Synthesis and Properties of Oligonucleotide (2-Aminoethyl)Phosphonates

Abstract

Oligonucleotides with novel, cationic backbone substituents have been prepared. Dinucleotide aminoethylphosphonates have been synthesized and the isomers were separated and used to prepare oligonucleotides with alternating positive and negative charges. The properties of these oligonucleotides have been examined.     

Second Generation Antisense Oligonucleotides—Inhibition of PKC-α and c-raf Kinase Expression by Chimeric Oligonucleotides Incorporating 6″-Substituted Carbocyclic Nucleosides and 2″-O-Ethylene Glycol Substituted Ribonucleosides

Abstract

6′-substituted carbocyclic deoxyribonucleosides and 2′-O-ethylene glycol substituted ribonucleosides have been evaluated as building blocks for antisense oligonucleotides. Within the former class 6′-hydroxy substituted building blocks in combination with internucleoside phosphorothioate linkages have the potential to enhance antisense activity. 2′-O-ethylene glycol substituted ribonucleosides generally allow for the construction of potent antisense oligonucleotides with reduced phosphorothioate content, but differences exist in their effects on biological activity in cell culture in spite of virtually identical effects on RNA-binding affinity. Activity enhancement was most pronounced for a 2′-O-methoxyethyl substituent.     

Biological Activities of Oligonucleotides Linked to Poly(L-Lysine)

Abstract

We have developped a method to couple oligonucleotides to poly(L-lysine). This tool has been tested with anti m-RNA synthetic oligodeoxyribonucleotides and 2′-5′(A)_n and allows oligonucleotides to enter intact cells.     

Inhibition of Influenza Virus Replication by Phosphorothioate and Liposomally Endocapsulated Oligonucleotides

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. Phosphorothioate and liposomally encapsulated oligonucleotides with two target sites (PB1 and PB2) were synthesized and tested for virus-induced cytopathogenicity effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODNs complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODNs targeted to PB1 was considerably decreased in comparison with the PB2 target sites. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. The activities of the modified oligonucleotides are effectively enhanced by using the liposomal carrier.     

Ultrafast Cleavage and Deprotection of Oligonucleotides Synthesis and Use of CAc Derivatives

Abstract

We have investigated the use of alkylamines as fast cleavage and deprotection reagents for the solid phase synthesis of oligonucleotides and found methylamine/ammonium hydroxide (or methylamine) as an efficient reagent. The transamination side product formed with the commonly used dC^bz has been eliminated by the use of dC^Ac phosphoramidite. This system has successfully been used in the synthesis of oligonucleotides and oligonucleoside phosphorothioates. DMT dC^Ac hydrogen phosphonate and DMT ribo C^Ac-2′-O Me phosphoramidite also have been prepared and used in the synthesis of oligonucleotides.     

The Reactions of H-Phosphonates with Bifunctional Reagents. V. Functionalization of Support-Bound Oligonucleotides and Synthesis of Nonradioactive Hybridization Probes a

Abstract

Three methods for the functionalization of oligonucleotides with aminoalkyl moieties have been developed and their efficiencies were evaluated in the preparation of non-radioactive hybridization probes.

     

Synthesis and Properties of New Fluorescein-Labeled Oligonucleotides

Abstract

2,4-Dinitroaniline is an efficient intramolecular fluorescence-quencher for fluorescein - labeled oligonucleotides and interacts with the heterocyclic bases on duplex formation. Consequently, intramolecular fluorescence quenching is disturbed in double labeled oligonucleotides of this type, and fluorescein shows strong fluorescence in a duplex form. There is a substential increase of the fluorescence-quantum yield when the marker and quencher is attached to a single guanosine residue. Two kinds of doubly labeled oligonucleotides have been synthesized, using the NPE/NPEOC strategy.     

A New Protecting Group Strategy for Amino Groups in Oligonucleotide Chemistry: CEOC Group

Abstract

A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.     

