Thermodynamics of Stacking and of Self‐Association of the Dinucleoside Monophosphate m⁶₂A‐U from Proton NMR Chemical Shifts:

Abstract

Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate Nh-dimethyladenylyl(3'-5')uridine (m4A-U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from -5 "C to 105 "C. An internal temperature calibration was used. The effects of intermolecular self-association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of mgA-U at the molecular level. The simple two-state approach for both self-association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least-squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self-association and stacking. However, the two-state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions : (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules ; (b) a temperature-dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the leastsquares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are : AH; = -28.8 kJ rnol-', A S ; = -93 J mol-' K-'. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are : AH; = -35 kJ mol-' and AS2 = -95 J mol-' K-'.The question of whether short oligonucleotides are really suitable as models for longer regions in nucleic acids has been raised [l], but cannot be answered until many careful systematic studies are carried out with experimental probes at atomic resolution. The most potent approach is offered by nuclear magnetic resonance (NMR) measurements, supplemented with thermodynamic information from independent techniques such as circular dichroism (CD) and ultraviolet hypochromism, and guided by information on geometrical details by X-ray methods.The shortest nuclei acid fragments available for studies of these properties, the dinucleoside monophosphates or dimers (L-M) represent at the same time 3'-terminal and 5'-terminal fragments of RNA (or DNA); therefore; these fragments constitute a limiting case. Therefore, attempts at This paper is part 24 in the series Nucleic Acid Cotzstituents from this laboratory; part 23 appeared elsewhere [4].Ahhrcviurions. CD, circular dichroism; NMR, nuclear magnetic resonance; Me4NC1, tetramethylammonium chloride; m$A...

Sequence‐dependent structural variation in single‐helical DNA Proton NMR studies of d(T‐A‐T‐A) and d(A‐T‐A‐T) in aqueous solution

“…The 2 mM and 4 mM samples were recorded at several temperatures in the low-temperature range in order to measure the concentration dependence of the observed chemical shifts. Exact sample temperatures were determined from the H 2 H 0 residual peak chemical shift as was described previously [54].…”

“…In order to derive information concerning intramolecular base-base stacking from chemical shift data, a correction for concentration dependence of these data is called for. The differential concentration temperature profile (DCTP) method, worked out by Hartel et al [54] was used to quantify the concentrationdependent chemical shift changes versus temperature in a least-squares calculation. Computer simulations of the experimental chemical shift curves are shown in Fig.…”

“…Program ASSTAK [54]' was used to fit two-state intramolecular stacking thermodynamics to the concentration-independent chemical shift contribution. This procedure was carried out for the base and 1' proton signals of both compounds.…”

Conformational analysis of the octamer d(G‐G‐C‐C‐G‐G‐C‐C) in aqueous solution

“…Inspection of Fig.1A reveals cross peaks corresponding to intraresidue base@) -Hl'(n-I) and interresidue base(n)-Hl'(n) NOEs. The H6(4)-H1'(4) NOE is lost in Note that the conversion factor a for the 0-52°C temperature range given in [24] should read a = 177.6.…”

“…In order to study the chemical shifts as a function of temperature, a series of spectra was recorded at 500 MHz (0-40°C) and at 300 MHz (40-100°C) at 5°C intervals. Exact temperatures were determined from the 'H'HO residual peak chemical shift relative to Me4NCl as described earlier [24]. '…”