2'-O-Methyloligoribonucleotide based artificial nucleases (2’-O-MeOBAN’s) cleaving a model of the leukemia related M-BCR/ABL m-RNA

Murtola, Merita; Strömberg, Roger

doi:10.3998/ark.5550190.0010.308

Cited by 12 publications

(22 citation statements)

References 19 publications

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“…Thus, the commercially available oligoether 2-[2-(2-methoxyethoxy)ethoxy]-acetic acid (TODA) (Scheme 3A), which carries a terminal O-methyl group, was coupled with of HATU-preactivated Fmoc-L-Dab-OH (N-α-Fmoc-L-2,4-diaminobutyric acid) to obtain the oligoether amino acid building block 8 . The amino acid 8 was then used in the synthesis of PNA 9 that is doubly conjugated with both an oligoether and neocuproine, which in the presence of a metal ion such as Zn 2+ [18,19,20,21] or Cu 2+ [22] turns into an RNA cleaver. The synthesis could be achieved by a standard PNA synthesis protocol on an automated synthesizer followed by removal of the Mtt from the Dapa unit and then post conjugation with 5-phenoxycarbonylaminoneocuproine [18,19] followed by removal of Fmoc, capping, cleavage from support and deprotection.…”

Section: Resultsmentioning

confidence: 99%

“…Complexes with bulged out nucleotides can be stabilized by inclusion of modified nucleosides [16] or conjugation to entities that interact with the unpaired region [17]. Bulged RNA is also known to be more susceptible to cleavage than RNA in a helical structure and has been interesting as targets for 2'-O-methyloligonucleotide artificial nucleases (OBAN’s) [18,19,20] as well as PNA-based artificial nucleases (PNAzymes) [21,22].…”

Section: Introductionmentioning

confidence: 99%

“…Therefore several different methods and constructs were developed as reported below. In addition co-conjugation of neocuproine that has been used in artificial nucleases [18,19,20,21,22] is performed as well as conjugation of an aminosugar.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of PNA Oligoether Conjugates

Ghidini

Steunenberg²,

Murtola

et al. 2014

Molecules

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Several different approaches have been explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. Single and double conjugation of 2-(2-(2-aminoethoxy)ethoxy)ethanol was obtained using carbonyldimidazole. Using a post PNA-assembly coupling procedure the building block 2-(2-(2-(benzoyloxy)ethoxy)ethoxy)acetic acid multiple attachment of 2-(2-(2-hydroxyethoxy)ethoxy)acetyl groups to both N-terminal and β-amino groups of inserted diaminopropionic acids residues was achieved. Use of a new oligoether functionalized amino acid allows inclusion of oligoether conjugates during on-line machine assisted synthesis which also allowed combination of methods for attachment of different oligoethers and co-conjugation of neocuproine as well as conjugation of an aminosugar.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Synthesis of PNA Oligoether Conjugates

Ghidini

Steunenberg²,

Murtola

et al. 2014

Molecules

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“…However, there may be situations where even low amounts of copper ions could have an influence, e.g. when conjugates to chelating agents are prepared such as those used in oligonucleotide-based artificial nucleases ( 45–47 ) and PNAzymes ( 48 , 49 ). An estimation of copper ions in the crude product (entry 17 in Supplementary Data ) gives that after cleavage from support, the POC has largely retained the copper [about 85–90% of the concentration expected if all copper added during reaction remained with the POC (the theoretical max concentration)].…”

Section: Resultsmentioning

confidence: 99%

An activated triple bond linker enables ‘click’ attachment of peptides to oligonucleotides on solid support

Wenska

Alvira

Steunenberg

et al. 2011

Nucleic Acids Research

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A general procedure, based on a new activated alkyne linker, for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition (‘click’ reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into ‘clickable’ azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.

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“…An alternative way to selectively degrade RNA target sequences could be to use synthetic oligonucleotides equipped with an intrinsic ability to catalyse the cleavage of their RNA targets (artificial ribonucleases) without the assistance of native enzymes [ 5 , 6 ]. Oligonucleotide-based artificial nucleases (OBANs) have been shown to catalytically cleave their RNA targets in a sequence- and site-selective manner [ 7 , 8 , 9 ]. Neocuproine, as a metal chelate, has also been incorporated into a peptide nucleic acid (PNA) backbone at a central position, giving rise to PNA-based artificial ribonucleases (PNAzymes) [ 10 , 11 , 12 , 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes

et al. 2019

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Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2′-hydroxyl nucleophile with 2′-H or 2′-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target.

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2'-O-Methyloligoribonucleotide based artificial nucleases (2’-O-MeOBAN’s) cleaving a model of the leukemia related M-BCR/ABL m-RNA

Cited by 12 publications

References 19 publications

Synthesis of PNA Oligoether Conjugates

Synthesis of PNA Oligoether Conjugates

An activated triple bond linker enables ‘click’ attachment of peptides to oligonucleotides on solid support

Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes

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