Binding Kinetics of Bisintercalator Triostin A with Optical Tweezers Force Mechanics

Abstract

The binding kinetics of the intercalative binding of Triostin A to lambda-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.

The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent

“…First, we showed that at equilibrium SYTOX green occupies a minimum of ~3.5 bp. These values are in agreement with those reported for other monointercalators, such as ethidium bromide (~2.4-3 bp) (Vladescu et al 2007), and YO-1 (~4 bp) (Murade et al 2009), or for bis-intercalators such as YOYO-1 (3.2 bp) (Günther et al 2010) or triostin A (4 bp) (Kleimann et al 2009). Secondly, we showed that upon intercalation of SYTOX green, the extension of the DNA molecule increases by ~43 %, which is consistent with fractional elongations reported for other intercalators (17-47 %) (Chaurasiya et al 2010).…”

“…By combining the binding site size and the fractional elongation, we can calculate that intercalation of SYTOX green increases the DNA length by 0.51 nm/dye = 1.5 bp/dye. This elongation of DNA upon intercalation is larger than that reported for other monointercalators, such as ethidium bromide (0.27-0.34 nm/dye) (Chaurasiya et al 2010;Coury et al 1996;Sischka et al 2005;Vladescu et al 2007) or YO-1 (0.32 nm/dye) (Chaurasiya et al 2010;Murade et al 2009), and similar to that reported for bis-intercalators such as YOYO-1 (0.54 nm/ dye) (Günther et al 2010) or triostin A (~0.44 nm/dye) (Kleimann et al 2009). Further information on the specific chemical structure and valence of SYTOX green will be needed to explain this discrepancy.…”

Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers

“…The binding site size was estimated as n ¼ 3:2250:3 bp/dye. This comparably large value reflects the fact that bis-intercalation inhibits the occupation of adjacent base pair voids (29). This negative cooperativity is referred to as ''nearest neighbor exclusion principle'' (30).…”

Biophysical characterization of DNA binding from single molecule force measurements

“…A recent study shows that bis-intercalator triostin [73] exhibits properties similar to YOYO, with very slow kinetics. Thus the force extension curves obtained are non-equilibrium even at very slow pulling rates.…”

“…Minor groove binders [58-59] and major groove binders do not change the contour length, while intercalators [58-59, 66] and the bis-intercalator YOYO [59, 66] exhibit an increase in contour length for DNA-ligand complexes. Concentration-dependent studies on intercalators and bis-intercalators show that the contour length increases with the increase in concentration until saturation at a specific concentration [54-55, 65, 73]. …”