Protein synthesis using poly(2′-halogeno-2′-deoxyadenylic acids) as messenger

Poly(2′-fluoro-2′-deoxyadenylic acid), poly(2′-chloro-2′-deoxyadenylic acid) and poly(2′-bromo-2′-deoxyadenylic acid) are used as messenger RNAs in protein synthesizing systems in vitro. All polynucleotides were active as messengers and [¹⁴C]lysine was incorporated into polypeptides. The initial velocity of polylysine formation was greater using poly(2′-fluoro-2′-deoxyadenylic acid) as messenger than in the case of poly(rA), and all synthetic messengers lived longer in the protein synthetic system.     

Substrate Specificity of Nuclease P1

Nuclease P₁ cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split.     

Binding geometries of triple helix selective benzopyrido [4,3-b]indole ligands complexed with double- and triple-helical polynucleotides

The binding modes of three benzopyrido [4,3-b]indole derivatives (and one benzo[-f]pyrido [4-3b] quinoxaline derivative) with respect to double helical poly(dA) · poly(dT) and poly[d(A-T)]₂ and triple-helical poly(dA) · 2poly(dT) have been investigated using linear dichroism (LD) and CD: (I) 3-methoxy-11-amino-BePI where BePI = (7H-8-methyl-benzo[e]pyrido [4,3-b]indole), (II) 3-methoxy-11-[(3′-amino) propylamino]-BePI, (III) 3-methoxy-7-[(3′-diethylamino)propylamino] BgPI where BgPI = (benzo[g]pyrido[4,3-b]indole), and (IV) 3-methoxy-11-[(3′-amino)propylamino] B f P Q where B f P Q = {benzo[-f]pyrido[4-3b]quinoxaline}. The magnitudes of the reduced LD of the electronic transitions of the polynucleotide bases and of the bound ligands are generally very similar, suggesting an orientation of the plane of the ligands' fused-ring systems preferentially perpendicular to the helix axis. The LD results suggest that all of the ligands are intercalated for all three polynucleotides. The induced CD spectrum of the BePI chromophore in the (II-BePI)-poly[d(A-T)]₂ complex is almost a mirror image of that for the (I-BePI)-poly(dA) · poly(dT) and (I-BePI)-poly(dA) · 2poly(dT) complexes, suggesting an antisymmetric orientation of the BePI moiety upon intercalation in poly[d(A-T)]₂ compared to the other polynucleotides. The induced CD of I-BePI bound to poly(dA) · 2poly(dT) suggests a geometry that is intermediate between that of its other two complexes. The concluded intercalative binding as well as the conformational variations between the different BePI complexes are of interest in relation to the fact that BePI derivatives are triplex stabilizers. © 1997 John Wiley & Sons, Inc. Biopoly 42: 101–111, 1997     

Methylated Polyl(L-lysine): Conformational Effects and Interactions with Polynucleotides

Abstract

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of e-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally- induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO₄)₂. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from β, to 50% α, to random coil at the maximum methylation.

Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)_n·(dAdT)_n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)_n·(dC)_n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative ψ-spectra are induced, which, in the case of (dGdC)_n·(dGdC)_n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration.

It is suggested that the biological effects of methylated proteins may be evoked by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Binding modes of V(O)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to various synthetic DNAs studied by polarized spectroscopy

