RESUMEN
OBJECTIVE: To evaluate the clinical importance of mixed mutans streptococci colonization in predicting caries in preschool children. METHODS: Caries prevalence was examined twice, with a 6-month interval, in 410 preschool children aged 3-4 years at baseline. A commercial strip method was used to evaluate the mutans streptococci score in plaque collected from eight selected interdental spaces and in saliva. Mutans streptococci typing polymerase chain reaction (PCR) assays (Streptococcus sobrinus and Streptococcus mutans, including serotypes c, e, and f) were performed using colonies on the strips as template. RESULTS: Twenty variables were examined in a univariate analysis to predict caries development: questionnaire variables, results of clinical examination, mutans streptococci scores, and PCR detection of S. sobrinus and S. mutans (including serotypes c, e, and f). Sixteen variables showed statistically significant associations (P < 0.04) in the univariate analysis. However, when entered into a logistic regression, only five variables remained significant (P < 0.05): caries experience at baseline; mixed colonization of S. sobrinus and S. mutans including S. mutans serotypes; high plaque mutans streptococci score; habitual use of sweet drinks; and nonuse of fluoride toothpaste. CONCLUSION: 'Mixed mutans streptococci colonization' is a novel measure correlated with caries development in their primary dentition.
Asunto(s)
Caries Dental/microbiología , Streptococcus mutans/fisiología , Streptococcus sobrinus/fisiología , Bebidas , Preescolar , Recuento de Colonia Microbiana , Índice CPO , Susceptibilidad a Caries Dentarias/fisiología , Placa Dental/microbiología , Carbohidratos de la Dieta/administración & dosificación , Femenino , Estudios de Seguimiento , Predicción , Encía/microbiología , Humanos , Masculino , Tiras Reactivas , Saliva/microbiología , Serotipificación , Streptococcus mutans/clasificación , Streptococcus sobrinus/clasificación , Diente Primario/microbiología , Pastas de Dientes/análisisRESUMEN
We analyzed the effect of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with 2',4'-BNA modified TFO was about 20 times larger than that observed with unmodified TFO. The observed increase in the binding constant at neutral pH by the 2',4'-BNA modification resulted from the considerable decrease in the dissociation rate constant.
Asunto(s)
ADN/química , Metano/análogos & derivados , Oligonucleótidos/química , Nucleótidos de Pirimidina/química , Hidrocarburos , Cinética , Metano/química , TermodinámicaRESUMEN
Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this paper, new DNA binding molecules; diamino-bistetrahydrofuran (bisTHF) and diamino-bisfuran are reported. The bisTHF ligand with RR configuration at the amino groups and C8 alkyl chains (RR8) stabilized GC-rich duplex. In contrast, bisfuran compounds stabilized AT-rich duplex. The binding affinity of RR8 with 12 mer duplex DNA was determined by isothermal titration calorimetry to be 3.3 x 10(8) M-1.
Asunto(s)
ADN/química , Furanos/química , Sitios de Unión , ADN/metabolismo , Furanos/síntesis química , Furanos/metabolismo , Cinética , Conformación Molecular , Conformación de Ácido NucleicoRESUMEN
The success or failure of 3D QSAR, particularly CoMFA, is most strongly dependent, especially for flexible compounds, on the conformation of the molecule used in the analysis, and on the orientation of the molecule relative to the other molecules in 3D space (i.e., alignment). The present study suggests a rational procedure for the estimation of binding conformation that uses the transferred nuclear Overhauser effect (TRNOE) experiment in combination with conformational analysis using CAMDAS (Conformational Analyzer with Molecular Dynamics And Sampling) program that is developed in our laboratory. In the next step the TRNOE-obtained conformation can be used as a reference template in order to obtain alignment of other ligands, that have a common binding site. In this step we used the SUPERPOSE program created in our laboratory, in order to estimate the binding conformation of other compounds, and to simultaneously obtain the alignment of compounds for CoMFA. The resulting CoMFA models could be expected to closely reproduce the interaction mode with protein represented by the reported X-ray results. In order to confirm the validity of our procedure described above, we show its application in obtaining CoMFA models of thermolysin inhibitors. We obtained twenty CoMFA models, and that with the highest q2 value (q2 = 0.701) was found to provide an interaction mode very similar to that represented by the X-ray results.
