Site-Specific Stabilization of DNA by a Tethered Major Groove Amine, 7-Aminomethyl-7-deaza-2′-deoxyguanosine

Abstract

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G1A2G3A4X5C6G7C8T9C10T11C12)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5′-neighboring base pairs, A4:T9 and G3:C10, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G3:C10 base pair, where αKop for the G3:C10 base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Base-pair Opening Dynamics of Nucleic Acids in Relation to Their Biological Function

“…(5) (Fig. 2B) [20,21]. The apparent relaxation rate constant ( R 1a ) for an imino proton determined by NMR experiments is the sum of the k ex value and the R 1 relaxation rate constant.…”

“…The Gibbs free energy difference (ΔG o bp ) between the closed and open states is calculated from the equilibrium constant for base-pair opening using Eq. (8):normalΔGbpogoodbreak=badbreak−normalΔGopeningogoodbreak=italicRTln()Kopwhere ΔG o opening is the Gibbs free energy change in the opening process, T is the absolute temperature and R is the universal gas constant [20,21,31]. The activation energies for base-pair opening (ΔG ‡ op ) and closing (ΔG ‡ cl ) are related to the k op and k cl values, respectively, by the Arrhenius equation.…”

“…A structural study of CPD-containing DNA duplexes suggested that, during nucleotide excision repair, the XPC-hHR23B complex recognizes DNA damage by directly searching for conformational distortions, such as a flexible backbone, a bent helix, or an unusual groove width [75]. NMR hydrogen exchange studies found that the two thymine bases of a CPD formed stable Watson-Crick base-pairs with the two opposite adenine bases, evident by a ΔΔG o bp ~ 0.2 kcal/mol relative to normal Watson-Crick T·A base-pairs [21]. When the CPD forms double T·G base-pairs, the ΔΔG o bp values are >1.9 kcal/mol [21].…”

“…The opening ( k op ) and closing ( k cl ) rate constants and/or equilibrium constant for base-pair opening ( K op = k op / k cl ) can be determined by measuring the exchange using an external catalyst. These experiments have been used to probe base-pair opening in various DNA duplexes [[3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]], DNAs containing modified base such as 5-flurourasil, N6-methyl adenine, or modified guanine [[19], [20], [21]], UV-induced photoadduct-containing DNA [22], IHF-complexed DNA [23], interstrand cross-linked DNA [24], i-motif structure formed by the complementary C-rich DNA [25,26], various RNAs [15,[27], [28], [29], [30], [31], [32], [33], [34], [35], [36]], peptide nucleic acids (PNAs) [37,38], and threose nucleic acid (TNA) [39]. NMR exchange and single molecule FRET experiments could be used to study the protonation/deprotonation of adenine bases and formation of A + ·C wobble pair [[40], [41], [42], [43], [44]].…”

NMR Analysis of Base‐Pair Opening Kinetics in DNA

“…NMR on the other hand provides a powerful tool to locally monitor the kinetics of each base pair opening under physiological conditions. The most common NMR approach to determine these local opening/closing rates relies on the 1 H NMR measurement of the proton exchange between water and the imino protons after magnetization transfer (Gueron, Kochoyan et al 1987, Gueron, Charretier et al 1990, Moe and Russu 1992, Folta-Stogniew and Russu 1996, Dhavan, Lapham et al 1999, Dornberger, Leijon et al 1999, Chen and Russu 2004, Every and Russu 2007, Every and Russu 2008, Lee, Lee et al 2009, Parker and Stivers 2010, Crenshaw, Wade et al 2011, Friedman, Jiang et al 2011, Every and Russu 2013, Szulik, Voehler et al 2013). Depending on the base pair stability, base pair opening occurs more or less frequently, allowing for the imino proton to exchange with water.…”