PubMed1978

Characterization and mapping of RNase T1-resistant oligonucleotides derived from the genomes of Akv and MCF murine leukemia viruses.

T1 RNA fingerprints of the genomes of Akv-1 and Akv-2 C-type viruses are indistinguishable and oligonucleotide maps of these viruses are probably the same. Akv-1 and -2 share 55--75% of their large T1-resistant oligonucleotides with four MCF viruses isolated from AKR mice or from NIH Swiss mice that inherit either the Akv-1 or Akv-2 virus-inducing locus of AKR. The majority of Akv oligonucleotides missing from T1 fingerprints of MCFs and the majority of oligonucleotides unique to MCF viruses are clustered and lie at corresponding positions in the 3' half of the oligonucleotide maps of Akv and MCF viruses. The RNA sequences present in different MCF isolates but not present in Akv-viruses are related. These results are consistent with a recombinational origin of MCF viruses, as proposed by Hartley and Rowe and their collaborators.

Chromosome Mapping↗

PubMed1977

RNase T1-resistant oligonucleotides of an N- and a B-tropic murine leukemia virus of BALB/c: evidence for recombination between these viruses.

We used two-dimensional gel electrophoresis to obtain fingerprints of 32P-labeled RNase T1-resistant oligonucleotides derived from the genomes of an N- and a B-tropic murine leukemia virus of BALB/c. These viruses share approximately 30 large T1-resistant oligonucleotides. In addition, there are eight large oligonucleotides unique to the N-tropic virus, and there are six B-trophic virus-specific oligonucleotides. Viruses, designated XLP-N, which appear by biological criteria and analysis of virion proteins to be recombinants between these N- and B-tropic viruses, possess some but not all of the N or B virus-specific oligonucleotides.

Drug Resistance, Microbial↗

PubMed1978

Sequence and location of large RNase T1 oligonucleotides in bacteriophage Qbeta RNA.

Twenty-nine oligonucleotides, 11 to 26 nucleotides in length, arising by complete RNase T1 digestion of bacteriophage Qbeta RNA and isolated by two-dimensional polyacrylamide gel electrophoresis, were sequenced. Their location within the genome was established with two methods. (a) In vitro synthesis of Qbeta RNA plus strands was started synchronously, using minus strands as template and nucleoside [alpha-32P]triphosphates as substrate; after various times, the reaction was stopped and the length of the products formed was correlated with their content of T1 oligonucleotides. (b) Qbeta [32P]RNA was elongated with poly(A) using terminal riboadenylate transferase; after mild treatment with alkali the fragments were fractionated by size and the poly(A)-containing molecules of each size class were isolated by chromatography on poly(U)-Sephadex and assayed for T1 oligonucleotides. The oligonucleotides in the 5' region were localized more precisely with method a, those near the 3' end with method b; in the middle region, the results of the two sets of analyses confirmed each other. The use of these oligonucleotides in the sequence determination of Qbeta RNA is discussed.

Base Sequence↗

PubMed1977

Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup.

The relationships among the genomes of various rhabdoviruses belonging to the vesicular stomatitis virus subgroup were analyzed by an oligonucleotide fingerprinting technique. Of 10 vesicular stomatitis viruses, Indiana serotype (VSV Indiana), obtained from various sources, either no, few, or many differences were observed in the oligonucleotide fingerprints of the 42S RNA species extracted from standard B virions. Analyses of the oligonucleotides obtained from RNA extracted from three separate preparations of VSV Indiana defective T particles showed that their RNAs contain fewer oligonucleotides than the corresponding B particle RNA species. The fingerprints of RNA obtained from five VSV New Jersey serotype viruses were easily distinguished from those of the VSV Indiana isolates. Three of the VSV New Jersey RNA fingerprints were similar to each other but quite different from those of the other two viruses. The RNA fingerprints of two Chandipura virus isolates (one obtained from India and one from Nigeria) were also unique, whereas the fingerprint of Cocal virus RNA was unlike that of the serologically related VSV Indiana.

Defective Viruses↗

PubMed1977

RNase T1-resistant oligonucleotides of B-tropic murine leukemia virus from BALB/c and five of its NB-tropic derivatives.

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides of a B-tropic murine leukemia virus from BALB/c and five NB-tropic viruses independently derived from this B virus by passage through NIH Swiss mouse embryo cells in vitro. The fingerprints of the B- and NB-tropic viruses were very similar: approximately 33 of 35 large T1-resistant oligonucleotides appeared to be shared by these viruses. However, the five NB-tropic viruses possessed an apparently common alteration relative to their B virus progenitor. This change involved the acquisition of one oligonucleotide and, tentatively, the loss of one oligonucleotide. We do not know whether these changes represent an alteration responsible for the change from B- to NB-tropism. Fingerprints of B- and NB-tropic viruses were not affected when the viruses were grown in cells of different Fv-1 type.

