RESONANCE RAMAN-SPECTROSCOPY OF 4-THIOTHYMIDINE AND OLIGODEOXYNUCLEOTIDES CONTAINING THIS BASE BOTH FREE IN SOLUTION AND BOUND TO THE RESTRICTION-ENDONUCLEASE ECORV

The resonance Raman spectra of 4-thiothymidine [T-4S] have been record ed (a) in the free deoxynucleoside form, (b) when incorporated into th e single stranded oligodeoxynucleotide d(AG[T-4S]TC), and (c) within t he double-stranded self-complementary dodecamer d(GACGA[T-4S]ATCGTC). Vibrational mode assignments of almost all the major Raman bands obser ved in each spectra have been made, mainly by comparison with the publ ished assignments of related nucleosides and nucleotides. Differences between the spectra were observed, particularly when [T-4S] and d(AG[T -4S]TC) were compared to d(GACGA[T-4S]ATCGTC). This is explained in te rms 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 mu M and this coupled with an a pparatus that uses small volumes (100 mu L) allowed measurement of the spectrum of d(GACGA[T-4S]ATCGTC) bound to the EcoRV endonuclease. Thi s well characterised nuclease, for which crystal structures are availa ble, recognizes d(GATAT) sequences. When this is replaced with d(GA[T- 4S]ATC), a poor substrate results but turnover can be prevented during data accumulation by omission of the essential cation Mg2+. Large shi fts in several of the Raman bands were observed, and these have been r elated to the environment of the [T-4S] base in the protein-bound olig onucleotide as deduced from the crystal structure. The wavenumber for the C=S stretch vibration in free d(GACGA[T-4S]ATCGTC) has been used t o calculate the strength of the Watson-Crick hydrogen bond between the sulphur atom in [T-4S] and the 6-NH2 group on its partner dA. On bind ing to the enzyme, the shift in the wavenumber of the C=S stretch indi cates this Watson-Crick hydrogen bond is weakened, in good agreement w ith X-ray structures. The advantage of using [T-4S] as a resonance Ram an probe is that it absorbs at 340 nm, a wavelength where other nuclei c acid and protein absorbance is minimal. Thus the spectra obtained ar e very simple and consist of signals that arise predominantly from the thiobase alone, and this facilitates data interpretation.