Triple helices formed by polyuridylic acid with some amino deoxyadenosine derivatives

Summary The stability of helical structures formed by polyuridylic acid with nucleosides and nucleotides derived from adenosine is not significantly affected by replacing hydroxyl groups by hydrogen, amino, or azido functions. Stability is affected by the position of the phosphate group.     

Effect of adenosine protonation on its interaction with polyuridylic acid

The effect of adenosine protonation on complex formation between poly(U) and adenosine has been studied by UV spectroscopy, titration and equilibrium dialysis techniques. A method has been developed to estimate the "misincorporation" of ionized monomer molecules into a polynucleotide--monomer complex. The method is based on combining the titration and binding data. Using this method it is shown that protonated adenosine interacts to some extent with poly(U) in the course of A.2 poly(U) dissociation at acidic pH. Qualitative differences between the effects of ionization of the polymer and monomer components on polynucleotide--monomer interaction are discussed.     

Cooperative binding of adenosine by polyuridylic acid: a further analysis

The dialysis data of Huang and Ts'o for the cooperative binding of adensine to polyuridylic acid are analyzed here using a grand-partition function Ising model method similar to that originally employed for polyelectrolytes by Rice and Nagasawa. An appropriate modification permitting the treatment of the sliding degeneracy of the two polyuridylic acid strands is also included. In addition to the previously estimated stacking energy of about ?6 kcal/mole one also obtains the free energy change F? for the transfer of a single adenosine molecule from a fixed site in solution to a fixed site on the polyuridylic acid. This binding energy falls in the range F? = ?140 to +620 cal/mole, indicating that binding in the 1:2 (purine: pyrimidine) complex is either very weak or repulsive. The absence of any comparable cooperative stacking of adenosines in solution at the same concentration together with the likely repulsive character of the binding implies that the stacking energy must contain a significant contribution from other processes than simple stacking of adenosines. A generalization of the theory to treat multicomponent binding and longer-range interactions is effected, and the form applicable to simultaneous binding of both adenosine and guanosine by polyuridylic acid is presented.     

Complex formation between polyuridylic acid and adenosine using spin-labeling

2 poly(u) . 1 adenosine complex formation was studied by spin-technique in the temperature range from 7 to 25 degrees C. It is shown that adenosine binding induces the conformational transition of poly(u) structure from flexible coil into rigid helix. The contribution of hydrogen bonding and stacking of adenosine to the total change of enthalpy and entropy upon complex formation was estimated.     

Simultaneous binding of adenosine and guanosine by polyuridylic acid: a further analysis

The dialysis data of Pitha, Huang, and Ts'o for the simultaneous binding of adenosine and guanosine to polyuridylic acid are analyzed here using a grand-partition function method described previously. The conclusion that the predominant mode of guanosine-binding cannot be a competition with adenosine for the primary hydrogen-bonding sites on the 2-polyuridylic acid complex emerges from this analysis. By setting a reasonable upper limit to the amount of competitive binding that might occur, it is found that the difference in standard free energies for the binding of guanosine and adenosine must be at least F _G ? F _A = 2400 cal/mole, provided the stacking energies for A ? A, A ? G, G ? G interactions are all equal. This difference in binding free energies implies a specificity of at least 80: 1 in favor of A on the primary sites at 5°C. Since this is a lower limit, the actual binding specificity may well be much greater. The desirability of achieving specificity through repulsion of incorrect bases, rather than via attraction of correct bases, is discussed.     

The measurement of plant polyadenylic acid by hybridisation with radioactive polyuridylic acid

Summary Saturation hybridisation of polyadenylic acid with [³H]polyuridylic acid is described. Under conditions of [³H]poly(U) excess, poly(A) is detected in the RNA of a number of higher plants. The ribonuclease resistant hybrids melt sharply when subjected to thermal denaturation. Plant RNA which contains poly(A) sequences detected by [³H]poly(U) hybridisation is polydisperse in molecular weight. Data presented shows that the amount of poly(A) in plant RNA is variable. This technique is useful for the qualitative and quantitative detection of poly(A) sequences in higher plant RNA.Abbreviations A.R. Analar Reagent - Poly(A) Polyadenylic acid - Poly(U) Polyuridylic acid - Oligo(dT)-cellulose oligo(deoxythymidylate)-cellulose - Tm melting temperature - SSC standard saline citrate     

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.     