Animals↗

PubMed1979

[Determination of the interaction of oligopeptides and oligonucleotides by a chromatographic technique (author's transl)].

Homologues of deoxyriboadenylic and deoxyribothymidylic acid show different degrees of retardation in a pentalysine gel. This chromatographic retardation is directly related to the strength of the interaction between the oligonucleotides and the immobilized pentalysine peptide residues. The strength of this interaction is determined by the total negative charge of the oligonucleotide, and by the nature of the constituent base of the oligonucleotide. The specificity of the interaction depends upon differences in conformation of the oligonucleotides.

Chromatography, Gel↗

PubMed2026

Cross-target and cell-preferential CRISPR-Cas9 inhibition with carbohydrate-tagged oligonucleotides.

Precise control of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) activity is important for limiting off-target effects and chromosomal rearrangements. Existing inhibitors, including anti-CRISPR proteins and spacer-targeting oligonucleotides, can be constrained by immunogenicity, target-sequence dependence, or delivery challenges. Here, we developed single-stranded DNA oligonucleotides that target conserved regions of the Streptococcus pyogenes Cas9 single-guide RNA (sgRNA) scaffold rather than the variable spacer. Screening identified single-stranded DNA 7 (ssDNA7), which targets stem-loop-1 and the adjacent linker and inhibits Cas9 activity across multiple tested spacer sequences and genomic loci without redesigning the inhibitor. Carbohydrate conjugation improved oligonucleotide stability and preferentially enhanced inhibitory activity in selected liver-derived or cancer cell models. Amplicon sequencing confirmed inhibition of endogenous editing in multiple cell models and normal liver-derived organoids, and supported cell-preferential inhibition in matched cell comparisons. These findings establish sgRNA-scaffold targeting as a strategy for cross-target inhibition of Cas9 and show that carbohydrate conjugation can tune its cellular activity.

CRISPR-Cas9↗

PubMed1976

Application of a spectrophotometric method to the determination of the composition of oligonucleotides obtained from cysteine transfer ribonucleic acid.

1. The applications of methods for determining the composition of oligonucleotides from u.v.-absorption spectra is described. 2. In the first method absorbances at selected wave-lengths were read from the spectra of oligonucleotides in solution in 7 M-urea which had been recorded at acid and alkaline pH values. 3. In the second method absorbances were sampled automatically at regular time-intervals during scans at acid and alkaline pH of each spectrum, converted into digital signals and recorded on paper take for computer processing. The holmium spectrum in the region of the holmium peak at 333.7 nm was superimposed on each nucleotide spectrum. The position of this peak maximum was used as a standard reference point in the computer-based analysis. 4. By using either method the composition was calculated by a least-squares procedure by using a library of values for five standard nucelotides obtained in a similar manner. 5. The methods gave satisfactory compositions for mixtures of mononucleotides as well as for five dinucleoside monophosphates. 6. Methods of minimizing the effects on the nucleotide composition of spectural changes due to base stacking are discussed. 7. The compositions of some oligonucleotides obtained during an investigation of the nucleotide sequence of tRNA (Cys) were determined and agreed with the sequences found by other methods.

Base Sequence↗

PubMed1977

Inhibition of pancreatic ribonuclease by 2'-5' and 3'-5' oligonucleotides.

Forty different oligonucleotides were investigated as possible inhibitors of the depolymerizing activity of RNase A. The strongest inhibitors among the diribonucleoside 2'-5' mono- phosphates were: G2'-5'G, C2'-5'G and U2'-5'G, and among the diribonucleoside 3'-5' monophosphates: ApU, ApC and GpU. Of the eight trinucleotides investigated, ApApUp, ApApCp and ApGpUp were the strongest inhibitors. All four dinucleotides studied (ApUp, ApCp, GpUp and GpCp) were very strong inhibitors, ApUp being the strongest one. The results show that the nature of the various bases in the oligonucleotide has an effect on the degree of inhibition, and that the 3' phosphomonoester group increases the binding of the oligonucleotide to RNase A. These inhibitors can be used in physicochemical and biochemical studies of ribonuclease.

Kinetics↗

PubMed1976

Allocation of 15 RNase T1-resistant large oligonucleotides of MS2 RNA.

Four large MS2 RNA fragments with the original 5'-end were obtained by limited RNase T1 digestion. The lengths of the fragments were 36, 30, 22, and 19% of the whole molecule. Whole MS2 RNA and the four fragments were completely digested with RNase T1 and analyzed by the two-dimensional "homochromatography fingerprint" technique. Sixteen oligonucleotides of MS2 RNA were separated and the oligonucleotides were assigned to the fragments. Fifteen of the sixteen oligonucleotides could be classified into three regions of the cistrons of bacteriophage MS2.

Base Sequence↗