Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA)

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell transcriptomics is crucial for identifying cell types, functions, and disease mechanisms.
• Current methods face challenges in isolating mRNA from individual cells.
• Transcriptome In Vivo Analysis (TIVA) is a photochemical method for single-cell mRNA isolation.

Purpose of the Study:

• To enhance the TIVA probe for improved stability and efficiency in single-cell mRNA isolation.
• To adapt TIVA for challenging environments, including whole organisms.

Main Methods:

• Development of modified TIVA probes with enhanced thermal stability and nuclease resistance.
• Utilized an extended 22mer polyU capture strand with two 9mer polyA blocking strands (22/9/9).
• Introduced a GC pair (22/9/9 GC) and a phosphorothioated backbone (PS-22/9/9) for further probe optimization.

Main Results:

• New TIVA probes exhibit higher thermal stability and improved mRNA capture affinity.
• The 22/9/9 GC probe reduced pre-photolysis interactions with mRNA.
• The PS-22/9/9 probe demonstrated extended serum stability (>48 h) and facilitated cellular uptake.

Conclusions:

• Modified TIVA probes offer enhanced performance for single-cell mRNA isolation.
• These advancements enable broader applications of TIVA in complex biological systems.
• Improved TIVA probes facilitate deeper insights into cellular function and disease.