Backbone Conformational Study of a Non‐Palindromic 16 Base Pair DNA Duplex Exploring 2D 31P–1H Heteronuclear Inverse Spectroscopy: Assignment of all NMR Phosphorus Resonances and Measurement of 3J 31P–1H3′ Coupling Constants

Assignment of all 31P resonances of a 16 base‐pair DNA duplex, 5′d(CTGCTCACTTTCCAGG)3′ · 5′d(CCTGGAAAGTGAGCAG)3′, related to the DNA κB site of the HIV‐1 LTR, was determined by 2D hetero‐nuclear inverse NMR spectroscopy (HSQC‐TOCSY and heteronuclear COSY). 3J(H3′–P) coupling constants for most of the oligomer phosphates were obtained using heteronuclear selective COSY. Theoretical considerations of the 31P relaxation in macromolecules in solution allow parameter optimization of two‐dimensional 1H–31P NMR experiments. Structural information can then be extracted to define the backbone conformation of large oligonucleotides in solution.