Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.     

Synthesis and Biophysical Studies of Modified Oligonucleotides Containing Acyclic Amino Alcohol Nucleoside Analogs

Abstract

Novel serine derivative of thymine was prepared and incorporated into oligonucleotides. These modified oligonucleotides were studied for their binding affinity with complementary DNA/RNA.     

Anti-Influenza Virus Activities of Nicked and Circular Dumbbell RNA/DNA Chimeric Oligonucleotides

Abstract

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.     

Application of Microchip Electrophoresis in the Analysis of RNA Aptamer-Protein Interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (< 3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Synthesis and Properties of 2′-O-Methylribonucleotide Methylphosphonate Containing Chimeric Oligonucleotides

Abstract

2′-O-methylribonucleoside methylphosphonamidites are synthesized and incorporated into oligonucleotides to obtain chimeric antisense oligonucleotides. The resulting oligonucleotide binds to their target RNA/DNA sequences efficiently and stable in a medium containing bovine serum.     

Targeted Gene Correction: Synthesis and Characterization of Double-Hairpin 2′-O-Methyl RNA/DNA Chimera Oligonucleotides

Abstract

A series of 2′-O-methyl RNA/DNA chimera oligonucleotides were synthesized with a double-hairpin structural motif. Liposome formulated delivery of the chimeras effected targeted, high conversion of mutant alleles in mammalian cell culture. The chimera oligonucleotides were prepared with DNA and 2′-OMe RNA phosphoramidite nucleoside monomers on the ABI 394 synthesizer.     

Optimization of Antisense Chimeric Oligonucleotides Containing α- and β-Anomeric Deoxynucleotides

Abstract

Various chimeric oligonucleotides containing α- and β-anomeric deoxynucleotide units and directed against the splice acceptor site of the HIV-1 tat RNA have been synthesized. Their hybridizing abilities to complementary DNA or RNA single strands, and their stability in cell culture medium or cell extracts were studied.     

Evaluation of Oligonucleotides Containing Two Novel 2′-O-Methyl Modified Nucleotide Monomers: A 3′-C-Allyl and a 2′-O-3′-C-Linked Bicyclic Derivative

Abstract

The two ribo-configured nucleosides 1-(3-C-allyl-2–0-methyl-β-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.     

KINETICALLY SELECTIVE BINDING OF SINGLE STRANDED RNA OVER DNA BY A PYRROLIDINE-AMIDE OLIGONUCLEOTIDE MIMIC (POM)

Replacing the sugar-phosphodiester backbone of nucleic acids with a pyrrolidine-amide backbone results in an oligonucleotide mimic POM 1 which binds with high affinity and specificity to complementary DNA and RNA. Unlike other modified oligonucleotides, POM binds much more rapidly to single stranded RNA than DNA.     

Use of Bioreversible Phosphate Protecting Groups for the Intracellular Delivery of Antisense Oligodeoxyribonucleotides

Abstract

The aim of this work was to design phosphorothioate oligonucleotides, the negative charges of which are temporarily masked by a group which facilitates the cellular uptake of these analogues. Then, selective removal of this group by enzymes normally present in the cells will deliver intracellularly the intact charged phosphorothioate oligomers able to hybridize to their complementary RNA targets and to elicit RNase H activity.     

Syntheses and Interactions of Oligodeoxyribonucleotides Containing 2′-Amino-2′-Deoxyuridine

Abstract

Oligodeoxyribonucleotides containing 2′-amino-2′-deoxy-uridine (dU) were synthesized and their ability to form duplexes with complementary DNA or RNA oligonucleotides was studied. Substitution of dU with dU in these oligomers results in lowered Tms of the duplexes.     

Synthesis,Hybridization, and Nuclease Resistance Properties of 2′-O-Aminooxyethyl Modified Oligonucleotides

Abstract

We have synthesized the novel 2′-O-AOE- and MIOE-5-methyluridine and -adenosine nucleosides and successfully incorporated them into oligonucleotides. The 2′-O-modifications significantly enhance hybridization against RNA (1.2 deg C/substitution) and furthermore, exhibits specificity for RNA vs. DNA. The nuclease resistance (SVPD) of 2′-O-AOE and MIOE modified oligonucleotides is comparable to that of 2′-O-MOE.     

