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Mapping RNase T1-resistant oligonucleotides of avian tumor virus RNAs: sarcoma-specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end.

The large RNase T1-resistant oligonucleotides of the nondefective (nd) Rous sarcoma virus (RSV): Prague RSV of subgroup B (PR-B), PR-C and B77 of subgroup C; of their transformation-defective (td0 deletion mutants: td PR-B, td PR-C, and td B77; and of replication-defective (rd) RSV(-) were completely or partially mapped on the 30 to 40S viral RNAs. The location of a given oligonucleotide relative to the poly(A) terminus of the viral RNAs was directly deduced from the smallest size of the poly(A)-tagged RNA fragment from which it could be isolated. Identification of distinct oligonucleotides was based on their location in the electrophoretic/chromatographic fingerprint pattern and on analysis of their RNase A-resistant fragments. The following results were obtained. (i) The number of large oligonucleotides per poly(A)-tagged ffagment increased with increasing size of the fragment. This implies that the genetic map is linear and that a given RNase T1-resistant oligonucleotides has, relative to the poly(A) end, the same location on all 30 to 40S RNA subunits of a given 60 to 70S viral RNA complex, (ii) Three sarcoma-specific oligonucleotides were identified in the RNAs of Pr-B, PR-C and B77 by comparison with the RNAs of the corresponding td viruses...

Avian Sarcoma Viruses

Dual recognition drives site-directed G-quadruplex stabilization: Oligonucleotide design in G4 ligand-oligonucleotide conjugates.

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among G4 motifs. G4 Ligand-Oligonucleotides conjugates (GL-Os) address this challenge by combining small-molecule G4 ligands with the sequence specificity of oligonucleotides, targeting sequences flanking the intended G4 target. Here, we systematically investigate how oligonucleotide length, backbone composition, and sequence complementarity govern GL-O binding, selectivity, and G4 stabilization. We show that effective G4 recognition depends on the interdependence between oligonucleotide hybridization and G4 ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Longer oligonucleotides promote more stable complexes and stronger G4 stabilization, whereas central mismatches disrupt this dual-recognition mechanism. Replacement of DNA with peptide nucleic acids (PNAs) enhances binding strength, thermal stability, and metabolic stability. Importantly, ligand conjugation redirects PNA oligonucleotides from nonspecific polymerase stalling toward selective G4 stabilization. Finally, we demonstrate receptor-mediated cellular uptake of modified GL-Os, supporting the feasibility of cellular delivery while highlighting remaining delivery barriers. Together, these findings show the molecular design principles governing GL-O behavior and provide a foundation for the future development and evaluation of selective G4-targeting therapeutics.

G-quadruplex DNA

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.

Genes, Viral

A Versatile Disulfide-Containing Solid-Support Strategy for 3'-Modifiers in Oligonucleotides: Introducing Modular Tandem Oligonucleotide Synthesis.

Chemical modifications of oligonucleotides are routinely employed to enhance their functional properties. Amino-modifiers serve as versatile chemical handles for postsynthetic (bio)conjugation, nucleic acid immobilization on solid supports, and investigations into nonenzymatic genome replication relevant to the origins of life, to name a few. Here, we report a cost-effective, disulfide-containing solid-support linkage that enables the on-column synthesis of nucleic acids with 3'-amino or 3'-phosphate modifications. The orthogonality of this solid-support linker facilitates an on-column protecting group strategy, enabling the synthesis of DNA and RNA containing 3'-amino-2',3'-dideoxyribosides from commercial unprotected mononucleosides. Additionally, we present an on-column deprotection protocol for DNA and RNA, prior to cleavage from the solid support, eliminating the precipitation step typically required in conventional RNA workflows, leading to higher recovery for certain strands. Expanding on our previous work, we introduce a versatile modular tandem oligonucleotide synthesis (mTOS) approach, allowing selective release of downstream strands from the one directly bound to the solid-support via the disulfide-containing linker. Together, these advances in solid-support design and oligonucleotide synthesis unlock new opportunities in bioconjugation, biotechnology, and the study of prebiotic replication mechanisms, broadening the utility of chemically modified nucleic acids across research disciplines.

