Photomodulating RNA cleavage using photolabile circular antisense oligodeoxynucleotides

Area of Science:

• Oligonucleotide chemistry
• Molecular biology
• Biochemistry

Background:

• Antisense oligodeoxynucleotides (asODNs) are used for gene silencing.
• Controlling asODN activity and specificity is crucial for therapeutic applications.
• Photocleavable linkers enable light-inducible control over oligonucleotide function.

Purpose of the Study:

• To develop caged antisense oligodeoxynucleotides (asODNs) with light-regulated RNA binding and hydrolysis.
• To investigate the effect of structural modifications (ring size) on the performance of caged asODNs.
• To demonstrate photo-modulation of RNA targeting and RNase H-mediated degradation.

Main Methods:

• Synthesis of caged asODNs with photocleavable linkers.
• Thermal stability assays (T(m)) to compare caged and uncaged structures.
• RNase H assays to quantify RNA digestion upon light activation.
• Gel shift assays to assess RNA/asODN binding affinity.

Main Results:

• Caged asODNs exhibited varying thermal stabilities and secondary structures.
• Light activation of H30, H40, C30, and C40 asODNs photo-modulated RNA digestion 2- to 3-fold.
• Caged asODN C20 showed a >20-fold increase in RNA digestion upon photoactivation.
• Binding affinities varied significantly among different caged asODN structures.

Conclusions:

• The ring size of caged asODNs can be adjusted to control mRNA hybridization.
• Light activation of caged asODNs enables photoregulated RNA hydrolysis by RNase H.
• This study presents a novel strategy for spatiotemporally controlled gene silencing using light.