Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima.

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.     

8-Diazoguanosine, 2,8-Diaminoadenosine and Other Purine Nucleosides Derived from Guanosine

Abstract

Diazotization of 8-aminoguanosine gave 8-diazoguanosine (2) which is stable in neutral and basic media, but decomposes to D-ribose and 8-diazoguanine in acidic conditions. 2-Amino-6,8-dichloro-9-(2,3,5-tri-0-acetyl-β-D-ribo-furanosyl)purine (5) was employed to synthesize 9-β-D-ribofuranosyl-2,6,8-triaminopurine (8) and a number of N6-alkyl-2-amino-8-chloro-9-β-D-ribofuranosylpurines.     

Physical and coding properties of poly(5-methoxyuridylic) acid.

The synthesis of poly(mo5U) requires a high concentration (2.7 mg/ml) of polynucleotide phosphorylase as well as a long reaction time (48 h). The resulting polynucleotide has a chain length of approximately 100 nucleotides. It shows no indication of a stable secondary structure. When poly(mo5U) is mixed with poly(A), a triple-stranded complex poly(A) . 2poly(mo5U) is formed. This complex has a melting temperature of 68.5 +/- 0.5 degrees C at 150 mMNa+ and exhibits a hysteresis loop between melting and reformation of the complex having a delta Tm of 11.5 degrees C. Poly-5-methoxyuridylic acid stimulates the binding of Phe-tRNA to 70-S ribosomes but is inactive in directing poly(Phe) synthesis.     

Calorimetric study of the complexes between polyuridylic acid and adenylic nucleotides.

Soluble complexes of poly (U) and adenylic nucleotides in NaCl solutions were studied by scanning microcalorimetry. The melting enthalpies, delta Hm, of poly (U) complexes with adenosine, 2',3' -cAMP, 2'(3')-AMP, 5-AMP, ADP, ATP in 1 M NaCl are 50.5; 45.0; 42.9; 28.6; 26.1 and 25.6 kJ/mole triplets, respectively. Delta Hm is independent of the complex melting temperature, Tm. The calorimetric enthalpies are considerably lower than the apparent delta Hv.H. obtained from Tm dependence on free monomer concentration. The enthalpy of complex formation in 1 M NaCl depends neither ob the number nor on the degree of ionization of the phosphate groups but is essentially determined by their 5' - or 2'(3')-position. In contrast to 2'(3')- AMP. 2 poly (U), delta Hm of 5'AMP. 2 poly (U) increases considerably at lowering Na+ concentration. The enthalpy of poly (U) double helix melting in 1 M NaCl is 8.8 kJ/mole pairs which is 2.5 times lower than that in MgCl2 solutions.     

Nonionic nucleic acid analogues. Synthesis and characterization of dideoxyribonucleoside methylphosphonates.

A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.     

Free energy of the binding of uridylic acid oligomers with double stranded poly(A) x poly(U)

The binding parameters (K, omega) and the free energy (DeltaG(0)) of triple helix formation have been estimated for complexes of oligo(U)(n) (n = 5, 7-10) with poly(A) . poly(U) on the basis of hypochromicity measurements. The data were treated according to the formula of McGhee and von Hippel [J. Mol. Biol. 86 (1974) 469] by a computer program ALAU [H. Schütz et al., Stud. Biophys. 104 (1984) 23] which takes absorbancies and total concentrations as input. In 1 mM cacodylate buffer pH 7.0 with 10 mM NaCl and 10 mM MgCl(2) at 5 degrees C the free energy of contiguous binding was found to be a linear function of the oligomer length with a slope of DeltaG(c,U)(0) = -0.72 (+/-0.03) kcal x mol(-1) per nucleotide. The mean cooperativity coefficient (omega) was 24.5 (+/- 5.6), and the corresponding free energy of interaction between the neighbouring oligonucleotides in the third strand was DeltaG(0(omega)) = -1.74 (+/-0.13) kcal x mol(-1).     

