Structure and dynamics of two elastin-like polypentapeptides studied by NMR spectroscopy

The structure and dynamics of two synthetic elastin-like polypentapeptides, poly(G(1)V(1)G(2)V(2)P) and poly(AV(1)GV(2)P), were studied in D(2)O and H(2)O at various temperatures by using (1)H, (2)H,(13)C, and (15)N NMR spectra, relaxations, and PGSE self-diffusivity measurement. Signal assignments were made using COSY, NOESY, HXCORR, HSQC, HMBC, and SSLR INEPT techniques. Temperature-induced conformation changes were studied using (3)J(NHCH) couplings, NOESY connectivity, chemical shifts, and signal intensities. Hydrodynamic radii were derived from self-diffusion coefficients measured by the pulsed-gradient spin-echo (PGSE) method. Selective hydration (hydrophilic or hydrophobic) was explored using NOESY and ROESY spectral methods and longitudinal and transverse (1)H relaxation of HOD and quadrupolar (2)H relaxation of D(2)O. Four different physical states were discerned in different temperature regions for both polymers: state I of a rather extended, statistically shaped and fully hydrated polymer below the critical temperature (approximately 299-300 K); state II, a relatively coiled and globular but disordered preaggregation state, developing in a rather narrow region, 300-303 K, in the case of poly(AV(1)GV(2)P) and in a broader region, overlapping with the next one, in poly(G(1)V(1)G(2)V(2)P); state III, a tightly coiled, more compact state in the region 303-313 K; and, finally, state IV, an aggregated (and eventually flocculating and sedimenting) state beyond 313 K. States II-IV coexist in varying proportions in the whole temperature range above 299 K. A structure characterized by a beta-turn stabilized by H-bonding between the Ala carbonyl and Val(2) NH groups of poly(AV(1)GV(2)P) was detected by NOESY just above the transition temperature. States II and III are progressively more stripped of their hydration sheath but retain some molecules of water confined and relatively immobilized in their coils.     

Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

(1)H NMR study of the states of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The spin-spin relaxation times, T(2), of hydrated samples of poly(hydroxymethyl methacrylate), PHEMA, poly(tetrahydrofurfuryl methacrylate), PTHFMA, and the corresponding HEMA-THFMA copolymers have been examined to probe the states of the imbibed water in these polymers. The decay in the transverse magnetization of water in fully hydrated samples of PHEMA, PTHFMA, and copolymers of HEMA and THFMA was described by a multiexponential function. The short component of T(2) was interpreted as water molecules that were strongly interacting with the polymer chains. The intermediate component of T(2) was assigned to water residing in the porous structure of the samples. The long component of T(2) was believed to arise from water residing in the remnants of cracks formed in the polymer network during water sorption.     

Raman spectroscopy of secondary structure of elastinlike polymer poly(GVGVP)

Raman spectra of the elastinlike polypentapeptide poly(GVGVP) were measured in H(2)O and D(2)O as solutions and, after increasing the temperature, as suspensions and sediments. In addition, spectra of the polypentapeptide in the solutions of increasing concentration and in the solid state were also investigated by gradually evaporating the water. Significant changes in band frequencies, intensities, and shapes were found for selected Raman bands in the measured spectra, particularly for the C-H stretching, the glycine CH(2) wagging, and some amide vibrations. The C-H stretching vibrations are influenced predominantly by the presence of water, the glycine CH(2) wagging vibrations are associated with conformational transitions. Three possible types of poly(GVGVP)s in the presence of water were indicated: polymer chains in a relatively extended state in the solution, a beta-spiral structure in the suspension, and irregularly bent chains in the sediment.     

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of ²H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of ³¹P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.     