Asunto(s)
Relación Estructura-Actividad Cuantitativa , Termolisina/antagonistas & inhibidores , Conformación MolecularRESUMEN
I have investigated the thermodynamic and kinetic effects of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation between a 23-bp target duplex and a 15-mer TFO using electrophoretic mobility shift assay, UV melting, isothermal titration calorimetry, and interaction analysis system. The thermodynamic and kinetic analyses have clearly indicated that the PN modification of TFO not only significantly increased the thermal stability of the pyrimidine motif triplex at neutral pH but also increased the binding constant of the pyrimidine motif triplex formation at room temperature and neutral pH by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters has suggested that the more rigidity of the PN TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant of the pyrimidine motif triplex formation at neutral pH. Kinetic data have also demonstrated that the observed PN modification-mediated promotion of pyrimidine motif triplex formation at neutral pH resulted from the considerable decrease in the dissociation rate constant rather than the increase in the association rate constant. This information will present an effective approach for designing chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, which may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
Asunto(s)
Amidas/química , Biotina/análogos & derivados , ADN/química , Conformación de Ácido Nucleico , Ácidos Fosfóricos/química , Pirimidinas/química , Amidas/síntesis química , Técnicas Biosensibles , Biotina/síntesis química , Calorimetría , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Cinética , Desnaturalización de Ácido Nucleico , Oligonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , Ácidos Fosfóricos/síntesis química , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
In the present study, we describe rapid formation of stable pyrimidine motif triplex at physiological pH. The triplex formation was achieved by the synergistic effect of poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). Either the PLL-g-Dex copolymer or the PN modification alone increased the binding constant by nearly two orders of magnitude for the triplex formation at neutral pH. The combination of both stabilizing factors that was the triplex formation with the PN TFO in the presence of the copolymer increased the binding constant by nearly four orders of magnitude. The kinetic study indicated that the copolymer increased the association rate constant, whereas the PN modification decreased the dissociation rate constant. No negative interference between these stabilizing effects was observed. The kinetically orchestrated effects in which the copolymer and the PN TFO contribute to distinct ingredients in triplex equilibrium achieved the rapid formation of the stable triplex.
Asunto(s)
Amidas/química , ADN/química , Dextranos/química , Ácidos Fosfóricos/química , Polilisina/análogos & derivados , Polilisina/química , Cationes/química , Cinética , Oligonucleótidos/química , Nucleótidos de Pirimidina/químicaRESUMEN
We examined the thermodynamic properties of 2',4'-bridged nucleic acid containing 2-pyridone as a nucleobase (PB) to recognize a C interruption in the homopurine strand of the target duplex for pyrimidine motif triplex formation at neutral pH. The triplex formation involving triplex-forming oligonucleotide with PB is highly sequence-selective to specifically recognize C:G target base pair rather than the other G:C, T:A, or A:T base pairs. PB.C:G triad gives significantly larger binding constant than T.C:G triad, which has been known to be the most stable combination in natural base.C:G triad. Our results certainly support the idea that PB could be a key nucleoside to recognize a C interruption in the homopurine strand of the target duplex with high binding affinity and selectivity, and reduce the restriction of target sequences for triplex formation.
Asunto(s)
ADN/química , Piridonas/química , Emparejamiento Base , Secuencia de Bases , Citosina/química , Guanina/química , Nucleótidos de Pirimidina/química , TermodinámicaRESUMEN
Triplex DNA has attracted considerable interest recently because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted mutagenesis (1-3). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopyrimidine or homopurine triplex-forming oligonucleotide (TFO) with the major groove of the homopurine-homopyrimidine stretch in duplex DNA (1-5). In the pyrimidine motif triplex, a homopyrimidine TFO binds parallel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T[Symbol: see text]A:T and C(+[Symbol: see text])G:C triplets ([Symbol: see text] and : represent Hoogsteen hydrogen bonding and Watson Crick base pairing, respectively). (1-5). Because the cytosine bases in a homopyrimidine TFO are to be protonated to bind with the guanine bases of the G:C duplex, the formation of the pyrimidine motif triplex needs an acidic pH condition, and is thus unstable at physiological pH (6-8). On the other hand, in the purine motif triplex, a homopurine TFO binds antiparallel to the homopurine strand of the target duplex by reverse Hoogsteen hydrogen bonding to form A[Symbol: see text]A:T (or T[Symbol: see text]A:T) and G[Symbol: see text]G:C triplets (1-5). Although the purine motif triplex is pH-independent, triplexes involving guanine-rich TFOs are inhibited by physiological concentrations of certain monovalent cations (M(+)), especially K(+) (9,10).
