Assuntos
Pareamento Incorreto de Bases , Cátions , Química Farmacêutica/métodos , Técnicas Genéticas , Análise Heteroduplex/métodos , Ácidos Nucleicos Heteroduplexes , Polimorfismo de Nucleotídeo Único , Sítios de Ligação , Linhagem Celular Tumoral , Desenho de Fármacos , Genótipo , Humanos , Concentração Inibidora 50 , Mercúrio , Modelos Químicos , Ácidos Nucleicos Heteroduplexes/química , Prata/química , Temperatura , Raios UltravioletaAssuntos
DNA/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Pirimidinas/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Purinas/química , TermodinâmicaRESUMO
We examined the effect of mercury (II) cation on the thermal stability of heteroduplex and homoduplex. Addition of mercury (II) cation increased the melting temperature of heteroduplex containing T:T mismatch base pair by about 4 degrees C. The thermal stability of homoduplex and heteroduplexes containing other kinds of mismatch base pairs was not significantly changed by the addition of mercury (II) cation. Isothermal titration calorimetric study demonstrated that mercury (II) cation directly bound to T:T mismatch base pair in hcteroduplex at a molar ratio of 1:1. The binding constant and the enthalpy change for the binding of mercury (II) cation to T:T mismatch base pair was approximately 10(6) M(-1) and -6 kcal mol(-1), respectively. We conclude that mercury (II) cation directly binds to T:T mismatch base pair in heteroduplex with high affinity and specificity. Our results certainly support the idea that the addition of mercury (II) cation to T:T mismatch base pair in heteroduplex could be a convenient strategy for heteroduplex analysis and may eventually lead to progress in single nucleotide polymorphism genotyping.
Assuntos
Pareamento Incorreto de Bases/efeitos dos fármacos , Mercúrio/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Dímeros de Pirimidina/metabolismo , Análise Heteroduplex , Ácidos Nucleicos Heteroduplexes/efeitos dos fármacos , Termodinâmica , Temperatura de TransiçãoRESUMO
We examined the effect of morpholino (MOR) backbone modification of triplex-forming oligonucleotide (TFO) on the 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 MOR-modified TFO was approximately 60 times larger than that observed with unmodified TFO. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the MOR backbone modification resulted mainly from the considerable increase in the association rate constant. The present results certainly support the idea that the MOR backbone modification of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
