Conformational dynamics of DNA bulge loops investigated by CE-LIF

Abstract

Capillary electrophoresis with laser induced fluorescence (CE-LIF) detection has been utilized to investigate the metastable states of DNA bulge loops during strand displacement reactions. DNA samples consisting of one of the oligonucleotides with sequences of X(CAG)_nY (n = 4–8, X = CACGAGCACGC and Y = CACACCGAAGC) and a 22-mer oligonucleotide with a sequence of Y′X′ (Y′ = GCGTGCTCGTG and X′ = GCTTCGGTGTG) were separated within 7 min by CE-LIF using 2.7% poly(ethylene oxide) (PEO) in the presence of EOF. Our CE data allowed calculation of the equilibrium constant (K) of the ds-DNA of 34-mer X(CAG)₄Y and 22-mer Y′X′ at 25 °C to be 1.3 (±0.1) × 10⁹ M⁻¹, with a rate constant (k) of 3.7 (±0.4) × 10⁵ M⁻¹ s⁻¹. We have further applied this CE-LIF technique to separately study the strand displacement of a ds-DNA of the 34-mer X(CAG)₄Y and 22-mer Y′X′ bulge loop DNA with a 22-mer XY and that of a ds-DNA of 22-mer XY and Y′X′ with the 34-mer X(CAG)₄Y. Our study reveals that the metastable DNA bulge loops and unbinding oligonucleotides were coexistent at room temperature, while they underwent displacement during a heating/cooling cycle. Our study reveals that this CE-LIF approach has great potential for the investigation of DNA related biological functions.