An investigation of single-site energetics and dynamics in nucleic acids by NMR spectroscopy

This thesis describes an investigation of dynamics and stability of individual base pairs in nucleic acid structures using nuclear magnetic resonance (NMR) spectroscopy.; The first part of the thesis describes the characterization of dynamics and stability of individual base pairs in a few nucleic acid structures using proton exchange. We have investigated three intramolecular triple-helical structures: a triple helix that contains only canonical YRY base triads [5 ′-d(AGAGAGAA-CCCC-TTCTCTCT-TTT-TCTCTCTT)-3′], an YRY triple helix that contains one non-canonical GTA base triad [5 ′-d(AGATAGAA-CCCC-TTCTATCT-TATA-TCT GTCTT)-3′] and a triple helix that contains canonical RRY triads: [5′-d(TCCTCCT-TTTTT-AGGAGGA-TTTTT-TGGTGGT)-3 ′]. For the canonical YRY triple helix, we have demonstrated the existence of enthalpy-entropy compensation in imino proton exchange and we have analyzed the energetic origin of this phenomenon. For the non-canonical YRY triple helix we have characterized the stability of the non-canonical GTA base triad and the energetic effects of this base triad upon the stability of canonical base triads in the triple helix. For the RRY triplex, we have found that formation of the triple-helical structure stabilizes the Watson-Crick double helix less effectively than for the YRY triple helix.; We have also synthesized and characterized the stability of a RNA structure using uniform 15N-labeling. The RNA is the 29-nt rat Sarcin-Ricin loop domain RNA 5′-r(G₁ G₂ G ₃ U₄ G₅ C₆ U₇ C₈ A₉ G₁₀ A₁₁ C₁₂ G₁₃ A₁₄ G₁₅ A₁₆ G₁₇ G₁₈ G₁₉ A₂₀ A₂₁ C₂₂ C₂₃ G₂₄ C₂₅ A₂₆ C₂₇ C₂₈ C₂₉)-3′. The molecule includes the RNA sequence 5′-r(A₉ G₁₀ U₁₁ A ₁₂ C₁₃ G₁₄ A₁₅ G₁₆ A ₁₇ G₁₈ G₁₉ A₂₀)-3′ which is conserved in all ribosomal structures and contains several important structural motifs that exists in many other RNA structures.; The second part of the thesis presents a characterization of the rotational dynamics of the adenine amino group in a canonical YRY DNA triple helix, using site-specific 15N-labeling and heteronuclear NMR technique. By comparing the activation enthalpy and entropy changes for amino groups rotation in the triple helix and in a DNA double helix we have concluded that the energetic origin of the decrease in the rotation rate for the amino group in the triple helix is entropic.; The third part of the thesis is an attempt to characterize changes in strength of hydrogen bonds involving imino protons in the A-tract DNA 5 ′-d(CGCAAAATTTTGCG)₂-3′, during the course of premelting transition using hydrogen/deuterium fractionation factors. The results have shown that the major changes in hydrogen bond strength occur at the junction of the A-tract and the non-A-tract region.