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Updated: Jul 9, 2026
Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression
Published on: November 4, 2025
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.
Abstract:
Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the 'caged' state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (T(m)) upon UV irradiation (DeltaT(m) = -29 degrees C). The most thermally stable conjugate, C6 (T(m) = 84 degrees C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex.
Insights
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.
- 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.