In vitro correction of antigen-induced immune suppression: effects of poly(A) poly(U) and prostaglandin E

Incubation of SJL or DBA/1 mouse spleen cells with poly(lTyr, lGlu)-polylPro—polylLys, (T, G)-Pro—L in vitro reduced the immune response potential of the cells to this immunogen as tested by adoptive transfer into irradiated, syngeneic recipients, followed by immunization with (T, G)-Pro—L in complete Freund's adjuvant. This reduction in immunocompetence was antigen-specific, since incubation with another antigen (rabbit immunoglobulin G) did not result in a suppression of responsiveness of the cells to subsequent in vivo immunization with (T, G)-Pro—L. Incubation of the spleen cell-(T, G)-Pro—L mixture in the presence of either prostaglandin E₁(PGE₁) or polyadenylic-polyuridylic acid (poly(A)·poly (U)) restored the immune response potential to the normal level. Incubation of (T, G)-Pro—L with spleen cells had no effect on cyclic AMP accumulation, whereas incubation of PGE₁ with the cells stimulated cyclic AMP production, irrespective of the presence of antigens. In contrast, the level of cyclic AMP was not affected by poly(A) · poly(U). The difference in cyclic AMP accumulation suggests that PGE₁ and poly(A) · poly(A) modify immune responsiveness by different mechanisms. The above observations were verified both in SJL and DBA/1 mice, which are the respective genetic high and low responders to (T, G) -Pro—L. This implies that the modifications of responsiveness described are not related to the genetic control of immune response to this immunogen.     

Intermediacy of poly(L-proline) II and beta-strand conformations in poly(L-lysine) beta-sheet formation probed by temperature-jump/UV resonance Raman spectroscopy

Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperature jump (T-jump) was used to investigate early steps in the temperature-induced alpha-helix to beta-sheet conformational transition of poly(L-lysine) [poly(K)]. Excitation at 197 nm from a tunable frequency-quadrupled Ti:sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although un-ionized poly(K) (pH 11.6) is mainly alpha-helical below 30 degrees C, there is a detectable fraction (approximately 15%) of unfolded polypeptide, which is mainly in the poly(L-proline) II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be beta-strand. Above 30 degrees C un-ionized poly(K) forms a beta-sheet at a rate (minutes) which increases with increasing temperature. A 22-44 degrees C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/beta-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, approximately 2% at the 3 micros time limit of the experiment. The rise time of the PPII signals is approximately 250 ns, consistent with melting of short alpha-helical segments. A model is proposed in which the melted PPII segments interconvert with beta-strand conformation, whose association through interstrand H-bonding nucleates the formation of beta-sheet. The intrinsic propensity for beta-strand formation could be a determinant of beta-sheet induction time, with implications for the onset of amyloid diseases.     

Inner-sphere complexes of divalent cations with single-stranded poly(rA) and poly(rU)

A combination of ultrasound velocimetry, density, and UV spectroscopy has been employed to study the hydration effects of binding of Mn(2+) and alkaline-earth cations to poly(rA) and poly(rU) single strands. The hydration effects, obtained from volume and compressibility measurements, are positive due to overlapping the hydration shells of interacting molecules and consequently releasing the water molecules to bulk state. The volume effects of the binding to poly(rA), calculated per mole of cations, range from 30.6 to 40.6 cm(3) mol(-1) and the compressibility effects range from 59.2 x 10(-4) to 73.6 x 10(-4) cm(3) mol(-1) bar(-1). The volume and compressibility effects for poly(rU) are approximately 17 cm(3) mol(-1) and approximately 50 x 10(-4) cm(3) mol(-1) bar(-1), respectively. The comparative analysis of the dehydration effects suggests that the divalent cations bind to the polynucleotides in inner-sphere manner. In the case of poly(rU) the dehydration effects correspond to two direct coordination, probably between adjacent phosphate groups. The optical study did not reveal any effects of cation on the secondary structure or aggregation of poly(rU). In the case of single-helical poly(rA) binding is more specific: dehydration effects correspond to three to five direct contacts and must involve atomic groups of adenines, and the divalent cations stabilize and aggregate the polynucleotide.     

The thermal induced helix--beta transition of poly(N epsilon-methyl-L-lysine) and poly(N delta-ethyl-L-ornithine) in aqueous solution

Uncharged poly(N^ε-methyl-L -lysine) (PMLL) and its isomer, poly(N^δ-ethyl-L -ornithine) (PELO), in alkaline solution (pH ca. 12) undergo a helix-to-β transition upon mild heating at 50°C or higher in a manner similar to that of poly(L -lysine) (PLL). The rate of conversion follows the order: PMLL < PELO < PLL. The helix can be regenerated upon cooling near zero degrees, for instance, after more than 12 hr at 2°C. At concentrations less than 0.02% the β form is intramolecular, but at higher concentrations both intra- and intermolecular β forms are generated. Poly(N^δ-methyl-L -ornithine) (PMLO), an isomer of PLL, behaves like poly(L -ornithine); uncharged PMLO in alkaline solution is partially helical and becomes disordered at elevated temperatures.     

Synthesis of poly(amidoamine) dendron-bearing lipids with poly(ethylene glycol) grafts and their use for stabilization of nonviral gene vectors

Recently, we developed a new type of cationic lipid that consists of an amine-terminated poly(amidoamine) dendron and two long alkyl groups. These dendron-bearing lipids achieved efficient gene transfection of cells through synergetic action of the proton sponge effect and membrane fusion in combination with fusogenic lipid dioleoylphosphatidylethanolamine. Using those dendron-bearing lipids as a base material, we developed in this study a functional component of gene vectors that stabilizes lipoplexes by multiple PEG chains and promotes gene transfection through the proton sponge effect. We combined a poly(ethylene glycol) (PEG, 550 Da) graft to each of four chain ends of the G2 dendron-bearing lipid (P4-DL). An analogous molecule having single PEG graft was also synthesized using the G0 dendron-bearing lipid (P1-DL). Inclusion of P4-DL decreased the size of the G3 dendron-bearing lipid-based lipoplexes more efficiently than P1-DL. In addition, P4-DL-containing lipoplexes exhibited two-orders-higher transfection efficiency than P1-DL-containing lipoplexes with the same PEG graft density. These results indicate the superiority of multiple attachments of PEG graft chains to a lipid for heightened ability to increase colloidal stability of lipoplexes while retaining their transfection activity. The lipoplexes stabilized by P4-DL were small, around 250 nm, and achieved efficient transfection in the presence of serum. Therefore, P4-DL and its analogues will form the basis for production of efficient nonviral vectors for in vivo use.     

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.     

Changes in poly(adenosine diphosphate-ribose) and poly(adenosine diphosphate-ribose) polymerase in synchronous HeLa cells.

An antibody has been prepared which is highly specific for poly(adenosine diphosphate-ribose). Neither poly(A), DNA, nor a variety of adenine-containing nucleosides or nucleotides were effective in competing with poly(ADP-ribose) for binding to the antibody. Of all compounds tested, only adenosine diphosphate-ribose competed for binding to the antibody. Unlabeled poly(adenosine diphosphate-ribose) was about 10 000 times more effective in competing with labeled polymer for antibody binding than was adenosine diphosphate-ribose. Using the antibody, the amount of poly(adenosine diphosphate-ribose) was found to increase from early S phase to a peak at mid S with a second, even larger increase seen at the S-G2 transition point in synchronously dividing HeLa cells. Pulse labeling of the polymer with [2-3H]adenosine was also maximal at the same time points. Changes in the levels of poly(adenosine diphosphate-ribose) polymerase activity measured in isolated nuclei coincided with the changes in amounts of polymer present in intact cells during progression from S phase into G2.     

The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B

The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins.     

