The Conformation of Single Stranded Oligonucleotides and of Oligonucleotide-Oligopeptide Complexes from their Rotation Relaxation in the Nanosecond Time Range

Abstract

The conformation of single stranded oligonucleotides is analysed by measurements of their rotation time constants. The oligomers are aligned to some degree by short electric field pulses; after pulse termination the transition to a random orientation is followed by measurements of the linear dichroism. An efficient deconvolution procedure is developed for evaluation of the experimental data obtained in the ns-time range. The increase of rotation time constants observed for chain lengths in the range from 14 to 22 residues are interpreted according to a weakly bending rod model providing a persistence length and a Stokes' diameter. The Stokes' diameters obtained for ribo- and deoxyriboadenylates are about 13Å, in approximate agreement with the expectation for a single stranded helix. The persistence length L = 53Å corresponding to ～16 nucleotide residues found for riboadenylates at 2°C appears to reflect relatively strong stacking interactions at this temperature. However, a comparison with the average length of stacked residues evaluated from available thermodynamic parameters of base stacking indicate that unstacked residues are not completely flexible. Apparently the ribose-phosphate chain provides an essential contribution to the stiffness of oligomers and polymers, even when the bases are unstacked. Addition of 100μM Mg²⁺ leads to an increase of the persistence length to 88Å. Corresponding measurements with deoxyriboadenylates show a slightly lower value of the persistence length than that found for riboadenylates. Addition of LysTrpLys and LysTyrLys to A(pA)₁₉ leads to an increase of the rotation time constant, which corresponds approximately to a length increment by one residue per bound peptide. Since controls performed with LysLeuLys do not show any similar effect, the increase of the time constants induced by LysTrpLys and LysTyrLys is attributed to intercalation of the aromatic amino acids.     

The reaction of aromatic peptides with double helical DNA. Quantitative characterisation of a two step reaction scheme

The binding of LysTrpLys and LysTyrLys to calf thymus DNA has been investigated by the field jump method using fluorescence detection. Two separate relaxation processes, clearly distinguished on the time scale and by opposite ampli- tudes, are observed for the binding of LysTrpLys to DNA with ~ 30000 base pairs. The concentration dependence of the relaxation time constants demonstrates a mechanism with a bimolecular step followed by a slow intramolecular transition with a forward rate of 6.4 X 103 s^?1 and an equilibrium constant of 11. Measurements at various degrees of peptide binding demonstrate that the binding mechanism associated with low binding rates is restricted to a rather low number of binding sites (roughly one site in 15 base pairs). The binding of LysTyrLys to the same DNA is not associated with relaxation pro- cesses of opposite amplitudes; nevertheless two processes could be identified and assigned to a two step mechanism corre- sponding to that observed in the case of LysTrpLys. In the presence of sonicated DNA both peptides show a single relaxa- tion process with characteristics similar to those observed for the slow process in the binding to high molecular DNA. The data indicate that the intramolecular step is faster for low than for high molecular DNA. These results suggest an assignment of the intramolecular step to an insertion of the aromatic residues into the DNA associated with bending of the helix. The increase in the rate of the intramolecular step with decreasing chain length of the DNA may then be explained by a higher flexibility of the double helix at lower chain lengths.     

Structure and dynamics of a tryptophanepeptide-polynucleotide complex

The binding of LysTrpLys to single stranded poly(A) was studied by measurements of fluorescence, UV-absorbance, electrodichroism and field jump relaxation. The van't Hoff enthalpy determined at constant degree of peptide protonation is -3.5 kcal/mol (delta S = 9 e.u.). The electrodichroism of bound tryptophane residues is negative; its absolute value decreases with increasing degree of binding theta. The magnitude of the dichroism at low theta indicates a preferential orientation of the tryptophane residues in the plane of the adenine bases, suggesting stacking of Trp with adenine bases. The overall degree of orientation decreases, however, to virtually zero at high theta. Relaxation measurements by low theta demonstrate the existence of two steps in the binding reaction of LysTrpLys to poly(A): a fast bimolecular step controlled by diffusion is followed by a slow intramolecular conversion with a forward rate of 1.5 x 10(5) s-1 and a backward rate of 2.7 x 10(3) s-1. The forward rate is close to that expected for an insertion reaction into stacked poly(A), yet the corresponding stability constant (approximately 55) is unexpectedly high.     