Human lysosomal sphingomyelinase: substrate efficacy of apolipoprotein/sphingomyelin complexes

Human apolipoprotein stimulation of sphingomyelin (SM) hydrolysis by sphingomyelinase from human skin fibroblasts has been studied. Apolipoproteins A-I, A-II, B, C-I, and E do not enhance sphingomyelin hydrolysis above control levels. In contrast, apoC-II stimulates sphingomyelin hydrolysis by approximately 2.5-fold. ApoC-III, the most potent apoprotein activator, stimulates hydrolysis by 3-4-fold. ApoC-III stimulation is not significantly different for the three different isoforms which carry 0, 1, or 2 sialic acid residues. The amino-terminal half of this apoprotein, C-III(1-40), which does not bind to phospholipid surfaces, does not activate sphingomyelinase. In contrast, the carboxyl-terminal half, C-III(41-79), which strongly binds to phospholipid surfaces, stimulates sphingomyelin hydrolysis to the same level as that produced by the intact, full-length apoprotein. Incubation of sphingomyelin vesicles with increasing proportions of apoC-III results in the formation of complexes of increasing apoC-III:SM ratio and decreasing radius. The hydrolysis of sphingomyelin in the 1:50 (mol/mol) complex was more than 2-fold greater than that of the 1:200 (mol/mol) complex. The rate of hydrolysis of egg yolk sphingomyelin in the 1:50 complex was maximal [0.9 mumol h-1 (mg of protein)-1] at the gel----liquid-crystalline phase transition temperature (Tm) of the complex (40 degrees C). The rate of hydrolysis fell markedly at either higher or lower temperature. Determination of the apparent Km and Vmax values below, at, and above Tm indicated that the temperature dependence of sphingomyelin hydrolysis was attributable primarily to changes in Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex formation between ribosomal protein S1, oligo-and polynucleotides: chain length dependence and base specificity.

In order to examine the nature of the complex formation between the ribosomal protein S1 and nucleic acids three methods were used: Inhibition of the reaction of n-ethyl[2.3 14C]-maleimide with S1 by the addition of oligonucleotides; adsorption of the complexes to nitrocellulose filters; and equilibrium dialysis. The complex formation is Mg2+ dependent at low salt concentrations and becomes Mg2+ independent at an ionic strength greater than 90 mM. Oligouridylates of increasing chain length reach an optimal KA of 3-3-10(7) M-1 at a chain length of n=13-14. Protein S1 contains one binding site for long chain oligouridylates, such as U12, and the standard-free-energy change on binding caused by one Pu increment is 0.41 kcal/mol, when n varies between five and fourteen. Complex formation is insensitive to the capacity of the homopolynucleotide bases to form hydrogen bonds. Homopolynuceotides, however, showing a Tm less than 250 in the buffer system used show an increased affinity for S1 compared to poly(A) and poly(C) (Tm greater than 40 degrees). The data are discussed with respect to the proposed binding of protein S1 to the 3-terminal end of the 16S RNA.     

Effects of internal nonbonded bases and a G.U base pair on the stability of a short ribonucleic acid helix.

Proton nuclear magnetic resonance has been used to examine the effect of both noncomplementary and G.U oppositions in the duplexes formed by the synthetic pentaribonucleotides CpApApUpG, CpApUpUpG, CpApGpUpG, and CpApCpUpG. The lack of any sigmoidal behavior in the chemical shift vs. temperature plots of the base protons in the individual pentaribonucleotides indicates that duplexes with noncomplementary base oppositions of the type: formula: (see text), (where X = A, U, G, or C) do not form. Variable temperature spectra of the mixture of CpApGpUpG and CpApUpUpG were recorded over the range of 70--10 degrees C. The chemical shift vs. temperature plot of the purine aromatic protons displayed sigmoidal curves. This demonstrated both duplex formation and the presence of a G.U. base pair. The average Tm of the duplex was found to be 23.4 +/- 2.0 degrees C. This is similar to that of the duplex formed by CpApUpG (24.0 +/- 1.0 degrees C) but less than the Tm of the following duplexes: CpApApUpG:CpApUpUpG (Tm = 28.5 +/- 2.1 degrees C), CpApGpUpG:CpApCpUpG (Tm = 38.4 +/- 0.6 degrees C) and CpApUpApUpG (Tm = 41.5 +/- 1.1 degrees C). The G.U base pair has a Tm (20.0 degrees C) significantly lower than the rest of the duplex (24 +/- 1 degree C) and is a region of local instability within the double helix. This 1H NMR study is the first to investigate both the formation and relative stability of an internal G.U. base pair neighboring regular Watson--Crick base pairs.     

