Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single-Molecule Fluorescence Applications.

Area of Science:

• Biochemistry
• Molecular Biology
• Organic Chemistry

Background:

• Sequence-specific fluorescence labeling is crucial for studying nucleic acid dynamics using methods like single-molecule Förster resonance energy transfer (smFRET).
• Current solid-phase synthesis methods for labeled nucleic acids yield diminishing returns with increasing strand length, limiting their application for long molecules.

Purpose of the Study:

• To develop a new postsynthetic labeling strategy for incorporating bioorthogonal groups into single-stranded DNA and RNA of any length.
• To enable precise labeling for advanced biophysical studies of nucleic acids.

Main Methods:

• DNA-templated synthesis to create a 12-alkyne-etheno-adenine modification.
• Copper-catalyzed azide-alkyne cycloaddition (CuAAC) for conjugating the Cy3 fluorophore.
• Application to DNA, short RNA, and a large 633-nucleotide RNA construct from Saccharomyces cerevisiae.

Main Results:

• Successful sequence-selective incorporation of a bioorthogonal group into single-stranded DNA and RNA.
• Efficient conjugation of the Cy3 fluorophore via CuAAC.
• Demonstrated utility in smFRET measurements and gel electrophoresis for verifying labeling on long RNA molecules.

Conclusions:

• The developed labeling strategy is effective for nucleic acids of unrestricted length, including large RNA constructs.
• This method significantly expands the possibilities for studying long, functional nucleic acids using fluorescence-based techniques.
• The approach is versatile and applicable to both DNA and RNA, paving the way for new research avenues.