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The chemical stability of abasic RNA compared to abasic DNA.

Küpfer PA, Leumann CJ - Nucleic Acids Res. (2006)

Bottom Line: We found that beta,delta-elimination was 154-fold slower compared to the same mechanism in abasic DNA.In the presence of aniline at pH 4.6, where only beta,delta-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed.Thus abasic RNA is significantly more stable than abasic DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.

ABSTRACT
We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2'-O-protecting group together with the analogous DNA and the 2'-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2'-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37 degrees C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via beta,delta-elimination and 2',3'-cyclophosphate formation. We found that beta,delta-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only beta,delta-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role.

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(A and B) Mechanisms of strand cleavage of abasic RNA at high pH.
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sch2: (A and B) Mechanisms of strand cleavage of abasic RNA at high pH.

Mentions: The presence of the 2′-OH group at an RNA- compared to a DNA abasic site substantially influences the mechanism of cleavage at high pH. The structurally and kinetically relevant intermediates for hydroxide induced strand scission are depicted in Scheme 2. As in the case of DNA, β-elimination of the oligonucleotide fragment 3′ to the abasic site can occur from the aldehyde form yielding a 5′-phosphorylated 3′-fragment and an enol intermediate which after enol-ketone tautomerization leads to a 4,5-dihydroxy-2-oxo-valeraldehyde unit attached to the 5′-fragment of the original RNA (Scheme 2A). Alternatively, the enol form is suited to a further δ-elimination step leading to complete excision of the abasic ribose unit and leaving behind a 3′-phosphorylated 5′ end. Besides this an alternative mechanism via cyclophosphate formation (Scheme 2B) does also apply under basic conditions producing a 5′-fragment with an abasic site that is phosphorylated in the 2′ or 3′ position and a 3′-fragment with a 5′-OH end. Cyclophosphate formation must not only occur in the hemiacetal form but can also happen in the alddehyde form of the abasic site (not shown in Scheme 2B).


The chemical stability of abasic RNA compared to abasic DNA.

Küpfer PA, Leumann CJ - Nucleic Acids Res. (2006)

(A and B) Mechanisms of strand cleavage of abasic RNA at high pH.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1761435&req=5

sch2: (A and B) Mechanisms of strand cleavage of abasic RNA at high pH.
Mentions: The presence of the 2′-OH group at an RNA- compared to a DNA abasic site substantially influences the mechanism of cleavage at high pH. The structurally and kinetically relevant intermediates for hydroxide induced strand scission are depicted in Scheme 2. As in the case of DNA, β-elimination of the oligonucleotide fragment 3′ to the abasic site can occur from the aldehyde form yielding a 5′-phosphorylated 3′-fragment and an enol intermediate which after enol-ketone tautomerization leads to a 4,5-dihydroxy-2-oxo-valeraldehyde unit attached to the 5′-fragment of the original RNA (Scheme 2A). Alternatively, the enol form is suited to a further δ-elimination step leading to complete excision of the abasic ribose unit and leaving behind a 3′-phosphorylated 5′ end. Besides this an alternative mechanism via cyclophosphate formation (Scheme 2B) does also apply under basic conditions producing a 5′-fragment with an abasic site that is phosphorylated in the 2′ or 3′ position and a 3′-fragment with a 5′-OH end. Cyclophosphate formation must not only occur in the hemiacetal form but can also happen in the alddehyde form of the abasic site (not shown in Scheme 2B).

Bottom Line: We found that beta,delta-elimination was 154-fold slower compared to the same mechanism in abasic DNA.In the presence of aniline at pH 4.6, where only beta,delta-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed.Thus abasic RNA is significantly more stable than abasic DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.

ABSTRACT
We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2'-O-protecting group together with the analogous DNA and the 2'-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2'-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37 degrees C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via beta,delta-elimination and 2',3'-cyclophosphate formation. We found that beta,delta-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only beta,delta-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role.

Show MeSH
Related in: MedlinePlus