Study of salt effects on adenosine–polyuridylic acid interaction

Complex formation between poly(U) and adenosine in solutions of salts that stabilize (Na₂SO₄), destabilize (NaClO₄), or have little effect on the water structure (NaCl), as well as the poly(U)·poly(A) interaction in NaClO₄, was studied by equilibrium dialysis and uv spectroscopy. At 3°C and neutral pH, Ado·2 poly(U) is formed in 1M NaCl and 0.33M Na₂SO₄. In NaClO₄ solutions under the same conditions, an Ado·poly(U) was found over the whole range of salt concentration investigated (10 mM?1M), which has not been previously observed under any conditions. The Ado-poly(U) was also found in a NaCl/NaClO₄ mixture, the transition from the triple- to the double-helical complex occurring within a narrow range of concentration of added NaClO₄. In the presence of 1M NaCl this transition is observed on adding as little as 10 mM NaClO₄, i.e., at a [ClO]/[Cl^?] ratio of about 1:100. However, when NaClO₄ is added to a 1M solution of the stabilizing salt Na₂SO₄, no transition occurs even at a [ClO]/[SO] ratio of 1:4. Investigation of melting curves and uv spectra has shown that in an equimolar mixture of the polynucleotides, only a double-helical poly(U)·poly(A) exists in 1M NaClO₄ at low temperatures; this also holds for 1M NaCl. This changes to a triple-helical 2 poly(U)·poly(A) and then dissociates as the temperature increases. At low temperatures and the poly(U)/poly(A) concentration ratio of 2:1, a mixture of 2 poly(U)·poly(A) and poly(U)·poly(A) was observed in 1M NaClO₄, in contrast to the case of 1M NaCl. Thus, sodium perchlorate, a strong destabilizer of water structure, promotes formation of double-helical complexes both in the polynucleotide–monomer and the polynucleotide–polynucleotide systems. Beginning with a sufficiently high ionic strength (μ ? 0.9), a further increase in the salt molarity results in an increase of the poly(U)·adenosine melting temperature in both stabilizing and neutral salts and a decrease in the destabilizing salt. In Na₂SO₄ concentrations higher than 1.2M Ado·2 poly(U) precipitates at room temperature. Analysis of the binding isotherms and melting profiles of the complexes between poly(U) and adenosine according to Hill's model shows that the cooperativity of binding, due to adenosine stacking on poly(U), increases in the order NaClO₄ < NaCl < Na₂SO₄. The free energy of adenosine stacking on the template is similar to that of hydrogen bonding between adenosine and poly(U) and ranges from ?1 to ?2 kcal/mol. The values of ΔH_t [the effective enthalpy of adenosine binding to poly(U) next to an occupied site, obtained from the relationship between complex melting temperature and free monomer concentration at the midpoint of the transition] are ?14.2, ?18.3, and ?16.8 kcal/mol for 1M solutions of NaClO₄, NaCl, and Na₂SO₄, respectively. The results indicate that the effects of anions of the salts studied are related to water structure alterations rather than to their direct interaction with the complexes between poly(U) and adenosine.     

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.     

Oligoribonucleotides containing 2',5'-phosphodiester linkages exhibit binding selectivity for 3',5'-RNA over 3',5'-ssDNA.

