Ignacio Tinoco

PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

The Watson-Crick double helix of DNA was first revealed in 1953. Since then a wide range of physical chemical methods have been applied to DNA and to its more versatile relative RNA to determine their structures and functions. My major goal is to predict the folded structure of any RNA from its sequence. We have used bulk and single-molecule measurements of thermodynamics and kinetics, plus various spectroscopic methods (UV absorption, optical rotation, circular dichroism, circular intensity differential scattering, fluorescence, NMR) to approach this goal.

Stability of ribonucleic acid double-stranded helices

Abstract The hypochromicity, as a function of temperature for 19 oligoribonucleotides capable of forming perfectly base-paired double helices, is used to extract thermodynamic parameters of helix formation. The data are analyzed by an all or none model of helix melting which permits assignment of Δ G , Δ H , and Δ S of formation to each of the ten possible Watson-Crick base-paired nearest-neighbor sequences. Helix stability is found to have a striking dependence on sequence, and formulae are provided to predict the T m of any RNA double helix of known sequence.

Absorbance melting curves of RNA

Publisher Summary This chapter discusses the experimental methods needed to acquire a melting curve and the analysis and interpretation of the data. Any standard commercial UV spectrophotometer can be equipped to measure melting curves. A useful instrument is a single-beam Gilford (Oberlin, OH) spectrophotometer (Model 2530) with an automated reference compensator that allows melting curves to be obtained on three separate samples simultaneously. One major advantage of using UV spectroscopy is the high sensitivity of the method. Normally, the absorbance of the sample used should be between 0.2 and 2.0. Sample preparation for UV melting studies is straightforward. The RNA stock solution is prepared by dialysis against the desired buffer and different concentrations are made by dilution. The high salt concentration is chosen to minimize electrostatic repulsion between strands and to avoid divalent ions, which catalyze hydrolysis of RNA and favor triple-strand formation. This solvent provides a standard condition for measuring melting curves and for comparing results with previously published data.

Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine·guanine base pairs

Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.

Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies

Atomic force microscopes and optical tweezers are widely used to probe the mechanical properties of individual molecules and molecular interactions, by exerting mechanical forces that induce transitions such as unfolding or dissociation. These transitions often occur under nonequilibrium conditions and are associated with hysteresis effects—features usually taken to preclude the extraction of equilibrium information from the experimental data. But fluctuation theorems allow us to relate the work along nonequilibrium trajectories to thermodynamic free-energy differences. They have been shown to be applicable to single-molecule force measurements and have already provided information on the folding free energy of a RNA hairpin. Here we show that the Crooks fluctuation theorem can be used to determine folding free energies for folding and unfolding processes occurring in weak as well as strong nonequilibrium regimes, thereby providing a test of its validity under such conditions. We use optical tweezers to measure repeatedly the mechanical work associated with the unfolding and refolding of a small RNA hairpin and an RNA three-helix junction. The resultant work distributions are then analysed according to the theorem and allow us to determine the difference in folding free energy between an RNA molecule and a mutant differing only by one base pair, and the thermodynamic stabilizing effect of magnesium ions on the RNA structure.

The stability of helical polynucleotides: base contributions.

Estimates have been made of the main contributions of the heterocyclic bases of DNA to the free energies of the two configurations in solution, the two-stranded helix and the single-strand random coil. The magnitude and directions of the dipole moments of the adenine, thymine, guanine and cytosine base groups have been calculated by a semi-empirical molecular orbital treatment. Dipole—dipole, dipole—induced-dipole and London force interactions among the bases in the helix are large, and make the free energy of the helix depend on the base composition and sequence. The helix stability (helix negative free energy) is proportional to the guanine + cytosine content. A comparison of calculated base-pair interactions with the nearest-neighbor base frequency data of Josse, Kaiser & Kornberg (1961) suggests that the base sequence in natural DNA may be influenced by the free energy. The free energy of the coil is difficult to estimate ; it tends to be more negative from base—solvent interactions for a greater guanine—cytosine content. The choice of solvent determines the magnitude of these interactions and thus determines how the melting temperature of the helix will depend on the base composition. In a solvent such as water, in which significant base—base interactions exist in the coil, the coil free energy depends on the base sequence and London forces may cause stacking of the bases into ordered arrays. The hydrogen bond energy of the bases and the strain energy in the helix probably contribute little to the enthalpy change of the helix—coil transition, although they ensure specific base-pairing in the helix. The net contribution of configurational and solvent entropy changes to the free energy change of the helix—coil transition is also probably small.

Following translation by single ribosomes one codon at a time

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,D

Thermodynamic parameters for double strand formation have been measured for the sixteen double helices of the sequence dCA3XA3G.dCT3YT3G, with each of the bases A, C, G and T at the positions labelled X and Y. The results are analyzed in terms of nearest-neighbors and are compared with thermodynamic parameters for RNA secondary structure. At room temperature the sequence (Formula: see text) is more stable than (Formula: see text) and is similar in stability to (Formula: see text) and (Formula: see text) are least stable. At higher temperatures the sequences containing a G.C base pair become more stable than those containing only A.T. All molecules containing mismatches are destabilized with respect to those with only Watson-Crick pairing, but there is a wide range of destabilization. At room temperature the most stable mismatches are those containing guanine (G.T, G.G, G.A); the least stable contain cytosine (C.A, C.C). At higher temperatures pyrimidine-pyrimidine mismatches become the least stable.

RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication, and therefore a potentially important drug target. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 ± 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 ± 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors.

Optical Rotation of Oriented Helices. III. Calculation of the Rotatory Dispersion and Circular Dichroism of the Alpha‐ and 310‐Helix

We have carried out a calculation of the optical rotatory dispersion (ORD) of α‐and 310‐helices which is essentially complete within the limitations of the Kirkwood approximation and its extensions. This treatment predicts that right‐handed helices will exhibit three rotatory bands at wavelengths greater than 190 mμ. The sign and position of these predicted bands are in good agreement with experiment. The predicted rotational strengths do not agree so well, but are in sufficiently good accord to provide strong support to the assignment of the longest‐wavelength band to the amide nπ* transition, and the shortest‐wavelength band thus far observed to a composite band due both to the perpendicular‐polarized exciton component and to the intrinsic rotational strength of the NV 1 transition. Calculation of the ORD and CD (circular dichroism) of the α‐helix from the calculated rotational strengths gives results in fair agreement with experiment, especially when the nπ* rotational strength is adjusted to agree with that observed in CD experiments. An analysis of the calculated ORD curves according to the Moffitt—Yang and Shechter—Blout procedures leads to values for the parameters in these equations which agree well with experiment in the case of the α‐helix. A similar treatment of the 310‐helix indicates that this helix can be distinguished from the α‐helix by: (a) appreciably more positive rotations in the visible region, (b) appreciably larger absolute values of b 0, A(α, ρ)225 and A(α, ρ)193. The dependence of various rotatory parameters on the length of the helix has been studied. The dispersion parameters b 0, A(α, ρ)225 and A(α, ρ)193, as well as the depth of the 233 mμ trough appear to be the least sensitive to chain length. The magnitude of the maximum at 198 mμ, however, seems quite chain‐length dependent, and therefore is probably not suitable as a measure of helix content.