Polyfluorophore labels on DNA: dramatic sequence dependence of quenching.
We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynucleoside fluorophores (ODFs)--a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3' end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl-labeled complementary strand resulted in strong quenching of fluorescence in 85% of the twenty ODF sequences. The high efficiency of quenching was also established by their large Stern-Volmer constants (K(SV)) of between 2.1 x 10(4) and 4.3 x 10(5) M(-1), measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a K(SV) that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the pi stack of fluorophores in the amplified quenching of fluorescence.
Teo
Yin Nah
YN
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Wilson
James N
JN
Kool
Eric T
ET
D001483:Base Sequence; D001580:Benzopyrenes / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010569:Perylene / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry; D013194:Staining and Labeling; D013267:Stilbenes / Q000737:chemistry
D001580:Benzopyrenes; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011721:Pyrenes; D013267:Stilbenes; D010569:Perylene; D004247:DNA