Cooperative interactions in single-strand oligomers of adenylic acid

Optical rotatory dispersion measurements were made on the oligonucleotides (pA)₂, (pA)₄, and (pA)₆ at neutral pH over the temperature range 5–85°C., and compared to similar data for polyriboadenylic acid. The data were interpreted in terms of a temperature-dependent stacking of the bases in the single-strand oligomers, with very little dependence of the degree of stacking on the chain length. These results can be explained by a theory of cooperative stacking. The degrees of freedom available per residue are rotations about the five backbone covalent bonds and the bond connecting the base to the ribose ring. To nucleate a stacking interaction between neighboring bases the backbone sequence must be ordered as must be the two bases. For this stack to grow by one base a backbone sequence must again be ordered, but only one additional base must be ordered. Thus, the degree of freedom of the base with respect to the ribose ring determines the extent of the cooperative effect and hence the effect of chain length. A matrix formulation of the partition function is presented which incorporates this cooperative nature of the interaction and is shown to be in fair agreement with the data. The entropy of ordering a base with respect to the ribose ring is found to be 0.68 e.u., which suggests that the purine has a torsional oscillation when unstacked, but does not have several isoenergetic positions of internal rotation available. The enthalpy of stacking is found to be ?6.5 kcal./mole. A model involving neighbor and next-nearest neighbor interactions could also account for the data. For all practical purposes, the stacking interactions of successive residues can be treated as independent, i.e., the state of one residue is essentially independent of the state of stacking of its neighbors.     

Structure of the Tet repressor and Tet repressor-operator complexes in solution from electrooptical measurements and hydrodynamic simulations

The Tet repressor protein and tet operator DNA fragments and their complexes have been analyzed by electrooptical procedures. The protein shows a positive linear dichroism at 280 nm, a negative linear dichroism at 248 nm, and a strong permanent dipole moment of 3.5 X 10(-27) C m, which is independent of the salt concentration within experimental accuracy. Its rotation time constant of 40 ns indicates an elongated structure, which is consistent with a prolate ellipsoid of 100 A for the long axis and 40 A for the short axis. The time constant can also be fitted by a cylinder of length 78 A and diameter 37 A, which is consistent with nuclease protection data reported on repressor-operator complexes, if the cylinder axis is aligned parallel to the DNA axis. Addition of tetracycline induces changes of the limit dichroism but very little change of the rotation time constant. The rotation time constants observed for the operator DNA fragments show some deviations from the values expected from their contour length; however, these deviations remain relatively small. Formation of repressor-operator complexes leads to some increase of the DNA rotation time constants. Simulations by bead models demonstrate that these time constants can be explained without any major change of the hydrodynamic dimension of the components. The data for the complexes are fitted by bead models with smooth bending of the DNA corresponding to a radius of curvature of 500 A, but at the given accuracy, we cannot rule out that the DNA in the complex remains straight or is bent to a smaller radius of approximately 400 A. Thus, binding of the Tet repressor, which is a helix-turn-helix protein as judged from its sequence, to its operator seems to induce minor bending but does not induce strong bending of the DNA double helix.     

The mechanism of DNA repair by uracil-DNA glycosylase: studies using nucleotide analogues

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.     

The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues

Abstract

2′,4′-Dideoxy-4′-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by T_m and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4′-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4′-oxygen in recognition and cleavage of dU residues in DNA.     

Thermodynamics and kinetics of the helix-coil transition of oligomers containing GC base pairs

