Topography of nucleic acid helices in solutions. The interaction specificities of optically active amino acid derivatives

The synthesis and interactions of the d- and l-enantiomers of the amino acid amide derivatives [Formula: see text] (I) and lysyl dipeptides [Formula: see text] (II) with poly rI.poly rC, poly rA.poly rU and calf thymus DNA is reported. The following results were found. (1) The degree of stabilization of the helices as measured by the T(m) (;melting' temperature) of the helix-coil transition was dependent on the nature of the amino acid. (2) For the poly rI.poly rC helix, the l-enantiomers of salts (I) and (II) stabilized more than the d-enantiomers. The same was true for calf thymus DNA in the presence of salts (II) and for poly rA.poly rU in the presence of salts (II) and the proline derivatives of salts (I). (3) As R increased in size and became more apolar, the amount of stabilization of the poly rI.poly rC helix in the presence of salts (I) decreased. On the other hand, the amount of stabilization increased with more polar substituents. An attempt was then made to determine whether the difference in stabilization of the double-stranded helices at the T(m) in the presence of the l- and d-enantiomers of salts (I) is due to the interaction with the helix, the random coil or both. A new method was developed for determining the binding of small ions to polyions that involves a competition between an insoluble polystyrene ion-exchange resin and the soluble polyion for the counterion. Dissociation constants are obtained for the complexes of single- and double-stranded helices with the salts (I). The results are illuminating and indicate that with certain helices, i.e. poly rA.poly rU, the interactions of salts (I) with the single strands may not be ignored. It is concluded that the high optical specificity found in Nature, i.e. d-ribose in nucleic acids and l-amino acids in proteins, cannot be attributed solely to monomer-polymer interactions described by Gabbay (1968).     

Topography of nucleic acid helices in solutions. 28. Evidence for a dynamic structure of DNA in solution

Proton magnetic resonance (pmr), ultraviolet absorption, induced circular dichroism (CD), and viscometric evidence is presented which show that reporter molecules $\text{1}$ and $\text{2}$ bind to DNA via an intercalation process. Preliminary kinetic studies show that the DNA·$\text{1}$ complex forms rapidly (i.e., <1 msec), whereas the DNA·$\text{2}$ complex forms at a considerably slower rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 100 msec}$). The kinetic results, and the steric requirements for intercalation of $\text{2}$ can be explained on the basis of a dynamic structure of DNA.     

Topography of nucleic acid helices in solutions. XXIV. Intercalation specificities of DNA and their possible role in the recognition process

Through the use of reporter molecules, I, it is shown that more than one type of intercalating site must exist in DNA. Although this finding is completely consistent with the DNA structure it has not yet been demonstrated. The significance of the presence of different intercalating sites in DNA may be of extreme importance in the recognition of nucleic acid-protein systems.     

Topography of nucleic acid helices in solutions. II. Structure of the double-stranded rA-rU, rI-rC, acid rA, and the triple-stranded rA-rU2 and rA-rI2 helices

Information concerning the structures of rA–rU, rA–rU₂ rI–rC, rA–rI₂, and acid rA helices in solutions is reported. Through the use of diquaternary ammonium salts of the general structure, \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}_1 {\rm R}_2 {\rm R}_3 \mathop {\rm N}\limits^ + ({\rm CH}_2 )n\mathop {\rm N}\limits^ + {\rm R}_1 {\rm R}_2 {\rm R}_3 \cdot 2{\rm Br}^ - $\end{document} (I), it is shown that (1) the distances between adjacent negatively charged oxygen atoms on the helix increases in the following order rA–rI₂ < rI–rC < rA–rU ? rA–rU₂; (2) the density of the helices increases in the order. rA–rI₂ < rA–rU < rA–rU₂ < rI–rC; (3) there is a large hydrophobia site in rA–rI₂ and possibly also in rA–rU, rA–rU₂, and rI–rC helices; (4) the results of the interactions between the salts of type I and the helices may be formulated in semi-quantitative terms by the use of two parameters, α, and β which are shown to be related to the charge separation and the density of the helices, respectively; (5) the studies in solutions compare favorably with the x-ray studies on the fibers; and (6) the acid rA helix differs significantly from the other helices by the fact that the electrostatic interstrand interactions between the negatively charged oxygen atom of a phosphate group and the positively charged 10-amino group of adenine contribute significantly to the stabilization of the helix, and thus it is found that the presence of the salts, I, leads to a significant destabilization of the acid rA helix.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells “proinhibitor” of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.     

Diacetylated derivatives of spermine and spermidine as novel promising tumor markers

N1,N12-diacetylspermine (DiAcSpm) and N1,N8-diacetylspermidine (DiAcSpd) are minor components of human urinary polyamine to which little attention has been paid until recently. HPLC analysis of urinary polyamines has revealed that the excretion of these diacetylpolyamines, in particular, into urine was frequently and markedly increased in association with every type of cancer so far examined. Remission was usually accompanied by recovery of urinary diacetylpolyamines to the normal level. DiAcSpm was more sensitive than CEA for detecting colorectal cancer patients, while DiAcSpd was highly specific for malignant conditions in that the excretion of the latter was scarcely elevated in cases of benign urogenital diseases. An ELISA procedure for rapid determination of DiAcSpm was developed to promote the clinical application of these new tumor markers, and subsequent studies indicated that DiAcSpm was elevated in 60% of colorectal cancer patients at early stages (stage 0 + I), whereas only 10% of these patients were CEA-positive. DiAcSpm may also be useful as a follow-up marker that is efficient for detecting recurrence and sensitive to changes in the clinical condition of patients. The evidence accumulated so far indicates that DiAcSpm and DiAcSpd are promising novel tumor markers. They deserve more intensive studies, including studies of their biochemistry and metabolism.     