Oligoribonucleotides containing 2',5'-phosphodiester linkages have been synthesized on a solid support by the 'silyl-phosphoramidite' method. The stability of complexes formed between these oligonucleotides and complementary 3',5'-RNA strands have been studied using oligoadenylates and a variety of oligonucleotides of mixed base sequences including phosphorothioate backbones. In many cases, particularly for 2',5'-linked adenylates, the UV melting profiles are quite sharp and exhibit large hyperchromic changes. Substituting a few 3',5'-linkages with the 2',5'-linkage within an oligomer lowers the Tm of the complex and the degree of destabilization depends on the neighboring residues and neighboring linkages. The 2',5'-linked oligoribonucleotides prepared in this study exhibited remarkable selectivity for complementary single stranded RNA over DNA. For example, in 0.01 M phosphate buffer--0.10 M NaCl (pH 7.0), no association was observed between 2',5'-r(CCC UCU CCC UUC U) and its Watson-Crick DNA complement 3',5'-d(AGAAGGGAGAGGG). However, 2',5'-r(CCC UCU CCC UUC U) with its RNA complement 3',5'-r(AGAAGGGAGAGGG) forms a duplex which melts at 40 degrees C. The decamer 2',5'-r(Ap)9A forms a complex with both poly dT and poly rU but the complex [2',5'-r(Ap)9A]:[poly dT] is unstable (Tm, -1 degree C) and is seen only at high salt concentrations. In view of their unnatural character and remarkable selectivity for single stranded RNA, 2',5'-oligo-RNAs and their derivatives may find use as selective inhibitors of viral mRNA translation, and as affinity ligands for the purification of cellular RNA.     

Effect of 5-alkyl substitution of uracil on the thermal stability of poly [d(A-r5U)] copolymers.

The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group).     

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).     

Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function

Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.     

Selectivity of spermine homologs on triplex DNA stabilization.

We synthesized seven homologs of spermine (H2N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-9; n = 4 for spermine) and studied their effects on melting temperature (Tm), conformation, and precipitation of poly(dA).2poly(dT). The triplex DNA melting temperature, Tm1 was 34.4 degrees C in the presence of 150 mM KCl. Addition of spermine homologs increased Tm1 in a concentration-dependent and structure-dependent manner, with 3-6-3 (n = 6) exerting optimal stabilization. The dTm1/dlog[polyamine] values were 9-24 for these compounds. The duplex melting temperature, Tm2 was insensitive to homolog concentration and structure, suggesting their ability to stabilize triplex DNA without altering the stability of the underlying duplex. Circular dichroism spectral studies revealed psi-DNA formation in a concentration-dependent and structure-dependent manner. Phase diagrams were constructed showing the critical ionic/polyamine concentrations stabilizing different structures. These compounds also exerted structural specificity effects on precipitating triplex DNA. These data provide new insights into the ionic/structural determinants affecting triplex DNA stability and indicate that 3-6-3 is an excellent ligand to stabilize poly(dA).2poly(dT) triplex DNA under physiologic ionic conditions for antigene therapeutics.     

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects.

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.     

Alpha-DNA VIII: thermodynamic parameters of complexes formed between the oligo-alpha-deoxynucleotides: alpha-d(GGAAGG) and alpha-d(CCTTCC) and their complementary oligo-beta-deoxynucleotides: beta-d(CCTTCC) and beta-d(GGAAGG) are different.

The temperature dependence of the formation of a complex between an alpha-d(CCTTCC) hexanucleotide and its complementary beta-d(GGAAGG) sequence was studied and compared to the formation of the beta-d(CCTTCC):beta-d(GGAAGG) complex. Such alpha-beta complex is more stable than the regular beta:beta complex. The Tm value for the alpha:beta complex is 28 degrees C (delta G degrees = -7.3 kcal/mole) while Tm = 20, 1 degree C (delta G degrees = -6.3 kcal/mole) for the beta:beta complex. The stoechiometry of the alpha:beta complex corresponds to the formation of a 1:1 duplex. However, when the alpha- strand is made of alpha-purines: alpha-d(GGAAGG), the stability of the alpha:beta complex, alpha-d(GGAAGG):beta-d(CCTTCC) is found to be lower (Tm = 13.8 degrees C) than the stability of the regular beta-beta complex, leading to the conclusion that the nature of the alpha-sequence is important in terms of stability when considering the synthesis of such a sequence for using it as antisense oligonucleotide.     

Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).