Circular dichroism of helices formed by purine monomers with pyrimidine polynucleotides

The CD spectra of a number of helical complexes formed by purine monomers and complementary pyrimidine polyribonucleotides have been observed over the range 200–400 nm. Each of these spectra is quite similar to that of the corresponding polymer–polymer helix. The spectra are evidently determined by the geometry of the asymmetric array of bases, largely unperturbed by the ribose–phosphate backbone. The helix structure (A-form), on the other hand, is determined by the backbone of the pyrimidine homopolymer. Data on the monomer–polymer complexes support the conclusion that the CD spectra of ribohomopolymer helices depend primarily on interastrand interactions of the same transition within a given base and are relatively unaffected by transitions of the complementary base.     

Topography of nucleic acid helices in solutions. 3. Interactions of spermine and spermidine derivatives with polyadenylic-polyuridylic and polyinosinic-polycytidylic acid helices

The synthesis of spermine derivatives (II), \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}_1 {\rm R}_{\rm 2} {\rm R}_{\rm 3} \mathop {\rm N}\limits^ + \left( {{\rm CH}_2 } \right)_3 \mathop {\rm N}\limits^ + {\rm R}_{\rm 1} {\rm R}_{\rm 2} \left( {{\rm CH}_2 } \right)_2 ]_2 \cdot 4{\rm X}^ - $\end{document}, and spermidine derivatives (III), \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm R}_1 {\rm R}_{\rm 2} {\rm R}_{\rm 3} \mathop {\rm N}\limits^ + \left( {{\rm CH}_2 } \right)_4 \mathop {\rm N}\limits^ + {\rm R}_{\rm 1} {\rm R}_{\rm 2} \left( {{\rm CH}_2 } \right)_3 \mathop {\rm N}\limits^ + {\rm R}_{\rm 1} {\rm R}_{\rm 2} {\rm R}_3 \cdot 3{\rm X}^ - $\end{document}, are reported. The effects of these salts on the helix–coil transition of rA–rU and rI–rC helices were examined. Increasing the size of the hydrophobic substituents, R¹, R², and R³ lowers the degree of stabilization of the helical structure. The disproportionation reaction, 2rA–rU→rA–rU₂ + rA occurs readily with salts II and III, especially when the substituents, R₁, R₂, and R₃ are small, i.e., H or Me. Spermine is found to stabilize the rA–rU² and rI–rC helices to approximately the same extent; however, large differences between the degree of stabilization of rA–rU₂ and rI-rC helices are observed when the substituents R₁, R₂, and R₃ are large hydrophobic groups. Similar results are also obtained for the spermidine series. Finally, differences in the interactions of the salts II and III with rA–rU₂ and rI–rC helices suggest that the latter helix is denser.     

The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered.

The binding of adenosine-¹⁴C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6-double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO₃^? addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S₀(1 ? αr)² to relate the fraction of uracil rings modified (r) to the number of binding “sites” remaining (S), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms (A_b versus C_a) of modified and unmodified poly(U) can be approximated closely by the following equation: ((1)) (1) where A_b = bound A, C_a = free A, n = minimum number of adjacent A′s in complex, S = concentration of sites on poly(U), and K₁ = (K_m)^1/m for all m ≥ n. The stacking energy of adenosine (w) can be calculated accurately using the following equation, where dθ/d ln C_a is obtained from Eq. (1). ((2)) (2) For unmodified poly(U), w is ?2.0 kcal/mole and ΔG° (?;RT ln K₁) is ?3.2 kcal/mole. The difference (?1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine-5′-phosphate.     

NMR investigation of chitosan derivatives formed by the reaction of chitosan with levulinic acid

Chitosan derivatives are obtained by reaction of chitosan with a low degree of acetylation and levulinic acid under different experimental conditions. The chemical structure of the different derivatives obtained is determined using ¹H and ¹³C NMR spectroscopies. The intrinsic viscosity is used to follow the molecular weight evolution. Finally, conditions are described in which water-soluble N-carboxybutylchitosan is obtained. In particular, the time of the reduction step and the ratio between reagents are investigated. Under mild conditions and short times of reduction there is a very low degree of substitution and only the monocarboxybutylchitosan is formed. The dicarboxylated form is never observed. The cyclic derivative (5-methylpyrrolidinone chitosan) is obtained when the reducing agent is added slowly to the reactants.     

Properties of triple helices formed by oligonucleotides containing 8-aminopurines

The synthesis of parallel hairpins carrying 8-aminopurines is described. These hairpins have a high affinity for specific polypyrimidine sequences resulting in the formation of very stable triplexes.     

Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence.