Quantitative Parametrs of Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template

Abstract

The cooperative interactions of oligonucleotides on the complementary template were studied using the quantitative analysis of the template alkylation with the oligonucleotides bearing covalently attached 4-[N-(2-chloroethyl)-N-methylamino]benzyl group at 5′-end. The influence of the mismatched nucleotides and the stabilizing N-(2-hydroxyethyl)phenazinium group at the 5′- and 3′-ends of the oligonucleotides on the parameters of cooperativity was evaluated.     

Mixed-Backbone Oligonucleotides Containing Phosphorothioate and Methylphosphonate Linkages as Second Generation Antisense Oligonucleotide

Abstract

Antisense oligonucleotides are being studied as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have synthesized phosphorothioate oligonucleotides containing methylphosphonate linkages at the 5′-end, the 3′-end, or in the center, and have evaluated the impact of these linkages on the biophysical properties, biological properties, and some of the safety parameters.     

Solid Phase Synthesis of 2′, 5′-Oligoadenylates Containing 3′-Fluorinated Ribose

Abstract

2′, 5′-phosphodiester bond-linked oligoadenylate trimers with 3′-fluoro-3′-deoxyadenosine residues incorporated at specific positions of the nucleotide sequence were synthesized by the solid phase phosphite triester (phosphoramidite) method. The syntheses were in the 2′ to 5′ direction and were performed manually using commercially available microcolumns. The oligonucleotides were 5′-end phosphorylated on the support before deprotection.     

Preparation and Properties of a New Type of Acyclic,Achiral Nucleoside Analogue

Abstract

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 T_m measurements showing a ΔT_m of ?2 to ?6.5°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.     

Fluorescent Labelling of Oligodeoxyribonucleotides by the Oxyamino-Aldehyde Coupling Reaction

Abstract

We describe the reaction of oligonucleotides containing an aldehydic group at the 5′-end or inside the sequence with an oxyamino label. The reaction was found to be highly selective and represents an efficient method for derivatization of oligonucleotides.     

Oligonucleotides Incorporating 7-(Aminoalkyn-1-yl)-7-deaza-2′-deoxyguanosines: Duplex Stability and Phosphodiester Hydrolysis by Exonucleases

Abstract

Self-complementary {[5′-d(G-C)₄]₂} and non-selfcomplementary oligonucleotides [5′-d(TAG GTC AAT ACT) ? 3′-d(ATC CAG TTA TGA)] containing 7-(ω-aminoalkyn-1-yl)-7-deaza-2′-deoxyguanosines (1a–c) (1) and 7-deaza-2′-deoxyguanosine instead of dG were studied regarding their thermal stability as well as their phosphodiester hydrolysis by either 3′ → 5′- or 5′ → 3′ – phosphodi esterase studied by MALDI-TOF MS.     

Synthesis and Properties of Oligonucleotides Involving a Perylene Unit Linked to a 2′-Deoxyribose Residue

Abstract

We report here the synthesis and binding properties of oligonucleotides involving a perylene unit linked to the anomeric position of a 2′-deoxyribose residue. Both anomers were separated and incorporated separately at either the 5′-end or the internal position of a pyrimidine sequence. In any case the presence of the perylene unit stabilizes the complexes formed with either the single or the double-stranded target.     

The Behaviour of 2′-Deoxy-2′-Fluorouridine Incorporated into Oligonucleotides by the Phosphoramidite Approach

Abstract

2′-Deoxy-2′-fluorouridine has been chemically incorporated into an oligodeoxynucleotide of the structure 5′ACGGAX 3′ (X=U(2′-F)) using the phosphoramidite method and the behaviour of the product has been studied. 5′-O-Monomethoxytrityl-2′-deoxy-2′-fluorouridine was fixed on silica gel at the 3′-end and the chain elongated on a DNA-synthesizer using nucleoside methoxyphosphoramidites. After alkaline work-up two products were observed. One was found to be the desired fluoro containing hexamer, whereas the other corresponds to an araU-hexamer (X=arabino-furanosyluridine). The latter compound is supposed to be a product of alkaline hydrolysis of the C-2′-F-bond. The oligomers containing 2′-fluoro- and ara-U at their 3′-end were chemically sequenced by a solid phase method on CCS-paper which confirmed the right primary structure.     

A versatile Solid Phase Method For the Synthesis of Masked 3′-Thiol Group Containing Oligonucleotides

Abstract

A new solid phase method for the introduction of masked thiol group on to the 3′-end of the oligonucleotide is reported. The modified oligonucleotides thus obtained can be better resolved by reverse phase column chromatography.     

Biotinylation of Oligonucleotides Using a Water Soluble Biotin Ester

Abstract

Oligonucleotides aminated at the 5′-end were bio-tinylated with a water soluble N-hydroxysuccinimide ester of biotin in large scale. The biotinylated oligonucleotides were purified by reversed phase HPLC.     

Use of a Base-Labile Protected Derivative of 6-Mercaptohexanol for the Preparation of Oligonucleotides Containing a Thiol Group at the 5′-End

Abstract

The preparation of a base-labile (Dnpe) protected derivative of 6-mercaptohexanol is described. The use of the phosphoramidite derivative of this compound improves both yields and the time needed for the preparation of oligonucleotides containing a thiol group at the 5′-end.     

Biologically Active Oligodeoxyribonucleotides. VII. Anti-HIV-1 Activity of Hexadeoxyribonucleotides Bearing 3′- and 5′-End-Modifications

Abstract

It has been determined that hexadeoxyribonucleotides (5′TGGGAG3′), which have modified aromatic groups such as the trityl group at the 5′-end, exhibit anti-HIV-1 activity in vitro. The 6-mer (S-1443) bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end and a 2-hydroxyethylphosphate group at the 3′end exhibited the most potent activity and the least cytotoxicity. Moreover, it was found that the S-1443 was the most stable, when the 6-mer analogues were incubated with mouse, rat, or human plasma.     