RESUMEN
Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
Asunto(s)
ADN/química , Pirimidinas/química , Electroforesis , Concentración de Iones de Hidrógeno , Cinética , Conformación de Ácido Nucleico , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
I examined the effect of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. Both thermodynamic and kinetic analyses have indicated that the PN modification of TFO increased the binding constant of the pyrimidine motif triplex formation at pH 6.8 by nearly 2 orders of magnitude. Kinetic data have also demonstrated that the observed increase in the binding constant at neutral pH by the PN modification resulted mainly from the considerable decrease in the dissociation rate constant rather than the increase in the association rate constant. The present results certainly support the idea that the PN modification of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
Asunto(s)
Amidas/química , ADN/síntesis química , Ácidos Fosfóricos/química , ADN/química , Cinética , Oligonucleótidos/química , TermodinámicaRESUMEN
We analyzed the thermodynamics of purine motif triplex formation by isothermal titration calorimetry. The signs of calorimetric enthalpy change, delta Hcal, and entropy change, delta S, of the triplex formation were negative in the temperature range between 15 and 35 degrees C. Since an observed negative delta S was unfavorable for the triplex formation, the triplex formation was driven by a large negative delta Hcal. delta Hcal decreased with increasing temperature, yielding a negative heat capacity change, delta Cp, of approximately -1.2 kcal mol-1 K-1. We found that the binding constant, Ka, increased with increasing temperature, leading to an apparent positive van't Hoff enthalpy change, delta Hvh, which was in sharp contrast with the large negative delta Hcal. The analyses of the observed temperature dependence of Ka and delta Hcal and the negative delta Cp suggest that the purine motif triplex formation near room temperature is not a simple two-state binding process but exhibits multiple states, which was previously observed for the pyrimidine motif triplex formation near room temperature.
Asunto(s)
Oligodesoxirribonucleótidos/química , Secuencia de Bases , Calorimetría , Entropía , Cinética , Sustancias Macromoleculares , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
We analyzed the effect of a fixed N-form sugar puckering of TFO (triplex-forming oligonucleotide) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. Both thermodynamic and kinetic analyses revealed that the binding constant of the pyrimidine motif triplex formation at pH 6.8 with modified TFO containing the fixed N-form sugar puckering was about 20-times larger than that observed with unmodified TFO. Kinetic data also demonstrated that the observed increase in the binding constant at neutral pH by the fixed N-form sugar puckering resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the fixed N-form sugar puckering of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
Asunto(s)
Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/síntesis química , Secuencia de Bases , Sitios de Unión , Carbohidratos/química , Concentración de Iones de Hidrógeno , Cinética , Estructura Molecular , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
We have investigated effects of chemical modifications of a third strand on the thermodynamic and kinetic properties of the triplex formation between a 23-bp duplex and each of four kinds of 15-mer chemically modified third strands using isothermal titration calorimetry and interaction analysis system. The chemical modifications of the third strand included one base modification, with replacement of thymine by uracil; two sugar moiety modifications, RNA and 2'-O-methyl-RNA; and one phosphate backbone modification, with replacement of phosphodiester by phosphorothioate backbone. The thermodynamic and kinetic parameters obtained were similar in magnitude at room temperature for the triplex formation with the base-modified and the sugar-modified third strands. By contrast, binding constant for the triplex formation with the third strand containing phosphorothioate backbone was much smaller by a factor of 10 than that for the other triplex formations. Kinetic analyses have also demonstrated that the third strand containing phosphorothioate backbone was much slower in the association step and much faster in the dissociation step than the other third strands, which resulted in the much smaller binding constant. The reason for the instability of the triplex with the third strand containing phosphorothioate backbone will be discussed. We conclude that, at least in the triplex formation with the chemically modified third strands studied in the present work, the modification of phosphate backbone of the third strand produces more significant effect on the triplex formation than the modifications of base and sugar moiety.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Calorimetría , Técnicas In Vitro , Cinética , Pirimidinas/química , Cloruro de Sodio , TermodinámicaRESUMEN
Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
Asunto(s)
ADN , Dextranos/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Polilisina/análogos & derivados , Concentración de Iones de Hidrógeno , Cinética , Polilisina/farmacología , Pirimidinas , TermodinámicaRESUMEN
We have previously reported that in the presence of poly (L-lysine)-graft-Dextran (PLL-g-Dex) copolymer, the binding constant of the pyrimidine-motif triplex formation at neutral pH is about 100-times higher than that observed without any triplex stabilizer. Here, to explore the mechanism of the promotion effect of the PLL-g-Dex copolymer at neutral pH, the sequence specificity and the ionic strength dependence of the pyrimidine-motif triplex formation was examined in the absence or presence of the copolymer. The sequence specificity of the pyrimidine-motif triplex formation at neutral pH in the presence of copolymer was almost similar to that at acidic pH without the copolymer. As the concentration of magnesium cation increased, the binding constant of the pyrimidine-motif triplex formation without the copolymer increased. On the other hand, the binding constant of the pyrimidine-motif triplex formation in the presence of the copolymer decreased upon the increase in the concentration of magnesium cation. Considering these results in light of counterion condensation (CC) theory, we conclude that the copolymer does not hinder the sequence specificity of the triplex formation, and isolates the triplex formation from the CC effect, which may lead to a net increase in entropy change upon the triplex formation, providing a favorable component to binding constant of the triplex formation.
