Nucleic Acids Research Assignment of the "C nuclear magnetic resonance spectrum of a short DNA-duplex with 'H-detected two-dimensional heteronuclear correlation spectroscopy

Proton-detected Hc heteronuclear correlated spectroscopy ([ H, C]COSY) was used to establish relations between the carbon-13 and proton nuclear magnetic resonance chemical shifts in the hexadeoxynucleoside pentaphosphate d-(GCATGC) . Using the previously established sequence-specific proton NMR assignments, sequence-specific assignments were thus obtained for nearly all proton-bearing carbons. This approach offers a new criterion for distinguishing between the proton NMR lines of purines and pyrimidines, based on the different proton-carbon-13 coupling constants. Furthermore, the adenine ring carbon 2 has a unique carbon-13 chemical shift, which enables a straightforward identification of the adenine C2H resonances by [ H, Cl-COSY. INTRODUCTION Most NMR studies of nucleotides have employed proton and phosphorous resonances, whereas C and N were little used because of the low sensitivity. With the introduction of heteronuclear 2D NMR experiments with proton detection (1-9), C and N resonances can now also be assigned conveniently and used to analyze nucleotides without isotope enrichment. In this report we demonstrate the use of heteronuclear correlated spectroscopy ([ H, C]-COSY) with studies of the hexadeoxynucleoside pentaphosphate duplex d-(GCATGC)2 : d G] C2 A3 T 4 G5 C6 C. -| G_2 T_3 A_4 C_5 G_6 d We use an experiment designed for ef f ic ient suppression of the signals from carbon-12 bound protons, enabling easy observation of the much weaker signals from the C sate l l i tes (1,10,11). Previously, sequence-specific C NMR assignments were obtained with [ H, C]-COSY for some oligoribonucleo© IR L Press Limited, Oxford, England. 2 6 7 Nucleic Acids Research t ides (12) and oligodeoxyribonucleotides (13) in single stranded form. For DNA fragments in duplex form ( e . g . , d-(CGCGCG)2,d-(CGCGCGCG)2; (14)) one-dimensional NMR methods were used and combined with empirical c r i t e r i a for resonance assignments. The presently described two-dimensional NMR approach has the advantage of superior r e l i a b i l i t y and speed. I t i s of part icular interest with regard to the potential of C NMR spectroscopy for studies of structure and dynamics of oligonucieotides (15), as well as i t s use as a complementary method to H NMR spectroscopy fo r studies of DNA-ligand interact ions (e .g. ,16) . MATERIALS AND METHODS The hexadeoxynucleoside pentaphosphate d-GCATGC was synthesized by the phosphotriester method in l iqu id phase as described elsewhere (17,18). The oligonucleotide was used as the Na sal t (obtained by running the DNA over a Dowex 50W X8 column in the Na-form, with H-O as the solvent, followed by desalt ing with Sephadex G-25). The NMR sample was prepared by dissolving the DNA in 500 j i l buffer solution in D? 0, containing 0.1 M NaCl and 50 mM 2 phosphate, p H = 7.0. This solut ion was twice lyophylized and redissolved in 500 JJI of 99.96% D-O. After a t h i r d lyophyl izat ion the f i na l solvent used was 500 y l of 99.996% D20 (Stohler Isotopes). The concentration of the DNA was 20 mM in duplex. The NMR spectra were recorded in a 5 mm sample tube on a Bruker AM 360 spectrometer at 297+0.5 K. A [V^C j -COSY experiment with 490 t ] values was recorded. The maximum t , value was 12.3 ms. Each free induction decay consisted of 1024 data points with a maximum t value of 140 ms. Prior to Fourier transformation the time domain data set was mul t ip l ied with a trapezoidal window along t , with the plateau reaching from 3 ms to 82 ms, and a cosine window was applied along to. Chemical sh i f ts are quoted re la t ive to in ter nal TSP (2,2,3,3tetradeutero-3-( t r imethyls i ly l )propionate, sodium s a l t ) . RESULTS AND DISCUSSION Figure 1 shows a survey of the [ H, C]-COSY spectrum of the hexadeoxynucleoside pentaphosphate d-(GCATGC) in duplex form. The H spectrum is along a>_ , the C spectrum along co. For each of the observed cross peaks the two doublet components have opposite sign. The assignments of the different groups of resonance peaks to particular CH or CHfragments in the mononucleotides follows from the chemical shift ranges generally observed for the corresponding C and ^H lines (e.g.,19-22).