The 5'-termini of the oligonucleotides from ribonuclease T 1 digested E. coli ribosomes

Oligonucleotides remaining in the 70s $\text{Escherichia}\text{coli}$ ribosomal particles after varying degrees of digestion with ribonuclease T₁ were phosphorylated with polynucleotide kinase in the presence of γ-labeled³²P-ATP. The resulting radioactively labeled RNA molecules were further digested with pancreatic ribonuclease and analyzed by a two-dimensional finger-printing technique. The numbers of labeled oligonucleotides were proportional to the duration of T₁ digestion; most of these oligonucleotides yielded ¹pAp and/or ¹pCp as their 5′-end groups upon alkaline hydrolysis.     

Internal initiation of polyuridylic acid translation in bacterial cell-free system

The task of the present work was to answer the question: is the free 5′-end needed for effective translation of a model polyribonucleotide template — polyuridylic acid — in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5′-end and the polyuridylic acid with blocked 5′-end were compared in the bacterial cell-free translation system. To block the 5′-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5′-end and uridylic oligoribonucleotide sequence at its 3′-end, schematically described as FAM(dC)₁₀(rU)₅₀, was covalently attached (ligated) to the 5′-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5′-blocked template and on the polyuridylic acid with free 5′-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5′-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.     

Progress in the Synthesis of Cyclic Deoxyribo- and Oligoribonucleotides

Abstract

The preparation of purine-rich sequences of cyclic DNA, up to a 28-mer, has been achieved. The products were purified by HPLC and PAGE (larger circles) and fully characterized. Cyclic RNA synthesis can be carried out using the same methodology as for cyclic DNA, provided that a single deoxynucleoside or a 2′-O-methylribonucleoside is placed at the 3′-end of the linear precursor.     

Introduction of 5′-Terminal Amino and Thiol Groups into Synthetic Oligonucleotides

Abstract

Oligonucleotides terminating in a 5′-primary amine group are synthesized using solid phase phosphoramidite chemistry. The 5′-terminal amine group in the deprotected oligonucleotide is further derivatized with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) followed by treatment with dithiothreitol (DTT) to produce 5′-thiol terminated oligonucleotides. Introduction of 5′-thiol group is further confirmed by reading the absorbance of the released chromophore, pyridine-2-thione at 343 nm; ?₃₄₃=8080/M.     

Nucleotides LXIV[1]: Synthesis,Hybridization and Enzymatic Degradation Studies of 2′-O-Methyl-Oligoribonucleotides and 2′-O-Methyl/Deoxy Gapmers

Abstract

2′-O-Methyloligoribonucleotides, deoxyoligonucleotides and 2′-O-methyl/ deoxy gapmers were synthesized using solid phase phosphoramidite chemistry employing the 2-(4-nitrophenyl)ethyl (npe) protection strategy. Melting temperatures of the synthesized oligonucleotides as well as their stability against degradation by several different nucleases were determined. 2′-O-Methyloligoribonucleotides showed the highest melting temperatures (T_m's) whereas 2′-O-methyl/deoxy gapmers revealed either slightly higher or surprizingly no thermal stabilities compared with their deoxy analogs when using selfcomplementary sequences. Gapmers with four 2′-O-methyl nucleotides on both ends showed about the same stability as all 2′-O-methyloligoribonucleotides against micrococal nuclease, nuclease S₁, and snake venom phosphodiesterase.     

Rapid in vitro labeling procedures for two-dimensional gel fingerprinting

Improvements of existing in vitro procedures for labeling RNA radioactively, and modifications of the two-dimensional polyacrylamide gel electrophoresis system for making RNA fingerprints are described. These improvements are (a) inactivation of phosphatase with nitric acid at pH 2.0 eliminated the phenol-chloroform extraction step during 5′-end labeling with polynucleotide kinase and [γ-³²P]ATP; (b) ZnSO₄ inactivation of RNase T₁ results in a highly efficient procedure for 3′-end labeling with T4 ligase and [5′-³²P]pCp; and (c) a rapid 4-min procedure for variable quantity range of ¹²⁵I and RNA results in a qualitative and quantitative sample for high-molecular weight RNA fingerprinting. Thus, these in vitro procedures become rapid and reproducible when combined with two-dimensional gel electrophoresis which eliminates simultaneously labeled impurities. Each labeling procedure is compared, using tobacco mosaic virus, Brome mosaic virus, and polio RNA. A series of Ap-rich oligonucleotides was discovered in the inner genome of Brome mosaic Virus RNA-3.     

A Practical Synthesis of N1-Methyl-2′-deoxy-ψ-uridine (ψ-Thymidine) and Its Incorporation into G-Rich Triple Helix Forming Oligonucleotides

Abstract

A convenient synthesis of N1-methyl-2′-deoxy-ψ-uridine (ψ-thymidine, ψT, 7a) has been accomplished in good yield. The structural conformation of 7a was derived by 2D NMR and 1D NOE experiments. The nucleoside 7a has been incorporated into G-rich triplex forming oligonucleotides (TFOs) by solid-support, phosphoramidite method. The triplex forming capabilities of the modified TFOs (S4, S5 and S6) containing ψT has been evaluated in antiparallel motif with a target duplex (duplex-31) 5′d(CTGAGACCGGGAAGGAGGAAGGGCCAGTGAC)3′-5′d(GACTCTGGCCCTTCCTCCTTCCCGGTCACTG)3′(D1) at pH 7.6. The triplex formation of modified homopyrimidine-oligomers (S1, S2 and S3) has also been studied in parallel motif with a duplex-10 (A₁₀:T₁₀) at pH 7.0.