Synthesis of new OBAN's and further studies on positioning of the catalytic group

Abstract

Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN's) have been synthesized. These consist of 2'-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine)via a urea linker. OBAN 4 carries the catalytic group on a linker extending from the C-4 of an internal cytosine moiety. OBAN 5 has two neocuproine units attached, each to linkers extending from the C-5 position of uridine moieties, one placed internally and the other at the at the 5'-end of the oligonucleotide. The key step in the synthesis of the OBAN systems is conjugation of the catalytic group to the respective amino linkers of the modified oligonucleotides. This is achieved by first converting the 5-amino-2,9-dimethylphenanthroline to the phenylcarbamate. The reaction of this neocuproine phenylcarbamate with the oligonucleotide carrying one or two primary aliphatic amines in aqueous buffer (at pH 8.5) leads to nearly quantitative formation of the urea-linked conjugates. Both OBAN systems were found to cleave RNA in the bulged out regions formed from the non-complementary part of the target sequences, in the presence of Zn(II) ions. Differences in efficiency between these and previously reported systems are discussed.

STUDIES IN OLIGONUCLEOTIDE-BASED ARTIFICIAL NUCLEASE SYSTEMS. INTRAMOLECULAR COPPER (II) COMPLEX FORMATION IN AN OLIGONUCLEOTIDE BIS-PHENANTHROLINE CONJUGATE

“…[2] Oligonucleotides I--III where synthesized using standard phosphoramidite methodology (on an Applied Biosystems 392 DNA/RNA Synthesizer) and preparation of the 3'-modified building block is described elsewhere. [3] Synthesis of the modified guanosine building block was carried from the isobutyryl protected 2'-O-methylguanosine by iodination [6] followed by substitution of the 5'-iodo with azide, [7] which was then converted to the 5'-amino derivative by hydrogenation.…”

“…[1,2] Our OBANs hybridize giving bulge formation in the central region of the RNA target. [3] In search of optimal combination of linker and linker attachment position we have prepared novel 2'-O-Me-oligoribonucleotide conjugates ( IV--VI) carrying dmp moieties.…”

Stability of a 2′‐O‐(Carbamoylmethyl)adenosine‐Containing Dinucleotide

“…We have developed oligonucleotide-based artificial nucleases (OBANs) with the aim of producing artificial enzymes that can cleave mRNA sequences arising from genetic or viral diseases. [4][5][6][7][8][9] In the development of oligonucleotides for biotechnology or therapy, a large number of modifications have been ex-ditions for oligonucleotide synthesis (conc. aq.…”

Hydrolysis of Bulged Nucleotides in Hybrids Formed by Rna and Imidazole-Derivatized Oligo-2′-O-Methylribonucleotides

“…The imidazole groups in these constructs target and cleave RNA outside the binding site of their oligonucleotide, while the oligonucleotide remains bound to the target after hydrolysis occurs. A potentially more effective strategy that has been developed that involves targeting metal based cleaving moieties to internal unpaired or bulged RNA bases formed when the oligonucleotide binds to its RNA target [18–20] . Hydrolysis at the site of these bases disrupts the oligonucleotide binding site thus freeing the oligonucleotide to bind another copy of the target.…”