Rapidly photocleavable triazene-functionalized nucleotides for DNA sequencing.

Area of Science:

• Chemical Biology
• Molecular Biology
• Biochemistry

Background:

• DNA sequencing by synthesis requires efficient nucleotide incorporation and rapid terminator removal.
• Current methods for terminator cleavage can be time-consuming, impacting overall sequencing speed.

Purpose of the Study:

• To develop a novel method for preparing triazene-functionalized nucleotides.
• To evaluate the rapid photocleavage of triazene linkers for DNA sequencing applications.
• To assess the utility of these nucleotides in single-molecule DNA sequencing by synthesis.

Main Methods:

• Synthesis of triazene-functionalized nucleotides via coupling of amine-modified nucleotides with aryl diazonium salts.
• Fluorescent labeling of triazene-modified nucleotides.
• Enzymatic incorporation of modified nucleotides into DNA.
• Photocleavage of the triazene linker using a reducing agent and light.
• Verification of four-color fluorescently labeled reversible terminator nucleotides in single-molecule sequencing.

Main Results:

• Triazene-functionalized nucleotides were successfully synthesized and exhibited high enzymatic incorporation efficiency.
• The triazene linker on DNA was rapidly photocleaved within 5 seconds.
• A radical pathway was identified as potentially accelerating photocleavage.
• The method demonstrated potential for reducing time costs in DNA sequencing by synthesis.

Conclusions:

• Triazene-functionalized nucleotides offer a rapid and efficient method for DNA sequencing by synthesis.
• The rapid photocleavage of the triazene linker is a key advancement for accelerating sequencing processes.
• This technology holds promise for improving the speed and efficiency of single-molecule DNA sequencing.