Absorbance-temperature profiles have been determined for the following self-complementary oligonucleotides or equimolar paris of complementary oligonucleotides containing GC base pairs: A₂GCU₂, A₃GCU₃, A₄GCU₄, A₆CG + CGU₆, A₈CG + CGU₈, A₄G₂ + C₂U₄, A₅G₂ + C₂U₅, A₄G₃ + C₃U₄, and A₅G₃ + C₃U₅. In all cases cooperative melting transitions indicate double-helix formation. As was found previously, the stability of GC containing oligomer helices is much higher than that of AU helices of corresponding length. Moreover, helices with the same length and base composition but different sequences also have quite different stabilites. The melting curves were andlyzed using a zipper model and the thermodynamic parameters for the AU pairs determined previously. The effect of single-strand stacking was considered separately. According to this model, the formation of a GC pair from unstacked single strands is associated with an ethalpy change of ?15 kcal/mole. Due to the high degree of single-strand stacking at room temperature the enthalpy change for the formation of GC pairs from unstacked single strands is only ?5 to ?6 kcal/mole. (The corresponding parameters for AU pairs are ?10.7 kcal/mole and ?5 to ?6 kcal/mole.) The sequence dependence of helix stability seems to be primarily entropic since no differences in ΔH were seen among the sequence isomers. The kinetics of helix formation was investigated for the same molecules using the temperature jump technique. Recombination of strands is second order with rate constants in the range of 10⁵ to 10⁷M^?1 sec^?1 depending on the chain length and the nucleotide sequence. Within a series of oligomers of a given type, the rates of recombination decrease with increasing chain length. Oligomers with the sequence A_nGCU_n recombine six to eight times slower than the other oligomers of corresponding chain length. The experimental enthalpies of activation of 6 to 9 kcal/mole suggest a nucleation length of one or two GC base pairs. The helix dissociation process has rate constants between 0.5 and 500 sec^?1 and enthalpies of activation of 25 to 50 kcal/mole. An increase of chain length within a given nucleotide series leads to decreased rates of dissociation and increased enthalpies of activation. An investigation of the effect of ionic strength on A_nGCU_n helix formation showed that the rates of recombination increase considerably with increased ionic strength.     

Structure and dynamics of double helices in solution: modes of DNA bending

The long range structure of DNA restriction fragments has been analysed by electro-optical measurements. The overall rotation time constants observed in a low salt buffer with monovalent ions is shown to decrease upon addition of Mg2+ or spermine. Since the circular dichroism and also the limiting value of the linear dichroism remain almost constant under these conditions, the effect is attributed to a change of the long range structure. According to a weakly bending rod model, the persistence length decreases from about 600 A in the absence of Mg2+ or spermine to about 350 A in the presence of these ions. The persistence length measured in the presence of Mg2+ is almost independent of temperature in the range of 10 to 40 degrees C. The nature of DNA bending is analysed by measurements of bending amplitudes and time constants from dichroism decay curves. The observed absence of changes in the bending amplitudes upon addition of Mg2+ or spermine, even though addition induces changes of the persistence length by a factor of 2, is hardly consistent with simple thermal bending. The combined results, including the remarkably small temperature dependence of persistence length and bending amplitude, can be explained by the existence of two bending effects: inherent curvature of DNA dominates at low temperature, whereas thermal bending prevails at high temperature. Analysis of bending amplitudes from dichroism decay curves according to an arc model provides an approximate measure for the degree of bending in restriction fragments. The model is consistent with the observed chain length dependence of bending amplitudes and provides an approximate curvature corresponding to a radius of about 400 A. Thus the curvature observed in restriction fragments is similar to that observed for high molecular DNA condensed into toroids by addition of ions like spermine. Particularly strong bending of DNA is induced by [Co(NH3)6]3+, indicated by an apparent persistence length of 200 A and an increased bending amplitude together with a reduced limit value of the linear dichroism. This effect is attributed to the high charge density of this ion and potential site binding.     

Molecular states in single-stranded adenylate chains by relaxation analysis

The single-strand helix-coil transition in various oligo- and polyadenylates is characterized by means of an improved cable temperature-jump technique. In all the polymers studied {poly(rA), poly(dA), poly[A(m^2′)] and poly[A(e^2′)]} helix-coil relaxation is observed in the time range from 30 to 1000 nsec. Relaxation-time constants observed at wavelengths λ<280 nm (τ_α) are different from those found at λ >280 nm (τβ), indicating the presence of more than two conformational states. The time constants τ_α increase in the series poly(dA), poly[A(m^2′)], constants τ_β/τ_α is approximately 2.5, except in poly(dA) where τ_β/τ_α ≈ 9. Relaxation measurements with r(A)_n- oligomers show a decrease in conformational mobility with increasing chain length. The relaxation curves also demonstrate that “internal” residues have lower reaction rates than residues at the ends of the oligomer chain. Measurement in D₂O reveal a solvent isotope effect for τ_α of +87% for poly(rA), and of +53% for poly(dA), whereas no isotope effect is found in τ_β. The absence of “slow” relaxation processes in the model compound 9,9′ -trimethylenebisadenine shows that the relatively low rate of the single-strand helix-coil transitions is due to the coupling of base stacking with the folding of the sugar–phosphate chain. The absence of a seprate relaxation process (corresponding to τ_β) in 9,9′-trimethylenebisadenine, as well as in the dinucleotides ApC and CpA, suggests that this relaxation process is dependent upon the presence of both the sugar–phosphate chain and of adjacent adenine bases. The experimental data provide evidence that there is more than one ordered conformation in various single-stranded oligo- and polyadenylates and that the transition between these conformations is influenced by the sugar conformation.     

