Quenching of Fluorescent Nucleobases by Neighboring DNA: The “Insulator” Concept

Abstract

Fluorescent nucleosides are widely used as probes of biomolecular structure and mechanism in the context of DNA, but they often exhibit low quantum yields because of quenching by neighboring DNA bases. Here we characterize the quenching by DNA of fluorescent nucleosides that have pyrene (Y), perylene (E), benzopyrene (B), or 2-aminopurine (2AP) as nucleobase replacements, and we investigate the effect of inserting varied nucleosides as potential "insulators" between the fluorescent nucleosides and other nearby DNA bases as a strategy for increasing quantum yields. The data show that the hydrocarbons are quenched by adjacent pyrimidines, with thymine being the strongest quencher. The quantum yield of pyrene is quenched 120-fold by a single adjacent T, that of benzopyrene tenfold, and that of perylene by a factor of 2.5. Quenching of excimer and exciplex dinucleoside labels (Y-Y, Y-E, E-E, etc.) was considerably lessened, but was strongest with neighboring thymine. 2-Aminopurine (2AP) is most strongly quenched (15-fold) by neighboring G. We tested four different insulator candidates for reducing this quenching by measuring the fluorescence of short oligonucleotides containing insulators placed between a fluorescent base and a quenching base. The insulators tested were a C(3) abasic spacer (S), dihydrothymidine nucleoside (DHT), terphenyl nucleoside (TP), and adenine deoxynucleoside (dA). Results showed that the abasic spacer had little effect on quenching, while the other three had substantial effects. DHT and terphenyl enhanced fluorescence of the fluorophores by factors of 5 to 70. Adenine base reduced the quenching of pyrene 40-fold. The results underscore the importance of the nearest neighbors in DNA-quenching mechanisms, and establish simple strategies for enhancing fluorescence in labeled DNAs.

2‐(1‐Ethynylpyrene)‐Adenosine as a Folding Probe for RNA—Pyrene in or out

“…They have proven useful in the context of recognition and as molecular probes, [6][7][8][9][10][11][12][13] electron injection, [14,15] bioengineering, [16,17] structure stabilisation [18] and structure determination [19][20][21][22][23] as well as nucleobase photophysics. [24,25] Their relatively high quantum efficiency, sensitivity to environmental factors [26,27] and capability to form excimers have opened many possibilities in the field of DNA and RNA hybridisation studies.…”

Detection of RNA Hybridization by Pyrene‐Labeled Probes

“…[6][7] Although pyrene has been used as a fluorescent probe to monitor the hybridization or tertiary folding of nucleic acids, [8] pyrene-oligonucleotide probes based on monomer emission intensity are usually disturbed by fluorescence quenching through an electron-migration process between excited pyrene and nucleotide bases. [9][10][11] The quenched fluorescence may decrease the detection ability of the probes. This means that for some DNA duplexes the fluorescence quenched by the flanking C/G base pair differs little from that of a single stranded probe.…”

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

“…This property enables them to generate a wide variety of emission colors with a single short-wavelength excitation (19). The emission from delocalized excited states that commonly occurs in ODFs results in their ability to be quenched with high efficiency (20)(21)(22), which has proven useful in the development of turn-on enzyme sensors (23). In addition, ODFs (which are commonly ∼1 nm in size) have much smaller size than other multicolor fluorescent tags such as FRET dye pairs, QDs, or fluorescent proteins.…”