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Updated: Aug 15, 2026
Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
Published on: November 1, 2019
Preparation and analysis of oligonucleotides containing the c4'-oxidized abasic site and related mechanistic probes
Jaeseung Kim1, Cortney R Kreller, Marc M Greenberg
1Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Abstract:
The C4'-oxidized abasic site (C4-AP) is produced by a variety of DNA damaging agents. This alkali labile lesion can exist in up to four diastereomeric cyclic forms, in addition to the acyclic keto-aldehyde. Synthetic oligonucleotides containing the lesion were prepared from a stable photochemical precursor. Chemical integrity of the lesion containing oligonucleotides was probed using phosphodiesterase lability. Analysis of the 3',5'-phosphate diester of the monomeric lesion released from single diastereomers of photolabile precursors by 1H NMR indicates that isomerization of the hemiacetal and/or hemiketal is rapid. The syntheses and characterization of oligonucleotides containing configurationally stable analogues of C4-AP, which serve as mechanistic probes for deciphering the structural basis of the biochemical and biological effects of the C4'-oxidized abasic lesion, are also described.
Insights
The C4'-oxidized abasic site (C4-AP) lesion, a DNA damage product, rapidly isomerizes between cyclic and acyclic forms. Stable analogues were synthesized to study the structural basis of C4-AP
Area of Science:
- DNA Damage and Repair
- Chemical Biology
- Organic Chemistry
Background:
- The C4"-oxidized abasic site (C4-AP) is a DNA lesion generated by various damaging agents.
- This lesion is alkali-labile and can exist as four diastereomeric cyclic forms and an acyclic keto-aldehyde.
- Understanding the structural and chemical properties of C4-AP is crucial for DNA repair mechanisms.
Purpose of the Study:
- To synthesize and characterize synthetic oligonucleotides containing the C4-AP lesion.
- To investigate the chemical integrity and isomeric forms of the C4-AP lesion.
- To develop configurationally stable analogues of C4-AP as mechanistic probes for biological studies.
Main Methods:
- Preparation of synthetic oligonucleotides containing the C4-AP lesion from a stable photochemical precursor.
- Assessment of chemical integrity using phosphodiesterase lability.
- Analysis of released monomeric lesion diastereomers using 1H NMR spectroscopy to study isomerization.
Main Results:
- Synthetic oligonucleotides containing the C4-AP lesion were successfully prepared.
- Analysis revealed rapid isomerization between hemiacetal and/or hemiketal forms of the C4-AP lesion.
- Configurationally stable analogues of C4-AP were synthesized and characterized.
Conclusions:
- The C4-AP lesion exists in rapidly interconverting cyclic and acyclic forms.
- The synthesized stable analogues provide valuable tools for mechanistic studies of C4-AP's biological effects.
- Further research using these probes will elucidate the structural basis of C4-AP's impact on DNA biochemistry and biology.
