Photoactivatable Circular Caged Oligonucleotides for Transcriptome In Vivo Analysis (TIVA)

Area of Science:

• Oligonucleotide chemistry
• Molecular biology
• Bioconjugation

Background:

• Light-activated oligonucleotide probes offer spatiotemporal control for gene expression studies.
• Cyclization with photocleavable linkers is an efficient and stable caging strategy.

Purpose of the Study:

• To introduce an improved protocol for circular oligonucleotide synthesis.
• To evaluate the stability and binding affinity of photocleavable caging strategies.
• To identify a suitable probe for transcriptome in vivo analysis (TIVA).

Main Methods:

• Circular oligonucleotide synthesis with a single HPLC purification step.
• Denaturing gel electrophoresis to assess pre-photolysis caging stability.
• Melting temperature measurements to determine post-photolysis target binding affinity.

Main Results:

• An optimized protocol for circular oligonucleotide synthesis was established.
• The stability and binding affinity of various oligonucleotide probes were characterized.
• A 14U 2'-OMe RNA probe demonstrated significant potential for TIVA applications.

Conclusions:

• The improved synthesis protocol simplifies the preparation of light-activated oligonucleotide probes.
• Photocleavable caging strategies can be fine-tuned for stability and target binding.
• The developed 14U 2'-OMe RNA probe is promising for mRNA isolation in transcriptome in vivo analysis.