Disulfides

Addition of short guanylyl blocks to oligonucleotide primers with a thermophilic polynucleotide phosphorylase. Its application to the synthesis of oligonucleotides containing guanylyl residues.

Polynucleotide phosphorylase from Thermus thermophilus catalyzed the addition of short guanylyl blocks from GDP to the 3'-hydroxyl termini of oligonucleotide primers at low temperature in a simple reaction mixture. Polyguanylic acid formation was inhibited at 37 degrees C, but the addition of one or two guanylyl residues to oligonucleotide primers proceeded in high yields. The reaction was applied to the synthesis of oligonucleotides containing guanylyl residues at the 3'-end. Using (Ap)2A and (Up)2U as primers, (Ap)3G, (Ap)3GpG, and (Up)3G were synthesized in yields of 25--52%. (Ap)2GpG was synthesized from ApA and GDP in a yield of 13%.

Guanine

Enzymatic synthesis of oligonucleotides of defined sequence. Addition of short blocks of nucleotide residues to oligonucleotide primers.

Polynucleotide phosphorylase from Escherichia coli can be used to catalyse the addition of short tracts of deoxyadenylate residues to the 3'-termini of deoxyribooligonucleotides of the type pdAn-dN (where dN = dC, dT or dG) using dADP as donor. Similarly, the enzyme can also be used to catalyse the addition of short tracts of adenylate residues to the 3'-termini of ribooligonucleotides of the type An-N (where N = C, U or G) using ADP as donor. In the ribooligonucleotide series, phosphorolytic cleavage of the primer oligonucleotides is significant and results in the concommitant production of oligoadenylates lacking the N residue. Oligomers of the same length, with and without the residue N, were readily separated by thermal elution from cellulose-pdT9 columns. This latter procedure therefore provides a simple method for purification of the oligoadenylates containing an internal base substitution and it also provides a convenient assay for oligonucleotide phosphorolysis.

Adenine Nucleotides

Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase.

Several oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.

Adenine Nucleotides

Immunochemical and oligonucleotide fingerprint analyses of Venezuelan equine encephalomyelitis complex viruses.

RNA oligonucleotide fingerprint analyses indicate that the genome RNA obtained from Trinidad donkey (TRD) Venezuelan equine encephalomyelitis (VEE) virus serotype I A, its vaccine strain derivative TC-83, and the VEE I B virus isolate PTF-39, have almost identical patterns of characteristic ribonuclease T1 resistant oligonucleotides. The TC-83 strain and the I B isolate can, on the basis of these analyses, be considered as variants of the TRD virus and categorized as I AB serotypes. Comparisons made by single and co-electrophoreses of the ribonuclease T1 digests of the RNA species of TC-83 and a VEE I C isolate P676 indicate that 16 of 37 large oligonucleotides of the TC-83 virus co-migrate with the oligonucleotides obtained from the I C isolate. Similar single and co-electrophoreses of ribonuclease T1 digests of the RNA species of TC-83 and a VEE I D isolate 3880 indicate that 18 of 41 TC-83 large oligonucleotides co-migrate with the oligonucleotides obtained from the I D virus isolate. At least nine of the TC-83 large oligonucleotides appear on the basis of these analyses, to be present in the digests of the genome RNA obtained from these selected I B, I C and I D virus isolates. The ribonucleast T1 digests of three I E virus isolates (Mina II, 63U2 and 71U388) give oligonucleotide fingerprints which, although comparable to each other, are more distinct from the I A and I B RNA fingerprints than are those of the I C and I D RNA species. The ribonuclease T1 resistant oligonucleotide fingerprints of VEE virus isolates belonging to serotypes (VEE subtypes) II, III and IV show little similarity to each other or to those of the serotype I virus isolates we have studied. The results obtained here agree with the reported close antigenic relationships of VEE, I A, I B, I C and I D virus isolates, and our studies suggest that these viruses have conserved nucleotide sequences. The I E virus isolates appear to have more distinct nucleotide sequences than do the other serotype 1 viruses. The results also agree with the serological differentiation of VEE, I, II, III and IV subtypes in that the oligonucleotide fingerprints of subtypes II to IV are different from each other and from those of the different serotype I virus isolates. On the basis of antigenic and genome relationships, VEE isolates can be classified as serotypes I to IV with serotype I viruses differentiated into the categories I AB, I C, I D and I E.

