Wilson James N, Cho Younjin, Tan Samuel, Cuppoletti Andrea, Kool Eric T
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Chembiochem. 2008 Jan 25;9(2):279-85. doi: 10.1002/cbic.200700381.
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.
荧光核苷在DNA研究中被广泛用作生物分子结构和机制的探针，但由于邻近DNA碱基的淬灭作用，它们的量子产率往往较低。在此，我们表征了以芘（Y）、苝（E）、苯并芘（B）或2-氨基嘌呤（2AP）作为碱基替代物的荧光核苷被DNA淬灭的情况，并研究了插入不同核苷作为潜在“绝缘体”，置于荧光核苷和其他邻近DNA碱基之间，以此提高量子产率的策略。数据表明，这些碳氢化合物被相邻的嘧啶淬灭，胸腺嘧啶是最强的淬灭剂。单个相邻的T可使芘的量子产率淬灭120倍，苯并芘淬灭10倍，苝淬灭2.5倍。准分子和激基复合物二核苷标记物（Y-Y、Y-E、E-E等）的淬灭作用明显减弱，但与邻近胸腺嘧啶时的淬灭作用最强。2-氨基嘌呤（2AP）被邻近的G淬灭作用最强（15倍）。我们通过测量含有置于荧光碱基和淬灭碱基之间的绝缘体的短寡核苷酸的荧光，测试了四种不同的绝缘体候选物以减少这种淬灭作用。测试的绝缘体有C(3)无碱基间隔物（S）、二氢胸腺嘧啶核苷（DHT）、三联苯核苷（TP）和腺嘌呤脱氧核苷（dA）。结果表明，无碱基间隔物对淬灭作用影响不大，而其他三种有显著影响。DHT和三联苯使荧光团的荧光增强了5至70倍。腺嘌呤碱基使芘的淬灭作用降低了40倍。这些结果强调了最近邻碱基在DNA淬灭机制中的重要性，并建立了增强标记DNA荧光的简单策略。