Dynamics of the RNA hairpin GNRA tetraloop

The dynamics of RNA hairpin tetraloops of the GNRA type [sequence G- any ribonucleotide (N)-purine (R)-A] was analyzed by fluorescence spectroscopy and by fluorescence-detected temperature-jump relaxation, using RNA oligomers with 2-aminopurine (2AP) substituted in two different positions of the loop sequence, Gp2APpApA (HP1) and GpAp2APpA (HP2), as indicator. The fluorescence of HP1 is much higher than that of HP2, indicating a lower degree of 2AP-stacking in HP1. Addition of Mg(2+) or Ca(2+) ions leads to an increase of fluorescence in HP1, whereas a decrease of fluorescence is observed in HP2. In both cases at least two ion-binding equilibria are required to fit titration data. T-jump experiments using fluorescence detection show a relaxation process with a time constant of 22 micros for HP1, whereas two relaxation processes with time constants 5 and 41 micros, are found for HP2. These results clearly demonstrate the existence of more than the single conformation state detected by NMR analysis. The T-jump amplitudes decrease with increasing bivalent ion concentration, indicating that one of the states is favored in the presence of bivalent ions. The loop relaxation processes are slower than standard stacking processes, probably because of activation barriers imposed by a restricted mobility of loop residues, and are assigned to a stacking rearrangement, probably between the 5' and the 3'-side. A similar process has been observed previously for the anticodon loop of tRNA(Phe). The rate constants of the transition are in the range of 10(4) s(-1) in the case of HP1. The data demonstrate the existence of structures that are not resolved by standard NMR because of fast exchange and are not found by X-ray analysis because of restrictions by crystal packing.     

A refined calculation of the solution dimensions of the complex between gene 32 protein and single stranded DNA based on estimates of the bending persistence length

The rotation diffusion coefficient of a complex of GP32, the single stranded DNA binding protein of the bacteriophage T4, with a single stranded DNA fragment with about 270 bases was determined to obtain further information on the flexibility of this particle. The rotation diffusion of these molecules is used as a sensitive measure of the flexibility of different DNA protein complexes. Using the theory of Hagerman and Zimm (Biopolymers 20, 1481 (1981)) and assuming a bending persistence length of about 35 nanometer it can be shown that the axial increment for GP32 complexes with single stranded DNA is close to 0.5 nm per base. The value for the bending persistence length is in agreement with values found for much larger DNA protein complexes using light scattering experiments. This value for the persistence length also implies that the complex is thin. The radius is estimated to be around 1.7 nm, which shows a moderate degree of hydration. With this set of parameters we can describe all the hydrodynamic experiments on GP32 complexes from 76 to more than 7000 bases obtained using electric birefringence, quasi-elastic light scattering and sedimentation experiments performed in our group over the last few years.     

Structure of the complex between lac repressor headpiece and operator DNA from measurements of the orientation relaxation and the electric dichroism.

The complex between lac repressor headpiece and short rodlike DNA fragments containing the lac operator sequence is characterised by measurements of the rotation diffusion. Using the method of electric dichroism we measure the rotation relaxation and determine changes in the length of the DNA upon ligand binding with high accuracy. According to these measurements any change in the length of the operator DNA upon binding of the first two headpiece molecules remains below 1A; the electric dichroism also remains virtually unchanged. At high degrees of (unspecific) binding we observe an increase in the rotation relaxation time, which is attributed to an increase of the apparent mean radius of the complex. As a control of our procedure for the determination of length changes we use the intercalation of ethidium bromide and arrive at an increase of the DNA length per bound ethidium of 3.2A (at 3.4A rise per base pair). The results obtained for the headpiece operator complex are not consistent with models assuming large changes of the DNA structure or intercalation of tyrosine residues.     

Spectroscopic properties of pyrene-containing DNA mimics

DNA mimics containing non-nucleosidic pyrene building blocks are described. The modified oligomers form stable hybrids, although a slight reduction in hybrid stability is observed in comparison to the unmodified DNA duplex. The nature of the interaction between the pyrene residues in single and double stranded oligomers is analyzed spectroscopically. Intra- and interstrand stacking interactions of pyrenes are monitored by UV-absorbance as well as fluorescence spectroscopy. Excimer formation is observed in both single and double strands. In general, intrastrand excimers show fluorescence emission at shorter wavelengths (approx. 5-10 nm) than excimers formed by interstrand interactions. The existence of two different forms of excimers (intra- vs. interstrand) is also revealed in temperature dependent UV-absorbance spectra.     

