Organ rejection is a major source of morbidity associated with transplantation. The current clinical treatment aimed at mitigating this phenomenon entails administrating immunosuppressive drugs to the recipient. Unfortunately, this induces a general immune suppression resulting in enhanced susceptibility to infectious agents and cancer. This program project proposes an alternative treatment protocol based on therapeutic oligonucleotides aimed at selective down-regulation of HLA class I and class II genes in the graft. This should result in a diminished ability of the graft to excite the immune response of the host, and thereby reduce or eliminate refection of the transplant. In this project, we present an experimental approach to the determination of the stability and structure of these therapeutic oligonucleotides. The oligonucleotides we will initially investigate have been designed to exert their gene-regulatory effect via one of several modes of action: antisense, antigene, aptamer or decoy. These modes of activity imply the ability to form complexes with other nucleic acids, such as RNA-DNA duplexes or DNA triplexes, or with transcription factor proteins. Furthermore, these DNA sequences may also form self- associated or internal structures mediated by guanine quadruplex formation, Watson-Crick base pairing or other interaction, This project focuses on elucidating and characterizing these interactions. Results from these studies will provide information necessary to develop structure-activity relations for this class of drugs and help establish their mechanisms of action. The techniques we will use include gel and capillary electrophoresis, UV and CD spectrophotometry, equilibrium ultracentrifugation, differential scanning calorimetry and two-dimensional NMR. With these methods, we will evaluate the molecularity, secondary structure and thermodynamic stability of complexes formed by these oligonucleotides. In addition, in selected cases we plan to carry out high resolution NMR studies to obtain a detailed structure of these complexes on a molecular level.
