Polyfluorophore labels on DNA: dramatic sequence dependence of quenching

Area of Science:

• Biophysical Chemistry
• Molecular Biology
• DNA Nanotechnology

Background:

• Oligodeoxynucleoside fluorophores (ODFs) are stacked, electronically interacting fluorophores built on a DNA scaffold.
• Dabcyl is a common fluorescence quencher used in various biological applications.

Purpose of the Study:

• To investigate the interaction between dabcyl and ODFs in a DNA context.
• To determine how the sequence and arrangement of fluorophore monomers within ODFs affect fluorescence quenching.
• To understand the role of excited states in the quenching mechanism.

Main Methods:

• Synthesis of twenty tetrameric ODF sequences with varied combinations of pyrene, benzopyrene, perylene, dimethylaminostilbene, and spacer monomers.
• Hybridization of ODF probes with dabcyl-labeled complementary DNA strands.
• Fluorescence spectroscopy to measure quenching efficiency and Stern-Volmer constants (K(SV)).

Main Results:

• 85% of the tested ODF sequences showed strong fluorescence quenching by dabcyl.
• High Stern-Volmer constants (K(SV) between 2.1 x 10^4 and 4.3 x 10^5 M^-1) indicated efficient quenching.
• Quenching efficiency exhibited strong sequence dependence, with anagrams showing orders of magnitude difference in K(SV).
• Excimer and exciplex emission bands were quenched more efficiently than monomer bands, suggesting involvement of delocalized excited states.

Conclusions:

• The sequence arrangement of ODFs is critical for efficient fluorescence quenching by dabcyl.
• Delocalized excited states within the fluorophore pi-stack play a significant role in amplified fluorescence quenching.
• This DNA-based system offers a tunable platform for fluorescence sensing and molecular probes.