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Photoactivatable Circular Caged Oligonucleotides for Transcriptome In Vivo Analysis (TIVA)
Linlin Yang1, Dora von Trentini1, HyunBum Kim2
1Department of Chemistry, University of Pennsylvania, 231 South 34 Street, Philadelphia, PA 19104-6323 (USA).
Abstract:
Light activation is an effective way to impart spatiotemporal control over oligonucleotide probes that are widely applied for gene expression regulation and target function investigation. Among the major oligonucleotide caging strategies, cyclization with a photocleavable linker is an elegant design, which affords both atom efficiency and stability in many biological environments. Here, we introduce an improved protocol for circular oligonucleotide synthesis requiring only one round of HPLC purification. With a series of poly-U oligonucleotide strands of different sizes and backbone modifications, the pre-photolysis caging stability and post-photolysis target binding affinity were studied through a denaturing gel assay and melting temperature measurements. A 14U 2'-OMe RNA probe was selected, with strong potential application in transcriptome in vivo analysis (TIVA) for mRNA isolation.
Insights
We developed a simpler method for creating light-activated oligonucleotide probes. These probes offer precise control for studying gene expression and biological targets, with potential for in vivo transcriptome analysis.
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.