Fundamental cryobiology of rat immature and mature oocytes: hydraulic conductivity in the presence of Me(2)SO, Me(2)SO permeability, and their activation energies

The hydraulic conductivity in the presence of dimethyl sulfoxide Me(2)SO (L(p)(Me(2)SO)), Me(2)SO (P(Me(2)SO)) permeability and reflection coefficient (sigma) of immature (germinal vesicle; GV) and mature (metaphase II; MII) rat oocytes were determined at various temperatures. A temperature controlled micropipette perfusion technique was used to conduct experiments at five different temperatures (30, 20, 10, 4, and -3 degrees C). Kedem and Katchalsky membrane transport theory was used to describe the cell volume kinetics. The cell volumetric changes of oocytes were calculated from the measurement of two oocyte diameters, assuming a spherical shape. The activation energies (E(a)) of L(p)(Me(2)SO) and P(Me(2)SO) were calculated using the Arrhenius equation. Activation energies of L(p)(Me(2)SO) for GV and MII oocytes were 34.30 Kcal/mol and 16.29 Kcal/mol, respectively; while the corresponding E(a)s of P(Me(2)SO) were 19.87 Kcal/mol and 21.85 Kcal/mol, respectively. These permeability parameters were then used to calculate cell water loss in rat oocytes during cooling at subzero temperatures. Based on these values, the predicted optimal cooling rate required to maintain extra- and intracellular water in near equilibrium for rat GV stage oocytes was found to be between 0.05 degrees C/min and 0. 025; while for rat MII oocytes, the corresponding cooling rate was 1 degrees C/min. These data suggest that standard cooling rates used for mouse oocytes (e.g., 0.5-1 degrees C/min) can also be employed to cryopreserve rat MII oocytes. However, the corresponding cooling rate required to avoid damage must be significantly slower for the GV stage rat oocyte. J. Exp. Zool. 286:523-533, 2000.     

Effect of virazole on the antiviral activity of poly(G) X poly(C) and other polyribonucleotide interferonogens

The effect of virazole on the antiviral activity of poly (G) X poly (C), poly (G, A) X X poly (C) and poly(G, I) X poly (C) was studied in cell cultures and on mice. It was shown that virazole in concentrations not sufficient for significant inhibition of the development of vesicular stomatitis virus or Sindbis virus in chick embryo cell cultures markedly increased the antiviral effect and allowed decreasing the minimum effective doses of the synthetic polyribonucleotide complexes with respect to the above viruses. Combined administration of poly (G) X poly (C) and virazole to mice 1-2 or 24 hours after infection with tick-borne encephalitis virus provided a much more pronounced decrease in the death rate of the animals than the use of the interferonogen alone. Virazole per se was little active and had no significant effect on the intensity of interferonogenesis promoted by the use of poly (G) X poly (C). A possibility of successful therapy of viral infections with polyribonucleotide interferonogens in combination with virazole or other chemotherapeutic drugs with broad antiviral spectrum is discussed.     

Direct determination of the π-helix structure and helix transition behavior of poly(β-phenethyl l-aspartate) via synchrotron X-ray diffraction

The helix-sense inversions of poly(β-phenethyl l -aspartate) (2P) and diblock copolymers (2P-3P), with 2P and poly(β-phenylpropyl l -aspartate) (3P) blocks, were studied in their solid states using synchrotron wide-angle X-ray diffraction and small-angle X-ray scattering. The characteristic parameters of the π-helix structure of 2P were directly determined in situ after the helix transition at a high temperature. In the 2P-3P block copolymers, the main chains of the 3P blocks initially convert from right- to left-handed α-helices, and then the 2P blocks convert irreversibly from right-handed α-helices to left-handed π-helices. The chemical structures of the side chains of poly(l -aspartic acid ester)s significantly affect their helix transition behaviors.     