Animals

T1 oligonucleotide maps of N-, B-, and B leads to NB-tropic murine leukemia viruses derived from BALB/c.

We previously described and characterized RNase T1 RNA fingerprints of an N-, a B-, and five B leads to NB-tropic murine leukemia viruses derived from BALB/c mice (Faller and Hopkins, J. Virol. 23:188-195, 1977, and J. Virol. 24:609-617, 1977). These viruses share the majority of their large RNase T1-resistant oligonucleotides, but each possesses some "unique" oligonucleotides relative to the others. We have ordered the large T1-resistant oligonucleotides of the N-, the B-, and one NB-tropic virus relative to the 3' end of their genomes to obtain oligonucleotide maps. These maps indicate that (i) the large T1 oligonucleotides shared by the N-, B-, and NB-tropic viruses probably occupy the same relative positions on their genomes; (ii) the 14 T1 oligonucleotides that differ between the N- and B-tropic viruses are derived from regions scattered along the genomes; and (iii) an oligonucleotide that is present in five NB-tropic viruses but not in their B-tropic virus progenitors lies toward the 5' end of the NB-tropic virus oligonucleotide map.

Animals

T1 oligonucleotides that segregate with tropism and with properties of gp70 in recombinants between N- and B-tropic murine leukemia viruses.

We have analyzed large RNase T1-resistant oligonucleotides derived from the genomes of 16 recombinants between N- and B-tropic murine leukemia viruses of BALB/c. The parental viruses, designated SP-N and LP-B, differ in several phenotypic or biochemically defined properties: N- or B-tropism; XC plaque morphology, electrophoretic mobility of three virion proteins (p15, p30, and gp70); ability to induce GIX antigen on infected cells; presence of 6 to 8 (out of 36 to 38 analyzable) large T1 oligonucleotides. One SP-N-specific T1 oligonucleotide was inherited by all 16 N-tropic recombinants and, thus, appears to be linked to N-tropism. This oligonucleotide lies in the 5' third of the oligonucleotide map of SP-N. One LP-B-specific T1 oligonucleotide was inherited by all 11 recombinants whose gp70 has an electrophoretic mobility like that of LP-B gp70 and that, like LP-B, fail to induce GIX antigen. This oligonucleotide lies in the 3' third of the oligonucleotide map of LP-B.

Animals

Nucleotide clusters in deoxyribonucleic acids. Comparison of the sequences of the large pyrimidine oligonucleotides of bacteriophages S13 and phiX174 deoxyribonucleic acids.

The large pyrimidine oligonucleotides from the DNAs of the two related bacteriophages phiX174 and S13 have been sequenced. The largest pyrimidine oligonucleotide present is unique to S13 DNA and is the undecanucleotide C5T6, sequence C-T-T-C-C-T-C-T-T-C-T. Considerable sequence homology has been found between the pyrimidine oligonucleotides of the two phage DNAs. Out of 14 oligonucleotide sequences from S13 DNA (120 bases) at least ten are identical with sequences of oligonucleotides from phiX174 DNA (92 bases) and two are closely related (17 bases), the only difference being a single thymine to cytosine transition in each sequence (a total of 107 identical bases). The pyrimidine oligonucleotides of each phage DNA show extensive internal sequence homology among each other with up to eight bases identical in sequence in pairs of different oligonucleotides. Another interesting observation is the occurrence of symmetrical sequences (true palindromes) which read the same forwards as backwards. The longest symmetrical sequence is the nonanucleotide C4T5 sequence, C-T-C-T-T-T-C-T-C, present in both S13 and phiX174 DNAs. The extensive sequence homology observed between the pyrimidine oligonucleotides of S13 and phiX174 supports the close relationship of the two phages and provides further evidence that they were derived from recent common ancestors.

