NMR Detection of NH···OC Hydrogen Bonds in ¹³C,¹⁵N-Labeled Nucleic Acids

Abstract

We describe the first direct observation of NsH‚‚‚OdC hydrogen bonding in nucleic acids via the four-bond 4h J NiNj coupling within an N1 i sH1 i ‚‚‚O6 j dC6 j -N1 j segment of a G‚G‚G‚G tetrad. The experiment, two-dimensional (2D) HN(N)-TOCSY, makes use of band-selective 15 N isotropic mixing to transfer magnetization exclusively via the 4h J NN couplings to yield correlations between N1H i (ω 2 ) and N1 j (ω 1 ) across the hydrogen-bonded segment. In a complementary experiment, 2D cross-polarization (CP)-H(N)CO-(NN)-TOCSY, employing band-selective heteronuclear 15 N-13 C cross-polarization sequences on either side of the 15 N-15 N TOCSY period, correlations are obtained between N1H i (ω 2 ) and C6 j (ω 1 ) nuclei across the hydrogen bond. The symmetric A 2 X 2 -type coupling topology of the four-spin nitrogen system in the G‚G‚G‚G tetrad permits accurate measurement of these couplings by a new procedure that fits the experimental data with known analytical isotropic mixing transfer functions. The techniques are demonstrated on a G‚G‚G‚G tetrad formed within a novel dimeric quadruplex fold of a uniformly 13 C/ 15 N- labeled d(G-G-G-T-T-C-A-G-G) DNA sequence. The value of the 4h J NN coupling constant for this system is estimated to be 0.136 ( 0.021 Hz.

A review with comprehensive data on experimental indirect scalar NMR spin–spin coupling constants across hydrogen bonds

“…To measure the coupling constant across an HB, several conditions have to be fulfilled: (i) The difficulty in measuring such couplings has limited them to 1/2 spin nuclei 1 H, 13 C, 15 N and 19 F, with very little data involving 31 P; biologically interesting nuclei such as 17 O and chemically important ones such as 35/37 Cl have, for the moment, to remain outside these studies. (ii) The HB strength must be from medium to high, and this limits most studies to NH, OH and FH as HB donors and O(sp 2 ) and N(sp 2 ) as HB acceptors.…”

An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs

“…Interestingly, hydrogen bonding between the adenine N3 nitrogen and the uridine H3N3 imino group brought one of the uridine carbonyl groups into close spatial proximity to the adenine H9N9 imino group. The direct detection of hydrogen bonds with imino groups as the donor and a carbonyl oxygen as acceptor is virtually impossible in larger RNAs because of the very small size of the corresponding 3h J NCO -and 4h J NN -coupling constants (27,28). However, the uridine C2 and C4 carbonyl carbon chemical shifts themselves are sensitive reporters of hydrogen bonding (29).…”

NMR Methods for Studying the Structure and Dynamics of RNA

“…[27,34] These are more challenging experiments due to the additional magnetization transfers and the smaller size of these couplings, and thus, the application of these methods to larger RNAs may be more limited.…”