The interactions between water-soluble cationic oxovanadyl[meso-tetrakis(4-N-methylpyridiumyl)]porphyrin (VOTMPyP) and various synthetic polynucleotide including poly[d(A–T)₂], poly[d(G–C)₂], and poly[d(I–C)₂] were studied using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy. When VOTMPyP formed a complex with poly[d(A–T)₂] and poly[d(I–C)₂], a positive CD signal at low [VOTMPyP]/[DNA] ratios (R ratios) and strong excitonic CD signals at above R ≥ 0.15 were induced. The appearance of the CD spectra of the VOTMPyP-poly[d(G–C)₂] complex were very different: a small negative CD at low R ratios and very small excitonic CD at high R ratios were observed. Considering the facts that the minor grooves of the former two polynucleotides resemble and the major groove of poly[d(I–C)₂] is similar with that of poly[d(G–C)₂], it is conclusive that VOTMPyP binds to the minor groove of all DNA at lower R ratios while they stack at the outside of DNA at higher R ratios. The binding geometry of VOTMPyP to all polynucleotides studied by LD seemed to be homogenous, irrespective of the R ratio. It has been found that VOTMPyP can have five- and six-fluxional coordination states. Comparing the absorption spectra of VOTMPyP complexed with poly[d(A–T)₂] and poly[d(G–C)₂], the distinctive absorptions of the five- and six-coordinated species were observed at lower R ratios which centered at 420–430 nm and 442 nm, respectively. While the six-coordinated VOTMPyP favored the poly[d(A–T)₂], the five-coordinated species favored the poly[d(G–C)₂] at the low R ratios. As the stacked species increased with an increasing R ratio, the six-coordinated species became the major bound species. These observations lead us to conclude that the guanine base′ amino group plays a crucial role not only in determining the binding mode of VOTMPyP but also in the conversion of the six-coordinated species to the five-coordinated species.     

Unusual Contribution of 2-Aminoadenine to the Thermostability of DNA

Abstract

The poly(dA-dU) and poly(dl-dC) duplexes have very similar thermostabilities (T_m). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m⁵dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m⁵C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n²dA-dU) and poly(dG-dC) as well as the poly(n²dA-dT) and poly(dG-m⁵dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40°C than the n²A.U-based ones. Poly(dA,n²dA-dT)-s containing various proportions of A and n²A as well as the natural DNA of S-2L cyanophage that contains n²A bases instead of A were also studied. It was found that dependence of T_m on the n²A-content was non-linear and that the lower T_m is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n²A:T base-pair as compared to the G:C are discussed.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. I. Structure and interactions of binding site I.

Escherichia coli ribosomal protein S1 plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. S1 protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds single-stranded RNA only (Draper et al., 1977). The nucleic acid binding properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper &; von Hippel, 1978).Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (K) of site I for poly(dA) and poly(dC) is ~3 × 10⁶m^?1 at 0.12 m-Na⁺, and the site size (n, the number of nucleotide residues covered per S1 bound) is 5.1 ± 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of S1 to single-stranded polynucleotides is ~10³ larger than K for binding to double-stranded polynucleotides, meaning that S1 (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na⁺] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affin is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2′-deoxyribose 5′-phosphate is approximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with S1-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed with an oligonucleotide.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

Number of binding sites of DNA and polynucleotides with tris(2,2'-dipyridyl)ruthenium(II) cations

The binding of tris(2,2′-bipyridyl)ruthenium(II) cations [Ru(bpy)] with single- and double-stranded (ss and ds) DNA, and the polynucleotides poly(A), poly(C), poly(G), poly(I), poly(I) · poly(C), and poly(U), was studied in aqueous solution. Steady-state electrical conductivity measurements with the polynucleotides, ssDNA, and dsDNA reveal that approximately three nucleotides offer one binding site. This may be compared with the ratio [nucleotide]/[Mg²⁺] of 2.4 : 1 for dsDNA. After laser excitation (353 nm), the luminescence of Ru(bpy) bound to nucleic acids shows two decay components. The contribution of the fast component, which is interpreted as resulting from quenching processes of the absorbed ruthenium complex, exhibits a maximum with increasing [nucleotide]/[Ru(bpy)] at a ratio of about three to one. Bound Ru(bpy) can be released from the strand by addition of NaClO₄ [half-concentration: 2.5 and ≤ 10 mM for poly(U) and dsDNA, respectively].     