Base Sequence

Location of envelope-specific and sarcoma-specific oligonucleotides on RNA of Schmidt-Ruppin Rous sarcoma virus.

Envelope-specific and sarcoma-specific nucleotide sequences have been located within the 10,000 nucleotides of the RNA of nondefective Schmidt-Ruppin Rous sarcoma virus (nd SR). For this purpose, about 30 RNase-T1-resistant oligonucleotides were ordered relative to the 3'-poly(A) terminus of the RNA, to construct an oligonucleotide map of the nd SR RNA. A cluster of seven envelope-specific oligonucleotides, identified by their absence from an otherwise very similar oligonucleotide map of an envelop-defective deletion mutant (which lacks the major viral glycoprotein), mapped at a distance of 2800-5000 nucleotides from the poly(A) end of nd SR RNA. A cluster of two sarcoma-specific oligonucleotides, identified by their absence from an otherwise nearly identical oligonucleotide map of a transformation-defective deletion mutant, mapped at a distance of 1000-2000 nucleotides from the poly(A) end of nd SR RNA. The oligonucleotide maps of nd SR and of the two deletion mutants were the same from the poly(A) end up to 650 nucleotides and included one terminal oligonucleotid, termed C, which is found in all avian tumor viruses tested so far. A possible gene order consistent with our data suggests that sarcoma-specific nucleotide sequences map between envelope-specific nucleotide sequences and the poly(A) end of the RNA.

Avian Sarcoma Viruses

Large T1 oligonucleotides of Moloney leukemia virus missing in an env gene recombinant, HIX, are present on an intracellular 21S Moloney viral RNA species.

HIX, a recombinant derived from Moloney leukemia virus, has an envelope glycoprotein different from that of the Moloney virus. HIX and Moloney viruses share the majority of the large T1 oligonucleotides derived from their genomes but each possesses a set of distinctive oligonucleotides that lie clustered in corresponding regions in the 3' halves of their oligonucleotide maps. These regions presumably contain envelope glycoprotein coding sequences. The type C viral envelope glycoprotein is believed to be translated from a 21S RNA. Thus, at least part of the region of the Moloney virus genome that is altered relative to HIX was expected to be present on such a species. To test this prediction, we purified an intracellular 21S Moloney viral RNA species and analyzed its large T1 oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. This RNA contains one T1 oligonucleotide that is probably derived from the 5' end of the Moloney virus genome, the Moloney virus T1 oligonucleotides that are missing in HIX, and those that lie to their 3' side on the Moloney virus T1 oligonucleotide map.

Genes, Viral

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.

Base Sequence

A two-dimensional thin layer chromatographic procedure for the sequential analysis of oligonucleotides employing tritium post-labeling.

Two dimensional PEI-cellulose thin layer chromatography can resolve sequentially degraded oligonucleotide fragments of tRNA. This technique entails the sequential degradation of the oligonucleotide with snake venom phosphodiesterase in the presence of bacterial alkaline phosphatase, and periodate oxidation followed by tritiated sodium borohydride reduction of the 3' terminal nucleoside. Subsequently the tritiated oligonucleotide fragments were resolved by two dimensional PEI-cellulose TLC. The results of these experiments indicate that, in some cases, the complete nucleotide sequence of a large oligonucleotide fragment may be determined by interpretation of the observed mobility shifts, thereby eliminiating the need for additional analysis of the oligonucleotide. In addition, the use of two-dimensional rather than one-dimensional resolution of the tritium labeled fragments allows for a complete separation of any interfering background spots from the sequentially degraded oligonucleotides. This procedure was applied to the complete nucleotide sequence analysis of several ribonuclease T1Val and ribonuclease A digestion products from human placenta tRNA.

Base Sequence

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.

Animals

OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.

Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.

Sequence Analysis, DNA

[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