New Pyrene Derivatives for Fluorescent Labeling of Oligonucleotides

Abstract

A series of pyrene-containing reagents have been synthesized and used for the fluorescent labeling of oligonucleotides.     

The Synthesis of Branched Oligonucleotides as Signal Amplification Multimers for Use in Nucleic Acid Assays

Abstract

Branched oligonucleotides have been synthesized using phosphoramidite derivatives with two protected hydroxyl functions. These molecules are employed for a label amplification strategy used in DNA probe diagnostics.     

Thin Layer Chromatography of Radiolabeled Oligonucleotide Analogues: A Rapid and Sensitive Purity Assay

Abstract

Oligonucleotide analogues are being developed for use in cell culture, animals and for therapy. Prior to use it is important to have an indication of the oligomers' purity. Thin layer chromatography (TLC) has been applied to analyze hosphoromonothioate and phosphorodithioate oligonucleotides radiolabeled with either ³²P Or ¹⁴C. TLC coupled with radioactivity has been compared to Polyacrylamide Gel Electrophoresis (PAGE) and High Pressure Liquid Chromatography (HPLC). TLC is a rapid and sensitive alternative to these methods and is particularly suited for chemically modified oligonucleotides.     

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.     

Conjugated Antisense Oligonucleotides for Inhibition of Human Cytomegalovirus In Vitro

Abstract

Several thiono triester containing oligonucleotide phosphorothioates linked with a lipophilic group have been synthesized. Some of these modified antisense oligonucleotides show potent anti-HCMV activity as well as improved cellular association and nuclease resistance.     

Time-of-flight Secondary Ion Mass Spectrometry of Branched RNA Fragrants: Messenger RNA Splicing Intermedistes

Abstract

The negative ion mass spectra generated by a reflecting time-of-flight mass spectrometer are reported for a series of protected oligonucleotides. Quasimolecular and sequence ions have been detected, and the location and nature of protecting groups have been confirmed.     

Dinucleoside Monophosphate Analogues Containing Disulfide Linkages

Abstract

Two dinucleoside monophosphate analogues containing disulfide linkages (1 and 2) have been prepared for incorporation into oligonucleotides. The modified oligomers will be tested for their potential as antisense agents.     

Nuclease Stability of LNA Oligonucleotides and LNA-DNA Chimeras

Abstract

The stabilizing properties of LNA and α-l-LNA oligonucleotides against endo- and 3′-exonucleases have been evaluated.     

Pteridine Nucleosides—New Versatile Building Blocks in Oligonucleotide Synthesis

Abstract

Chemical syntheses of 1-(2-deoxy-β-D-ribofuranosyl)lumazines and isopterins as well as 8-(2-deoxy-β-D-ribofuranosyl)-4-amino-7(8H)pteridones and -isoxanthopterins have been developed to make the structural analogs of the naturally occurring 2′-deoxyribonucleosides in the pteridine series available. The corresponding phosphoramidites have been used in machine-aided solid-support syntheses leading to new types of fluorescence labeled oligonucleotides. The effects of the various fluorophors on duplex formation and as labels for enzyme reactions is demonstrated.     

Cleavage of Leishmania Mini-exon Sequence by Oligonucleotides Conjugated to a Dimidazole Construction

Abstract

RNA sequences derived from the Leishmania amazonensis mini-exon and pre-mini-exon sequences have been targeted with complementary oligonucleotides bearing a diimidazole construction mimicking active center of ribonuclease A. The conjugates were shown to cleave the target RNAs at specific positions.     

In Search of Acyclic Analogues as Universal Nucleosides in Degenerate Probes

Abstract

Five acyclic nucleoside analogues with unnatural base moieties have been synthesized of which three successfully were incorporated into oligonucleotides. The acyclic analogue containing the base 5-nitroindazole was the least discriminating and should be further pursued for use as a universal nucleoside analogue.