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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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Related in: MedlinePlus

left: HPLC traces (280 nm) of the β-elimination reaction of the 2′-OMe heptamer 5b at different time intervals; controls: trace A: protected heptamer 5a; trace B: protected 5a after treatment with 0.1 M NaOH for 48 h at 37°C; right: linear fit (R2 > 0.999) of ln[S]/[S0] versus t calculated from the HPLC traces.
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fig1: left: HPLC traces (280 nm) of the β-elimination reaction of the 2′-OMe heptamer 5b at different time intervals; controls: trace A: protected heptamer 5a; trace B: protected 5a after treatment with 0.1 M NaOH for 48 h at 37°C; right: linear fit (R2 > 0.999) of ln[S]/[S0] versus t calculated from the HPLC traces.

Mentions: The time course of strand cleavage of oligonucleotide 5b is depicted in Figure 1. From the HPLC traces it becomes clear that the abasic heptamer 5b (peak 4) decreases while at the same time three major fragments with lower retention times appear (peaks 1–3). Lane B is a control with protected oligonucleotide 5a proving that no cleavage occurs if the anomeric center is still protected. The T1/2 and ks for 5b under these conditions were calculated to be 594 min and 1.96 × 10−5 s−1, respectively. Peaks 1–3 were isolated and analyzed by ESI-MS (Table 1). Peaks 2 and 3 contain the two 5′-RNA-fragments after β-elimination and β,δ-elimination, respectively, while peak 1 corresponds to the phosphorylated 3′-fragment. Thus it is clear that both β-and δ-elimination steps take place, however at much slower rates compared to DNA 7b (Table 4).


The chemical stability of abasic RNA compared to abasic DNA.

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

left: HPLC traces (280 nm) of the β-elimination reaction of the 2′-OMe heptamer 5b at different time intervals; controls: trace A: protected heptamer 5a; trace B: protected 5a after treatment with 0.1 M NaOH for 48 h at 37°C; right: linear fit (R2 > 0.999) of ln[S]/[S0] versus t calculated from the HPLC traces.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: left: HPLC traces (280 nm) of the β-elimination reaction of the 2′-OMe heptamer 5b at different time intervals; controls: trace A: protected heptamer 5a; trace B: protected 5a after treatment with 0.1 M NaOH for 48 h at 37°C; right: linear fit (R2 > 0.999) of ln[S]/[S0] versus t calculated from the HPLC traces.
Mentions: The time course of strand cleavage of oligonucleotide 5b is depicted in Figure 1. From the HPLC traces it becomes clear that the abasic heptamer 5b (peak 4) decreases while at the same time three major fragments with lower retention times appear (peaks 1–3). Lane B is a control with protected oligonucleotide 5a proving that no cleavage occurs if the anomeric center is still protected. The T1/2 and ks for 5b under these conditions were calculated to be 594 min and 1.96 × 10−5 s−1, respectively. Peaks 1–3 were isolated and analyzed by ESI-MS (Table 1). Peaks 2 and 3 contain the two 5′-RNA-fragments after β-elimination and β,δ-elimination, respectively, while peak 1 corresponds to the phosphorylated 3′-fragment. Thus it is clear that both β-and δ-elimination steps take place, however at much slower rates compared to DNA 7b (Table 4).

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