"Fingerprinting" high molecular weight RNA by two-dimensional gel electrophoresis: application to poliovirus RNA.

Conditions are described under which complete RNase T1 digests of high molecular weight RNA can be separated into numerous components by two-dimensional gel electrophoresis. Small and large oligonucleotides (n = 1 - 2c0) can be resolved without losses. The procedure yields fingerprints which are diagnostic for a particular species of RNA and an index of its purity as will be shown for the genomes of poliovirus type 1 and 2.     

A fluorimetric assay for available lysine in proteins

A two-dimensional fingerprinting gel system that provides sensitive analyses with high resolution of T1-resistant oligonucleotides of large RNA molecules is described. Unique oligonucleotides less than 30 bases in length are recovered quantitatively while longer oligonucleotides are recovered in very large (～90%) yields by active transfer of the fingerprint to DEAE paper. After elution of the oligonucleotides from DEAE paper, secondary analysis is performed by digestion of oligonucleotides with pancreatic RNase and separation of the products by high-voltage electrophoresis on polyethyleneimine cellulose. The complete analysis of up to 40 oligonucleotides can be accomplished within 4 days.     

Direct detection of small RNAs using splinted ligation

This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5'-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ligation. Detection of the labeled small RNAs is performed by denaturing gel electrophoresis and autoradiography or phosphor-imaging. This protocol has been successfully used to study expression of various classes of biological small RNAs from nanogram to microgram amounts of total RNA without an amplification step. It is significantly simpler to perform and more sensitive than either northern blotting or ribonuclease protection assays. Once the oligonucleotides have been synthesized and total RNA has been extracted, the procedure can be completed in 6 h.     

RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane.

Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3'-end of the 16S RNA).     

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.     

The use of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for regiospecific cleavage of RNA by RNase H from Escherichia coli.

Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.     

A method to select chemically modified aptamers directly

In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.     

Assignment of the large oligonucleotides of vesicular stomatitis virus to the N, NS, M, G, and L genes and oligonucleotide gene ordering within the L gene.

Analyses of prototype vesicular stomatitis (VSV, Indiana serotype) mRNA-32P-labeled viral RNA duplexes have established the assignments of 65 of the 72 large oligonucleotides that are recovered by two-dimensional electrophoresis of RNase T1 digests of the viral RNA. Fifty of the oligonucleotides are recovered in the L RNA duplex, four each in the N, M, and NS duplexes, and three in the G RNA duplex. Studies of three small defective-particle RNA species indicate that they have only L gene oligonucleotides in addition to three of the seven unassigned oligonucleotides. Some L gene ordering of oligonucleotides can be postulated from the defective-particle RNA sequence analyses. Analyses of naturally occurring alternate isolates of VSV Indiana have established that by comparison to the prototype virus strain, the alternate isolates minimally have genome sequence differences in L, G, N, NS and/or unassigned regions of the genome. Changes in the genome have also been induced by vitro high-level mutagenesis of the prototype virus.     

Detection of gastrin-specific mRNA using oligodeoxynucleotide probes of defined sequence.

Three oligodeoxynucleotides have been chemically synthesized and used as hybridization probes to detect gastrin-specific mRNA within an heterogeneous population of RNA molecules. Using these oligonucleotides whose nucleotide sequences were deduced from the amino acid sequence of the hormone, 0.2 fmol of gastrin mRNA can be detected/microgram of poly(A)-RNA from hog antrums. The 32P-labeled oligonucleotides hybridize specifically to RNA covalently coupled to DBM-paper (Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354). Labeled oligonucleotides also hybridize specifically to RNA separated by gel electrophoresis in the presence of CH3HgOH and covalently coupled to DBM-paper. Using this procedure, we have found that the mRNA coding for gastrin contains about 620 nucleotides, which is in agreement with results obtained using gastrin cDNA to determine mRNA size.     

Oligonucleotides Quality Control Analysis in Free Solution by Capillary Electrophoresis at Acidic pH

Abstract

A new method for fast, automated and inexpensive oligonucleotides analysis by capillary electrophoresis at low pH is presented. This method does not need any sieving media to resolve a mixture of polynucleotides which are analysed in free solution and separated on the base of composition and not length. This technique has been used to test a large set of standard and modified oligonucleotides thus to be applied in oligos routine quality control.     

Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.     

Large-scale purification of synthetic oligonucleotides and carcinogen-modified oligodeoxynucleotides on a reverse-phase polystyrene (PRP-1) column

A procedure is described for the large-scale purification of synthetic oligonucleotides using a polystyrene (PRP-1, Hamilton Co.) high-performance liquid chromatography (HPLC) column with a phosphate/methanol/acetonitrile solvent system. Pure oligonucleotides are obtained with a three-step procedure that involves only one column purification step. The dimethoxytrityl group is left on the oligomer for the HPLC purification. The use of the PRP-1 polystyrene column with a phosphate/methanol/acetonitrile solvent system provides excellent separation of the desired dimethoxytrityl-bearing oligonucleotide from failure sequences. The dimethoxytrityl group is removed by treatment with acetic acid and the oligonucleotide is desalted on a C-18 Sep-Pak cartridge. The oligodeoxynucleotides obtained are shown to be essentially pure by HPLC, polyacrylamide gel electrophoresis, and 500-MHzNMR spectroscopy. This procedure is especially useful for the large-scale purification of oligonucleotides required for NMR studies. The PRP-1 column and the phosphate/methanol/acetonitrile solvent system is useful for purifying modified oligonucleotides containing lipophilic groups such as the carcinogen 2-(acetylamino)fluorene.     

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines. 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.     

Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus.

The site of recombination of a mink cell focus-inducing strain (Mo-MuLV83) derived from an ecotropic Moloney murine leukemia virus (Mo-MuLV) was mapped by fingerprint analysis of the large RNase T1-resistant oligonucleotides, employing a two-dimensional gel electrophoresis method. Mo-MuLV83, in contrast to the ecotropic Mo-MuLV, demonstrated a broadened host range, i.e., growth not only on mouse cells but also on mink cells, and recombination involved the env gene function. The genomic RNA of these two viruses shared 42 out of a total of 51 to 53 large T1 oligonucleotides (81%) and possessed a similar subunit size of 36S. Most of these T1 oligonucleotides were mapped in their relative order to the 3' polyadenylic acid end of the viral RNA molecules. There were 10 common oligonucleotides immediately next to the 3' termini. A cluster of 7 (in Mo-MuLV83) or 10 (in Mo-MuLV) unique T1 oligonucleotides were mapped next to the common sequences at the 3' end, and they all appeared concomitantly in a polyadenylic acid-containing RNA fraction with a sedimentation coefficient slightly larger than 18S. Therefore, the env gene of Mo-MuLV was situated at a location approximately 2,000 to 4,000 nucleotides from the 3' end of the genomic RNA, and the gene order of Mo-MuLV appeared to be similar to that of the more rigorously determined avian oncornaviruses. cDNA(SFFV) specific for the xenotropic sequences in the spleen focus-forming virus RNA hybridized to the cluster of unique oligonucleotides of Mo-MuLV83 RNA. This suggests that the loci of recombination involve the homologous env gene region of a xenotropic virus.     

The use of nuclease P1 in sequence analysis of end group labeled RNA.

A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.     

Preparation and properties of a new type of acyclic, achiral nucleoside analogue

Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by Tm measurements showing a deltaTm of -2 to -6.5 degrees C per modification. An oligonucleotide with two modifications at the 3'-end showed considerable resistance towards cleavage by a 3'-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.     

2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: an analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA

A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.     

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.