Anti-cooperative interactions in single-strand oligomers of deoxyriboadenylic acid

Optical rotatory dispersion measurements were made on the deoxyribo nucleotides d(pA)₂, d(pA)₄, d(pA)₆ and poly(deoxyriboadenylic acid) at neutral pH over the temperature range 5–80°C. and were compared to similar data for the analogous oligoriboadenylic acids. The data were interpreted in terms of a temperature-dependent stacking of the bases in the single-strand deoxyribo oligomers. The thermal transition curves show an inverted chain-length dependence compared to the ribo oligomer curves. These results are explained by a theory of anti-cooperative interaction, where the nucleation parameter σ is >1. The theory, based on a one-dimensional Ising model involving both attractive nearest-neighbor and repulsive next-nearest-neighbor interactions, predicts the inverse chain length dependence and agrees rather well with the experimental data. At and above the transition temperature, the deoxyribo polymer is seen to consist of isolated stacked base pairs separated by at least one unit of random coil, there being only a very small probability for the existence of sequences of stacked residues longer than one. The partition function is seen to undergo an irregular behavior as a function of chain length because of the anti-cooperative phenomenon. It is necessary to use an enthalpy of stacking of ?5.0 kcal./mole in order to fit the experimental data with the theory. This value, 1.5 kcal./mole more positive than the ΔH found for the ribo oligomers, is reasonable, since the 2′ hydroxyl group would be expected to stabilize the stacking interaction in the ribo oligomers. Various kinds of distribution functions are calculated and plotted graphically for this theoretical model. A physical rationale is presented for the use of a repulsive next-nearest-neighbor term in this theory for the deoxyribo oligomers.     

Specific recognition by the tripeptide lysyl-tryptophyl-alpha-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558-563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by trans Pt are required to observe a change in stacking efficiency. In contrast modification by cis Pt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cis Pt.     

Electric, optical and hydrodynamic parameters of lac repressor from measurements of the electric dichroism. High permanent dipole moment associated with the protein

Lac repressor and its tryptic core have been investigated by electro-optical methods. The reduced dichroism measured as a function of the electric field strength is not consistent with an induced dipole, but indicates the existence of a strong permanent dipole moment (approximately 4 X 10(-27) C m) for the holo-repressor, which is almost independent of ion concentration and pH. A dominant contribution of a permanent dipole is also demonstrated by the shape of the dichroism rise curve. The experimental data are not consistent with a counterion polarization phenomenon and also do not indicate a major contribution from proton fluctuations. Probably the nature of the dipole is similar to that found for compounds with a tetrahedral substitution by angular residues. Other potential models involve large conformational fluctuations or inherent asymmetry of the lac repressor. Rotation time constants obtained from the dichroism decay are not consistent with a spherical shape, for either the holo- or core repressor. A simple interpretation of the data by prolate ellipsoids suggests a short diameter of 6 nm for both holo- and core repressor and long diameters of 14 and 12 nm for holo- and core repressor, respectively. Addition of the inducer isopropyl-beta-D-thiogalactopyranoside leads to a change of the limit dichroism, but does not affect the rotation time constants within experimental accuracy.     

Enhanced stiffness of silk‐like fibers by loop formation in the corona leads to stronger gels

We study the self‐assembly of protein polymers consisting of a silk‐like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C‐terminal end. The silk blocks self‐assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self‐consistent‐field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk‐like blocks in the core, resulting in a more rigid fiber.     

Duplex structure formation between oligo(dA)''s and oligo(dT)''s generated by thymine-specific interaction with netropsin.

The formation of oligomeric duplex molecules in the presence of the antibiotic netropsin in the series p(dA)n-p(dT)n is demonstrated using low-temperature CD measurements. Addition of Netropsin to mixtures of oligomers generates the same type of CD spectra as observed for poly(dA)-poly(dT) and maintains the duplex structure at temperatures at which base pairing of free oligomers is thermodynamically unstable. The shortest chain length forming a netropsin complex by thymine-specific interaction with the oligopeptide is represented by p(dA)4-p(dt)4. Studies with sequence isomers show that adjacent thymine residues strongly favour the complex formation with the oligopeptide.