Site-Specific Introduction of Functional Groups onto Bases in Synthetic Oligonucleotides for Biological Applications

Abstract

Postsynthetic functionization of bases in oligonucleotides provides a useful approach to the preparation of DNA and RNA containing modified bases. In this communication is presented our recent work on this subject.     

DNA Probes: Applications of the Principles of Nucleic Acid Hybridization

Abstract

Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.     

Formation of the double helix: a mutational study

To investigate the mechanisms by which oligonucleotides hybridize to target molecules, the binding of two oligodeoxynucleotide probes to RNA targets was measured over a broad range of temperatures. Mutations were then scanned across each DNA/RNA hybrid to map, at single base resolution, sequences important for hybridization. Despite being unrelated in sequence, each hybrid formed by a similar mechanism. In the absence of secondary structure, two stretches of bases, termed nucleation regions, cooperated with one another by a looping mechanism to nucleate hybridization. Mutations inside each nucleation region strongly decreased hybridization rates, even at temperatures well below the melting temperature (T_m) of the hybridized duplex. Surprisingly, nucleation regions were detected in a RNA target but not a corresponding DNA target. When either nucleation region was sequestered in secondary structure, the hybridization rate fell and the mechanism of hybridization changed. Single-stranded bases within the nucleation region of the probe and target first collided to form a double helix. If sufficiently G + C rich, the double helix then propagated throughout the oligonucleotide by a strand invasion process. On the basis of these results, general mechanisms for the hybridization of oligonucleotides to complementary and mutant targets are proposed.     

Single strand targeted triplex-formation. Destabilization of guanine quadruplex structures by foldback triplex-forming oligonucleotides.

Oligonucleotides that can hybridize to single-stranded complementary polypurine nucleic acid targets by Watson-Crick base pairing as well as by Hoogsteen base pairing, referred to here as foldback triplex-forming oligonucleotides (FTFOs), have been designed. These oligonucleotides hybridize with target nucleic acid sequences with greater affinity than antisense oligonucleotides, which hybridize to the target sequence only by Watson-Crick hydrogen bonding [Kandimalla, E. R. and Agrawal, S. Gene(1994) 149, 115-121 and references cited therein]. FTFOs have been studied for their ability to destabilize quadruplexes formation by RNA or DNA target sequences. The influence of various DNA/RNA compositions of FTFOs on their ability to destabilize RNA and DNA quadruplexes has been examined. The ability of the FTFOs to destabilize quadruplex structures is related to the structurally and thermodynamically stable foldback triplex formed between the FTFO and its target sequence. Antisense oligonucleotides (DNA or RNA) that can form only a Watson-Crick double helix with the target sequence are unable to destabilize quadruplex structures of RNA and DNA target sequences and are therefore limited in their repertoire of target sequences. The quadruplex destabilization ability of FTFOs is dependent on the nature of the cation present in solution. The RNA quadruplex destabilization ability of FTFOs is -20% higher in the presence of sodium ion than potassium ion. The use of FTFOs, which can destabilize quadruplex structure, opens up new areas for development of oligonucleotide-based therapeutics, specifically, targeting guanine-rich sequences that exist at the ends of pro- and eukaryotic chromosomes and dimerization regions of retroviral RNA.     

RecA-mediated joint molecule formation between O-methylated RNA/DNA hairpins and single-stranded targets

Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events. Received: 2 June 1997 / Accepted: 29 September 1997     

Synthesis of Fused Oligoribonucleotides with Trideoxyribonucleotide Containing Phosphorothioate to Stabilize Against Nuclease Activity

Abstract

Fused oligonucleotides(21mer) consisting of RNA(18mer) and DNA(3mer) were synthesized by combined use of the phosphotriester and phosphoramidite methods. The RNA(18mer) corresponds to the leader sequence of phage fl coat protein mRNA containing initiation codon. The RNA was stabilized against 3′-exonucleases by joining with trideoxy-ribonucleotides containing phosphorothioate linkages and it would be applied to the studies on the initiation complex formation in prokaryotic translation.