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Free-Radical Ring Closure to Conformationally Locked α-l-Carba-LNAs and Synthesis of Their Oligos:: Nuclease Stability, Target RNA Specificity, and Elicitation of RNase H
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry. (Prof. Jyoti chattopadhyaya)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry. (Prof. Jyoti Chattopadhyaya)
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2010 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 75, no 18, 6122-6140 p.Article in journal (Refereed) Published
Abstract [en]

A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T(m) by 3-5 °C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the β-D-LNA-T or β-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.

Place, publisher, year, edition, pages
U. S. A: American Chemical Society (ACS), 2010. Vol. 75, no 18, 6122-6140 p.
Keyword [en]
conformationally constrained nucleoside, free-radical intramolecular cyclization, antisense oligonucleotide, RNA target therapeutic
National Category
Organic Chemistry
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-176562DOI: 10.1021/jo100900vOAI: oai:DiVA.org:uu-176562DiVA: diva2:535923
Available from: 2012-06-20 Created: 2012-06-20 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Conformationally Constrained Oligonucleotides for RNA Targeting
Open this publication in new window or tab >>Conformationally Constrained Oligonucleotides for RNA Targeting
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems.

In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application.

AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate.

Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 66 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 948
Keyword
conformationally constrained nucleoside, antisense oligonucleotide, RNA affinity, nuclease stability, RNase H, RNA degradation
National Category
Biochemistry and Molecular Biology Organic Chemistry
Research subject
Chemistry with specialization in Bioorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-179069 (URN)978-91-554-8414-9 (ISBN)
Public defence
2012-09-22, B7:101a, BMC, Husargatan 3, Uppsala, 13:00 (English)
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Supervisors
Available from: 2012-08-30 Created: 2012-08-07 Last updated: 2013-01-22Bibliographically approved

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Publisher's full texthttp://pubs.acs.org/doi/abs/10.1021/jo100900v

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Li, QingPlashkevych, Oleksandr

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