Methods in three-dimensional NOE-NOE nuclear magnetic resonance spectroscopy, three-dimensional diffusion-ordered NOESY nuclear magnetic resonance spectroscopy, and studies of methylphosphonate antisense oligonucleotides

In an effort to increase the limits on molecular size and system complexity currently found in NMR spectroscopy, many methods and experiments have been proposed. A hybrid-hybrid rate matrix approach was found to provide an accurate and computationally efficient method for structural refinement based on 3D NOE-NOE data. The hybrid-hybrid method began by simulating the 3D data required to deconvolute experimental 3D data into 2D NOESY-like data. The deconvoluted data was then merged with a complete volume matrix simulated from the starting model. Structural refinement proceeded using an established 2D relaxation matrix procedure (2D MORASS) to produce distance constraints for restrained molecular dynamics refinement. Historically, structural determination by NMR was limited to single component samples. A new experiment, called 3D DOSY-NOESY, was proposed that will enable structural determination of multi-component systems. This experiment yielded diffusion labeled NOESY cross peaks which produced DOSY-NOESY sub-planes (2D NOESY-like planes that were resolved based on diffusion coefficient). The sub-planes could be used for spectral assignment and structural determination. Finally, NMR studies on two methylphosphonate modified oligonucleotide drugs, bound to their target DNA, were found to have unusual melting characteristics. Normally, as a duplex oligonucleotide approached the melting temperature there would be a corresponding loss of internucleotide connectivity in the 3-D NOESY spectrum. In these drug-target duplex systems, however, it was found that the drug strand lost internucleotide connectivity 5 to 10 degrees before the target strand. This unusual behavior was attributed to multiple conformations of the drug strand, which were distinct by NMR, binding to a single target structure or a family of target structures which were not distinguishable by NMR.