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Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression
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Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides.

XinJing Tang1, Jyothishmathi Swaminathan, Alan M Gewirtz

  • 1The Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.

Nucleic Acids Research
|December 7, 2007
PubMed
Summary
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Light-activated antisense oligodeoxynucleotides (asODNs) enable controlled mRNA degradation. Conjugates showed significant c-myb mRNA knockdown in leukemia cells after UV activation, demonstrating photomodulation of protein levels.

Area of Science:

  • Molecular Biology
  • Oligonucleotide Chemistry
  • Gene Regulation

Background:

  • Antisense oligodeoxynucleotides (asODNs) are used to target specific mRNA sequences for degradation.
  • Controlling the activity of asODNs in living cells remains a challenge.
  • RNase H is an enzyme that degrades RNA when hybridized to an antisense oligonucleotide.

Purpose of the Study:

  • To develop light-activated asODNs for spatiotemporal control of mRNA degradation.
  • To investigate the efficacy of photocleavable linker-conjugated asODNs targeting c-myb mRNA.
  • To assess the photomodulation of c-myb mRNA and protein levels in human leukemia cells.

Main Methods:

  • Synthesis of asODN-photocleavable linker-sense ODN (asODN-PL-sODN) conjugates.
  • Biochemical assays to measure RNase H activity and RNA degradation upon UV irradiation.

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  • Evaluation of phosphorothioated analogs in K562 cells for c-myb mRNA and protein knockdown.

    • Main Results:

    • Six asODN-PL-sODN conjugates (C1-C6) were synthesized, with C5 and C6 showing promising characteristics.
    • Conjugate C6 exhibited low background RNase H activity and a significant increase in RNA digestion post-UV irradiation.
    • Phosphorothioated analogs S-C5 and S-C6 demonstrated effective c-myb mRNA knockdown and S-C5 showed photomodulation of c-MYB protein levels in K562 cells.

    Conclusions:

    • Light-activated asODNs offer a method for precise control over gene silencing.
    • The developed conjugates, particularly S-C5, show potential for therapeutic applications in targeting specific gene expression.
    • Further optimization may be needed to achieve protein level photomodulation with more stable conjugates like S-C6.