Highly multiplexed single-cell in situ RNA and DNA analysis with bioorthogonal cleavable fluorescent oligonucleotides

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Comprehensive single-cell analysis of RNA and DNA in situ is crucial for understanding biological processes and diseases.
• Existing methods may have limitations in multiplexing capabilities and sensitivity.

Purpose of the Study:

• To develop a highly multiplexed single-cell in situ analysis approach for both RNA and DNA.
• To enable sensitive detection and quantification of numerous nucleic acid targets within individual cells.

Main Methods:

• Utilized bioorthogonal cleavable fluorescent oligonucleotides for in situ hybridization.
• Employed an iterative cycle of hybridization, fluorescence imaging, and efficient cleavage of fluorophores.
• Integrated analysis of DNA, RNA, and protein using cleavable probes and antibodies.

Main Results:

• Demonstrated detection of different nucleic acids in each hybridization cycle using multi-color staining.
• Achieved at least ten continuous hybridization cycles on the same specimen without compromising nucleic acid integrity.
• Showcased the potential for single-molecule sensitivity in quantifying hundreds to thousands of targets.

Conclusions:

• The developed method offers a powerful, highly multiplexed platform for in situ single-cell analysis of nucleic acids and proteins.
• This approach significantly advances systems biology and biomedical research by enabling detailed cellular profiling.