Ring current shielding effects in nucleic acid double helices.

Values of ring current shielding in parts-per-million have been calculated for double helical nucleic acids in the A-RNA (RNA-11), A' -RNA (RNA-12) and B-DNA geometries. Atomic coordinates determined previously from x-ray diffraction of fibers were used to calculate the positions of protons relative to both nearest and second nearest neighboring bases, including those on the complementary strand. The magnitude of the diamagnetic shielding was then calculated for each aromatic ring. From these calculations tables were constructed for use in determining the shielding expected for carbon-bound and ring nitrogen-bound protons of any double helical nucleic acid sequence. The results are compared with available experimental data for several oligonucleotides and with previous ring current shielding calculations where differences of up to 0.4 ppm are found.     

Free energy of imperfect nucleic acid helices. 3. Small internal loops resulting from mismatches

Physical studies of enzymioally synthesized oligoribonucleotides of defined sequence are used to evaluate quantitatively the destabilizing influence of mismatched bases in a double helix. The series (A-)₄G(-C)_n(-U)₄, N = 1 to 6, exist as imperfect dimer helices when N is equal to or less than 4, and as monomolecular hairpin helices when N is 5 and 6. Internal loops become progressively more destabilizing as their size increases from 2 to 4 to 6 nucleotides resulting from 1, 2 and 3 consecutive mismatched base pairs. However, the stability of a helix will generally be greater if a given number of mismatched pairs occur consecutively rather than in isolation from one another.These data may be used for improved calculations of stability of RNA secondary structure, to estimate the frequency of structural fluctuations in a double helix and to assess the stability of modified polynucleotide helices. An unmodified double helix of one million randomly arranged base pairs should contain on the time average approximately 10 G.C and 500 A.U pairs in non-hydrogen bonded, unstacked conformations at 25 °C. Our estimate of the effect of mismatching on T_m values of high polymers is less precise because of the long temperature extrapolation required. However, we estimate that DNA or RNA treated with mutagens which interrupt up to 20% of the nucleotide pairs will show a drop of about 1.2 deg. C in melting temperature with each unit per cent of modification.     

Effects of spermine and spermidine on the inorganic pyrophosphatase of Streptococcus faecalis. Interactions between polyamines and inorganic pyrophosphate.

We have shown a dual role for Mg2+ in the hydrolysis of PPi catalysed by inorganic pyrophosphatase (PPase; EC 3.6.1.1) of Streptococcus faecalis; Mg2+ is necessary for the formation of the substrates, Mg1PPi2- and Mg2PPi0, and it also acts as an allosteric activator [Lahti + Jokinen (1985) Biochemistry 24, 3526-3530]. No activity can be observed with S. faecalis PPase in the absence of bivalent cations, which indicates that free PPi cannot serve as a substrate for this enzyme. However, significant activities were observed in the presence of spermine and spermidine, even though no bivalent cations were present. It was shown by particle-induced gamma-ray emission and particle-induced X-ray-emission analysis that the polyamines used were not contaminated with Mg2+ or any other bivalent cations that could support PPase activity. Hence it is obvious that polyamines are able to form a complex with PPi that serves as a substrate for PPase. The apparent stability constants for the 1:1 adducts of spermine and spermidine were estimated by a resin competition method. The values obtained at pH 7.5 were 2.7 X 10(3) M-1 and 6.4 X 10(2) M-1 respectively. Kinetic results further suggested that polyamines can also substitute for Mg2+ as an activator in vitro. The physiological significance of these polyamine effects were discussed.     

Interaction of the ruthenium red cation with nucleic acid double helices

The hexapositive complex cation ruthenium red very effectively stabilizes DNA and RNA double helices against thermal denaturation. In the presence of nucleic acid helices, this symmetric cation acquires an extrinsic CD spectrum near the wavelength of the dye's maximum absorbance. Competition experiments with single-stranded polyd(T) show this induced CD to be the result of selective binding to helical sites. The preferential affinity of ruthenium red for double helical binding sites is so great that it brings about biphasic absorbance- temperature profiles of polyd(A-T) at low [cation]: [polynucleotide phosphate]. The visible CD signal and fraction of helix melting at the upper transition increases with ruthenium red concentration until approximate charge neutrality is reached. These interactions, which have been studied in detail with the poly(U-U) helix as well as polyd(A-T), are likely largely electrostatic, since sufficient [NaCl] eliminates the bipliasic melting of polyd(A-T), renders the ultraviolet absorbance of poly (U) insensitive to ruthenium red, and abolishes the induced CD effects. The bipliasic melting of polyd(A-T) at intermediate [dye] is attributed to saturation of remaining double helical segments by cation migration from newly melted regions- Furthermore, virtually no change was observed in the induced CD upon melting through the first transition, whereas the effect is destroyed upon inciting through the second transition. A quantitative treatment of the data is used to obtain binding site size and association constant for the complex. The induced effect may prove useful in the exploration of exposed nucleic acid helical structure in such complex particles as nucleosomes or ribosomes.     

Parallel nucleic acid helices with hoogsteen base pairing: Symmetry and structure

Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2′-endo region and the phosphodiester conformations are (trans, gauche^?), and the RNA helices have sugars in the C3′-endo region and the phosphodiester conformations are (gauche^?, gauche^?). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix. © 1994 John Wiley & Sons, Inc.     

Free energy of imperfect nucleic acid helices. II. Small hairpin loops

Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap)₄G(pC) _N(pU)₄, N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair.Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers.We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m-Na⁺ concentration). As an example, we apply the rules to an isolated fragment of tRNA^Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.