Asunto(s)
ADN/química , Dextranos , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Polilisina/análogos & derivados , Secuencia de Bases , Calorimetría , Concentración de Iones de Hidrógeno , Concentración Osmolar , Relación Estructura-Actividad , TermodinámicaRESUMEN
We have developed diamino-bistetrahydrofuran compounds (diamino-bisTHF) as new DNA binding molecules. Diamino-bisTHF (3:RR8) stabilized GC-rich duplex DNA with sequence specificity. DNA binding affinity increased as the alkyl chain was lengthened, indicating that the hydrophobic interaction is essential for DNA binding. It was also found that DNA binding affinity of the ligands depends on the stereochemistry of the amino group. In thermodynamic evaluation, diamino-bisTHF (3:RR8) showed a high affinity to the 12 bp duplex at a molar ratio of 1:1.
Asunto(s)
ADN/química , Furanos/química , Furanos/síntesis química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Diseño de Fármacos , Ligandos , Estructura Molecular , Desnaturalización de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
The solution structure of the B domain of staphylococcal protein A (FB) complexed with the Fc fragment of immunoglobulin G (IgG) is reported. A previous NMR analysis has shown that in solution FB is composed of a bundle of three alpha-helices, helix I, helix II, and helix III [Gouda, H., Torigoe, H., Saito, A., Sato, M., Arata, Y., and Shimada, I. (1992) Biochemistry 31, 9665-9672]. In contrast, the crystal structure of FB in the FB-Fc complex lacks helix III. Uniformly 15N- and 15N/13C-labeled FB were prepared, and the backbone 13C resonances were assigned. The spectral data obtained in the present study indicated that in solution all three helices including helix III are preserved in the FB-Fc complex. The mode of interaction of FB with the Fc fragment was discussed on the basis of the combined data of hydrogen-deuterium exchange experiments and 1H-15N correlation spectroscopy. It was concluded that a contiguous surface shaped by F14, Y15, E16, L18, and H19 in helix I, and N29, Q33, L35, and K36 in helix II is responsible for the binding.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Estructura Terciaria de Proteína , Proteína Estafilocócica A/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Deuterio , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas de Mieloma/química , Proteínas de Mieloma/metabolismo , Estructura Secundaria de Proteína , Protones , Soluciones , Proteína Estafilocócica A/químicaRESUMEN
To investigate the effect of chemical modification of the third strand on the stability of triplex DNA, we have examined the thermodynamic properties of the triplex formation between a 23-mer double-stranded homopurine-homopyrimidine and each of five kinds of 15-mer chemically modified single-stranded homopyrimidines by isothermal titration calorimetry, and the kinetic properties by interaction analysis system. The modifications of the third strand included two base modifications, two sugar moiety modifications, and one phosphate backbone modification. The thermodynamic and kinetic parameters for the triplex formation were similar in magnitude among the two base-modified and two sugar-modified single strands. By contrast, the binding constant for the triplex formation with the single strand with phosphorothioate backbone was more than ten times as small as that for the other triplex formation. On the basis of the kinetic analyses, the single strand with phosphorothioate backbone was more difficult to associate with and easier to dissociate from the target double strand than the other single strands, which resulted in the much smaller binding constant.
Asunto(s)
ADN/química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , ADN de Cadena Simple/química , Cinética , Nucleótidos de Pirimidina , TermodinámicaRESUMEN
To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 degrees C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Nitrofenoles/inmunología , Animales , Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Calorimetría , Haptenos , Fragmentos Fab de Inmunoglobulinas/química , Técnicas In Vitro , Ratones , Nitrofenoles/química , Concentración Osmolar , Fenilacetatos , Temperatura , TermodinámicaRESUMEN
In order to investigate the role of the constant domains on the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalpy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.