Spectroscopic studies on the structure of poly(8-bromoadenylic acid): Effect of glycosidic torsion angle on the conformation and flexibility in polyribonucleotides

The helix–coil transition and conformational structure of poly(8-bromoadenylic acid) [poly(8BrA)] have been investigated using ¹H- and ¹³C-nmr, CD, and ir spectroscopy. The results have been compared with the structure of the related 5′-mono- and polynucleotides. The chemical shifts of H(2′), H(3′), C(2′), and C(3′) nmr signals show an interesting correlation with both the puckering of ribose ring and glycosidic bond torsion angle. Poly(8BrA) shows an upfield shift of the C(3′) signal and a downfield shift of the H(3′) signal compared to the chemical shifts in poly(A). These shifts are consistent with a C(3′) endo-syn conformation for poly(8BrA). A similar effect has been reported previously and is also observed here on the C(2′) and H(2′) signals when the preferred conformation is C(2′)endo-syn (e.g., in 5′-8BrAMP). The chemical-shift parameters thus act as a probe for studying syn ? anti and N ? S equilibria in solutions. The three-bond ¹H-′¹³C coupling constants between H(1′) and C(8) and C(4) have been measured in poly(8BrA) and 5′-8BrAMP and their structural implications have been discussed. The observed preference of a C(3′)endo-syn conformation for poly(8BrA), coupled with other evidence, throws doubt on the validity of a correlation previously reported whereby a syn conformation is associated with a C(2′)endo ribose pucker. The backbone conformation of randomly coiled poly(8BrA) is very similar to the structures found in polyribonucleotides: poly(A) and poly(U). All three polymers show strong preferences for the backbone angles found in RNA helices. The CD spectrum of poly(8BrA) has a striking relationship to that of poly(A). The signs of all extrema are inverted, and the magnitudes are related by a constant factor. We suggest that these differences result from a change in the angle between coupled transition moment vectors in the two polymers. Infrared spectra of poly(8BrA) in H₂O and D₂O solution are reported for the frequency range below 1400 cm^?1. The antisymmetric >PO stretching vibration is observed at an unusually low frequency in the helix (1214 cm^?1). The symmetric >PO stretch occurs at ～1095 cm^?1 but is not resolved from a ring vibration near this frequency. A conformationally sensitive band, characteristic of helical RNA structures, is observed at 817 cm^?1 and disappears when the helix is melted. This observation confirms the conclusion that ordered poly(8BrA) has a regular helical structure with an RNA backbone conformation. A stereochemical explanation is provided for the failure of poly(8BrA) (or other syn polymers) to form double helices with anti-polyribonucleotides.     

Polynucleotide conformation from flow dichroism studies

We demonstrate that the isotropic absorption and linear dichroism in an unknown flow field can be used to determine base tilt in polynucleotides if three transitions are measured and the directions of the corresponding dipoles are known. The method is applied here to reach conclusions about the base tilt in poly(rA), poly(rA)⁺·poly(rA), and poly(rC). The respective values are: 28° tilt about the axis + 50° toward C8 from the C1′ → N9, and 25° tilt about the axis + 118° toward C5 from C1′ → N1. The results for poly(rA)⁺·poly(rA) are consistent with the accepted model. Spectra were measured for poly(rC)⁺·poly(rC), but definite conclusions must await reliable directions for transition dipoles. The dipole direction for the 218-nm transition in rC is found to be +13° or +43° toward C5 from C1′ → N1. The CD spectra to about 168 nm are presented and discussed.     

Spatial configurations of polynucleotide chains. 3. Polydeoxyribonucleotides

The virtual bond scheme set forth in preceding papers for treating the average properties of polyriboadenylic acid (poly rA) is here applied to the calculation of the unperturbed mean-square end-to-end distance of polydeoxyriboadenylic acid (poly dA). The modifications in structure and in charge distribution resulting from the replacement of the hydroxyl group at C_2′ in the ribose residue by hydrogen in deoxyribose produce only minor modifications in the conformational energies associated with the poly dA chain as compared to those found for poly rA. The main difference is manifested in the energy associated with rotations about the C_3′–O_3′ bond of the deoxyribose residue in the C_2′-endo conformation; accessible rotations are confined to the range between 0° and 30° relative to the trans conformation, whereas in the ribose unit the accessible regions comprise two ranges centered at approximately 35° and 85°. The characteristic ratio 〈r²〉0/nl² calculated on the basis of the conformational energy estimates is ≈9 for the poly dA chain with all deoxyribose residues in the C_3′-endo conformation and ≈21 with all residues in the C_2′-endo form. Satisfactory agreement is achieved between the theoretical values and experimental results on apurinic acid by treating the poly dA chain as a random copolymer of C_3′-endo and C_2′-endo conformational isomers present in a ratio of ～1 to 9.     

Binding properties of Ru(II) polypyridyl complexes with poly(U)·poly(A)*poly(U) triplex: the ancillary ligand effect on third-strand stabilization

The binding properties of [RuL₂(mip)]²⁺ {where L is 1,10-phenanthroline (phen) or 4,7-dimethyl-1,10-phenanthrollne (4,7-dmp) and mip is 2′-(3″,4″-methylenedioxyphenyl)imidazo[4′,5′-f][1,10]phenanthroline} with regard to the triplex RNA poly(U)·poly(A)*poly(U) were investigated using various biophysical techniques and quantum chemistry calculations. In comparison with [Ru(4,7-dmp)₂(mip)]²⁺, remarkably higher binding affinity of [Ru(phen)₂(mip)]²⁺ for the triplex RNA poly(U)·poly(A)*poly(U) was achieved by changing the ancillary ligands. The stabilization of the Hoogsteen-base-paired third strand was improved by about 10.9 °C by [Ru(phen)₂(mip)]²⁺ against 6.6 °C by [Ru(4,7-dmp)₂(mip)]²⁺. To the best of our knowledge, [Ru(phen)₂(mip)]²⁺ is the first metal complex able to raise the third-strand stabilization of poly(U)·poly(A)*poly(U) from 37.5 to 48.4 °C. The results reveal that the ancillary ligands have an important effect on third-strand stabilization of the triplex RNA poly(U)·poly(A)*poly(U) when metal complexes contain the same intercalative ligands.     

Density functional study of the electronic and optical properties of fluorene-thieno[3,2-b]thiophene-based conjugated copolymers

Quantum mechanical techniques are applied to investigate a family of π-conjugated copolymers: poly(9,9′-dimethylfluorene-alt-thiophene) (PFT), poly(9,9′-dimethylfluorene-alt-thieno[3,2-b]-thiophene) (PFTT), poly(9,9′-dimethylfluorene-alt-bithiophene) (PFT2), and poly(9,9′-dimethylfluorene-alt-α,α′-bisthieno[3,2-b]-thiophene) (PFTT2). Linear extrapolation is employed to obtain polymers' properties from oligomer calculations. That is, the HOMO–LUMO gaps (Δ_H–Ls), band gaps (E _g s), ionisation potentials and electron affinities of the copolymers are obtained by plotting the corresponding quantities of the oligomers as a function of the inverse chain length (1/n) and extrapolating them to infinite chain length. The electronic properties of the neutral, positive and negative oligomers are determined using the density functional theory (DFT) at B3LYP/6-31G* approximation. The lowest singlet excitation energies of the oligomers of PFT, PFTT, PFT2, and PFTT2 are also determined with the use of the time-dependent DFT again at B3LYP/6-31G* approximation. Comparisons are made with experimental values when possible.     

Fluorescent 2-aza-epsilon-adenosine derivatives of polyadenylic acid, polyuridylic acid, and polyinosinic acid.

A new fluorescent analog of adenosine, 1,N⁶-etheno-2-aza-adenosine, has been incorporated into polynucleotides by polynucleotide phosphorylase polymerization of 1,N⁶-etheno-2-aza-adenosine-5′-diphosphate and adenosine-5′-diphosphate, uridine-5′-diphosphate, or inosine-5′-diphosphate. These new oligonucletides possess high fluorescence when excited at 358 nm and emit at 495 nm. The ratio of the fluorescent and nonfluorescent portions of the copolymer can be controlled by the initial composition of the 2-aza-ε-adenosine-diphosphate and the corresponding nucleoside diphosphate. Fluorescent copolymers with a ratio varying from 1.6 to 35 have thus been synthesized. The physicochemical study of copolymers containing less than 10% of the 1,N⁶-etheno-2-aza-adenosine moiety showed that they are similar to poly(A), poly(U), or poly(I). Therefore, fluorescence and polarization study of the 1,N⁶-etheno-2-aza-adenosine residues that have been incorporated into the copolymer provides a sensitive indicator for the structure of the copolymer. Potentially these new copolymers may provide unique roles in probing the structure of poly(C) and poly(A) in cellular mRNA.     

DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258

The complexes of Hoechst 33258 with poly[d(A-T)₂], poly[d(I-C)₂], poly[d(G-C)₂], and poly[d(G-m⁵C)₂] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)₂] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)₂] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45° relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity—leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)₂]. By contrast, when bound to poly[d(G-C)₂] and poly[d(G-m⁵C)₂], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)₂] and poly[d(G-m⁵C)₂] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed. © 1996 John Wiley & Sons, Inc.     

Secondary structure in polyuridylic acid: Non-classical hydrogen bonding and the function of the ribose 2′-hydroxyl group

The role of non-classical hydrogen bonding in RNA structure has been investigated using polyuridylic acid, which has a labile ordered structure at temperatures near 0 °C, as a model system. By comparing the proton nuclear magnetic resonance spectrum of poly(U) in the transition region with that of uridine and the dimer UpU we find evidence that both the imino N(₃)-H and the ribosyl 2′-OH protons are hydrogen bonded. The characteristics of the former are consistent with participation in N(₃)-HOC bonding primarily between residues in the same strand. As yet we cannot unambiguously assign the acceptor for the 2′-OH in ordered poly(U): because of its apparent stability and the acceptable stereochemistry, we presently favor a bond between ribose 2′-OH and O(1′) connecting adjacent nucleotides of the same strand. This arrangement could contribute to the co-operativity of the poly(U) helix formation. The recently proposed 2′-OHO(1′) interactions in crystalline yeast transfer RNA^Phe suggest similar interactions might play a role in the conformational stability of natural RNAs. A second conformational transition below the major transition in the ultraviolet can be detected in poly(U) by monitoring the H(₆) proton of uracil.     

Experimental thermodynamics of the helix--random coil transition. 3. Determination of the transition enthalpies of the helical complexes poly(I+C) and poly I in solution

The enthalpies of the helix-coil transitions of the ordered polynucleotide systems of poly(inosinic acid)–poly(cytidylic acid) [poly(I + C)], (helical duplex), and of poly (inosinic acid) [poly(I + I + I)], (proposed secondary structure: a triple-stranded helical complex), were determined by using an adiabatic twin-vessel differential calorimeter. Measuring the temperature course of the heat capacity of the aqueous polymer solutions, the enthalpy values for the dissociation of the helical duplex poly (I + C) and the three-stranded helical complex poly(I + 1 + 1), respectively, were obtained by evaluating the additional heat capacity involved in the conformational change of the polynucleotide system in the transition range. The ΔH values of the helix-coil transition of poly (I + C) resulting from the analysis of the calorimetric measurements vary between the limits 6.5 ± 0.4 kcal/mole (I + C) and 8.4 ± 0.4 kcal/mole (I + C). depending on the variation of the cation concentration ranging from 0.063 mole cations kg H₂O to 1.003 mole cations/kg H₂O. The calorimetric investigation of an aqueous poly I solution (cation concentration 1.0 mole/kg H₂O) yielded the enthalpy value ΔH = 1.9 ± 0.4 kcal/mole (I), a result which has been interpreted qualitatively following current models of inter- and intramolecular forces of biologically significant macromolecules. Additional information on the transition behavior of poly(I+ C)Was obtained by ultraviolet and infrared absorption measurements.     

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.