Increased expression of the oncogene c-erbB-2 is associated with malignant cell transformation and poor prognosis in several tumors such as breast and ovarian cancer and in adenocarcinoma and possibly in astrocytic brain tumors. The overall goal of this project is to understand the structural and thermodynamic parameters of novel c_erbB-2 antisense oligonucleotides that contain inverted anomeric centers and polarity reversals. We plan to carry out a systematic study of the structural and thermodynamic properties of the systems described below in order to be able to predict and optimize, position and length of beta anomeric DNA stretches, modified bases and sugar residues in the alpha anomeric components, for optimal stability, nuclease resistance, and Rnase H activity, that will result in the design of potent second generation antisense agents. The main tool used is NMR spectroscopy, supported by UV and CD spectroscopy and nuclease sensitivity studies. 1) Study and design of alpha anomeric ODN/RNA duplexes which activate Rnase H. Determination of the length and requirement of a beta anomeric stretch flanked by alpha anomeric components vs. Rnase H activity nd DNA exo- and endonuclease resistance. 2) Determination of the solution structure of DNA/DNA decamer duplexes that contain alpha anomeric nucleotides and a spacer at the 3'-3' phosphodiester linkage. Alpha anomeric bases are embedded in a parallel manner in an overall antiparallel helix. Each base is flanked by an unusual 3'3' and 5'5' linkage. The stability of each of these duplexes will be determined by UV melting studies. 3) NMR analysis of the solution structure of DNA/RNA duplexes were the DNA strand contains alpha anomeric nucleotides and spacers at the 3'-3' phosphodiester linkage. Each DNA strand will contain a single alpha anomeric base in order to clearly distinguish among the individual base substitutions. The stability of each of these duplexes will be determined using UV melting profiles. 4) Improvement of the thermodynamic stability of the ODN/RNA hybrids. Several chemical modifications will be explored to further imrpove the ODN-RNA binding. 5) Design, properties and structure of third generation ODNs. Utilizing the results obtained from Aims 1,3 and 4, more potent anti-c-erbB-2 ODNs with superior thermodynamic and Rnase H activation properties. NMR will be used to verify our design strategy.
