Thorogood H, Waters T R, Parker A W, Wharton C W, Connolly B A
Department of Biochemistry and Genetics, University of Newcastle, Newcastle upon Tyne, U.K.
Biochemistry. 1996 Jul 2;35(26):8723-33. doi: 10.1021/bi960230j.
The resonance Raman spectra of 4-thiothymidine [4ST] have been recorded (a) in the free deoxynucleoside form, (b) when incorporated into the single stranded oligodeoxynucleotide d(AG[4ST]-TC), and (c) within the double-stranded self-complementary dodecamer d(GACGA[4ST]ATCGTC). Vibrational mode assignments of almost all the major Raman bands observed in each spectra have been made, mainly by comparison with the published assignments of related nucleosides and nucleotides. Differences between the spectra were observed, particularly when [4ST] and d(AG[4ST]TC) were compared to d(GACGA[4ST]ATCGTC). This is explained in terms of the variations in structure between single-and double-stranded DNA. Good quality spectra were obtained at nucleotide/oligonucleotide concentrations of between 100 and 500 microM and this coupled with an apparatus that uses small volumes (100 microL) allowed measurement of the spectrum of d(GACGA[4ST]ATCGTC) bound to the EcoRV endonuclease. This well characterised nuclease, for which crystal structures are available, recognizes d(GATAT) sequences. When this is replaced with d(GA[4ST]ATC), a poor substrate results but turnover can be prevented during data accumulation by omission of the essential cation Mg2+. Large shifts in several of the Raman bands were observed, and these have been related to the environment of the [4ST] base in the protein-bound oligonucleotide as deduced from the crystal structure. The wavenumber for the C = S stretch vibration in free d(GACGA[4ST]ATCGTC) has been used to calculate the strength of the Watson-Crick hydrogen bond between the sulphur atom in [4ST] and the 6-NH2 group on its partner dA. On binding to the enzyme, the shift in the wavenumber of the C = S stretch indicates this Watson-Crick hydrogen bond is weakened, in good agreement with X-ray structures. The advantage of using [4ST] as a resonance Raman probe is that it absorbs at 340 nm, a wavelength where other nucleic acid and protein absorbance is minimal. Thus the spectra obtained are very simple and consist of signals that arise predominantly from the thiobase alone, and this facilitates data interpretation.
已记录了4-硫代胸苷[4ST]的共振拉曼光谱：(a) 以游离脱氧核苷形式；(b) 掺入单链寡脱氧核苷酸d(AG[4ST]-TC)时；(c) 在双链自互补十二聚体d(GACGA[4ST]ATCGTC)中。主要通过与已发表的相关核苷和核苷酸的归属进行比较，对每个光谱中观察到的几乎所有主要拉曼带进行了振动模式归属。观察到了光谱之间的差异，特别是当将[4ST]和d(AG[4ST]TC)与 d(GACGA[4ST]ATCGTC)进行比较时。这可以根据单链和双链DNA之间结构的变化来解释。在核苷酸/寡核苷酸浓度为100至500微摩尔时获得了高质量的光谱，再加上使用小体积(100微升)的仪器，使得能够测量与EcoRV内切核酸酶结合的d(GACGA[4ST]ATCGTC)的光谱。这种特征明确且有晶体结构的核酸酶识别d(GATAT)序列。当用d(GA[4ST]ATC)取代时，底物效果不佳，但在数据积累期间通过省略必需阳离子Mg2+可以防止周转。观察到几个拉曼带发生了大的位移，这些位移与从晶体结构推断出的蛋白质结合寡核苷酸中[4ST]碱基的环境有关。游离d(GACGA[4ST]ATCGTC)中C = S伸缩振动的波数已用于计算[4ST]中的硫原子与其配对dA上的6-NH2基团之间的沃森-克里克氢键的强度。与酶结合时，C = S伸缩波数的位移表明这种沃森-克里克氢键被削弱，这与X射线结构非常吻合。使用[4ST]作为共振拉曼探针的优点是它在340 nm处吸收，在这个波长下其他核酸和蛋白质的吸光度最小。因此获得的光谱非常简单，主要由仅来自硫代碱基的信号组成，这便于数据解释。
