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Targeted gene knockout by 2'-O-aminoethyl modified triplex forming oligonucleotides
N Puri1, A Majumdar, B Cuenoud
1NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
The Journal of Biological Chemistry
|June 5, 2001
Summary
Modified oligonucleotides show promise for gene targeting. While extensive modifications enhance in vitro binding, moderate modifications yield superior in vivo gene knockout activity, suggesting a balance is key for therapeutic applications.
Area of Science:
- Molecular Biology
- Oligonucleotide Chemistry
- Gene Targeting
Background:
- Triplex forming oligonucleotides (TFOs) offer potential for gene targeting but face limitations in binding at physiological conditions.
- Pyrimidine TFOs with 2 -O-aminoethyl (AE) substitutions demonstrate improved triplex formation kinetics and stability.
Purpose of the Study:
- To evaluate the impact of varying 2 -O-aminoethyl (AE) modifications on psoralen-linked TFOs.
- To assess the in vitro and in vivo performance of AE-modified TFOs for gene targeting and HPRT gene knockout.
Main Methods:
- Preparation of psoralen-linked TFOs with varying AE substitutions.
- Biochemical assays in vitro to determine target affinity and stability under different Mg(2+) concentrations.
- In vivo stability and HPRT gene knockout assays.
Main Results:
- AE TFOs exhibited higher in vitro target affinity compared to 2 -OMe substituted TFOs, with minimal affinity loss in reduced Mg(2+).
- AE TFOs demonstrated enhanced stability in physiological buffer, with maximal affinity and stability seen in TFOs with near-complete AE substitution.
- In vivo, TFOs with moderate AE modification showed the highest stability and HPRT gene knockout activity.
Conclusions:
- The 2 -O-aminoethyl (AE) modification can enhance the biological activity of pyrimidine TFOs for gene targeting.
- Extensive AE substitution is detrimental to in vivo efficacy, indicating an optimal level of modification is required for biological applications.