The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA.
Biochem Biophys Res Commun
; 403(2): 225-9, 2010 Dec 10.
Article
em En
| MEDLINE
| ID: mdl-21073861
ABSTRACT
The cyanine dye, YOYO-1, has frequently been used in single DNA molecule imaging work to stain double-stranded DNA as it fluoresces strongly when bound. The binding of YOYO-1 lengthens the DNA due to bis-intercalation. We have investigated the kinetics of binding, via this increase in DNA length, for single, hydrodynamically-stretched molecules of lambda DNA observed via Total Internal Reflection Fluorescence (TIRF) microscopy. The rate and degree of lengthening in 40mM NaHCO(3) (pH 8.0) buffer depend upon the free dye concentration with the reaction taking several minutes to reach completion even in relatively high, 40nM, concentrations of YOYO-1. In the absence of overstretching of the DNA molecule, we determine the second order rate constant to be 3.8±0.7×10(5)s(-1)M(-1), the dissociation constant to be 12.1±3.4nM and the maximum DNA molecule extension to be 36±4%. The intercalation time constant (inverse of the pseudo-first order rate constant), τ, decreased from 309 to 62s as YOYO-1 levels increased from 10 to 40nM. The kinetics of binding help with interpretation of the behavior of DNA-YOYO-1 complexes when overstretched and establish defined conditions for the preparation of DNA-YOYO-1 complexes.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Compostos de Quinolínio
/
Benzoxazóis
/
DNA
/
Corantes Fluorescentes
/
Substâncias Intercalantes
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article