Laser-excited raman spectroscopy of biomolecules. XII. Thermally induced conformational changes in poly(L-glutamic acid)

The α-helical from of poly(L -glutamic acid) [α-poly(Glu)] gives rise to the same amide I and III lines as α-poly(γ-benzyl-L -glutamate) at 1652 and 1296 cm^?1, respectively. The latter is a superposition of the amide III line near 1290 cm^?1 and a line deu to vibrational made of CH₂ groups of the side chain near 1300 cm^?1. A line at 924 cm^?1 is tentatively identified as characteristics of α-poly(Glu). Both the β₁- and β₂- forms of poly(Glu) give rise to characteristic of β-amide. III frequencies that are similar because of their similar backbone structures. Differences in the conformations of their side chains and in the environments of the backbone are reflected in the region 800–1200 cm^?1 and in the amide I. A line at 1042 cm^?1 and a pair at 1021 and 1059 cm^?1 are tentatively assigned as characteristic of β₁-poly(Glu) and β₂-poly(Glu), respectively. The α-β₂ transition in poly(L -Glu⁷⁸L -Val²²) is shown by the appearance of all the β₂-characteristic lines in the thermally transformed sample. The same features observed in poly(L -Glu⁹⁵L -Val⁵) also indicate that the α-β₂ transition of poly(Glu) is facilitated by the presence of L -valine and that the content of L -valine is not critical for this purpose. Investigation of the Raman spectra of the calcium, strontium, barium and sodium slats of poly(Glu) shows that these salts, under the conditions of preparation used, all the have random-coil conformations.     

Protonated polynucleotide structures. IX. Disproportionation of poly (G)-poly (C) in acid medium

The interaction between poly (G) and poly (C) was investigated in neutral and acid medium by optical methods. Three main points arise from this investigation. (1) The formation of poly (G)·poly (C) was complete only above an ionic strength of about 0.6M [Na⁺]. Lowering the ionic strength increased the amounts of free poly (G) and free poly (C) that could be detected. (2) When titrating towards acid pH values a transition took place which was characterized by potentiometry, mixing curves, and circular dichroism: a three-stranded poly (G)·poly (C)·poly (C⁺) complex was formed analogous to the transition observed for the acid titration of poly (I)·poly (C). (3) Even when the poly (G)·poly (C) complex was incompletely formed (at low ionic strength) in neutral medium all poly (C) entered the triple-stranded complex.     

Temperature dependence of the Raman spectrum of DNA. II. Raman signatures of premelting and melting transitions of poly(dA).poly(dT) and comparison with poly(dA-dT).poly(dA-dT).

The temperature dependence of the Raman spectrum of poly(dA).poly(dT) (dA: deoxyadenosine; dT: thymidine), a model for DNA containing consecutive adenine.thymine (A.T) pairs, has been analyzed using a spectrometer of high spectral precision and sensitivity. Three temperature intervals are distinguished: (a) premelting (10 < t < 70 degrees C), in which the native double helix is structurally altered but not dissociated into single strands; (b) melting (70 < t < 80 degrees C), in which the duplex is dissociated into single strands; and (c) postmelting (80 < t degrees C), in which no significant structural change can be detected. The distinctive Raman difference signatures observed between 10 and 70 degrees C and between 70 and 80 degrees C are interpreted in terms of the structural changes specific to premelting and melting transitions, respectively. Premelting alters the low-temperature conformation of the deoxyribose-phosphate backbone and eliminates base hydrogen bonding that is distinct from canonical Watson-Crick hydrogen bonding; these premelting perturbations occur without disruption of base stacking. Conversely, melting eliminates canonical Watson-Crick pairing and base stacking. The results are compared with those reported previously on poly(dA-dT).poly(dA-dT), the DNA structure consisting of alternating A.T and T.A pairs (L. Movileanu, J. M. Benevides, and G. J. Thomas, Jr. Journal of Raman Spectroscopy, 1999, Vol. 30, pp. 637-649). Poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) exhibit strikingly dissimilar temperature-dependent Raman profiles prior to the onset of melting. However, the two duplexes exhibit very similar melting transitions, including the same Raman indicators of ruptured Watson-Crick pairing, base unstacking and collapse of backbone order. A detailed analysis of the data provides a comprehensive Raman assignment scheme for adenosine and thymidine residues of B-DNA, delineates Raman markers diagnostic of consecutive A.T and alternating A.T/T.A tracts of DNA, and identifies the distinct Raman difference signatures for premelting and melting transitions in the two types of sequences.