Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers

Abstract

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nickfree dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.

DNA interaction with DAPI fluorescent dye: Force spectroscopy decouples two different binding modes

“…[9,15,21,22] Such result strongly suggests that groove binding is the main mode of interaction between DAPI and k-DNA, a conclusion also achieved by these cited works. [22] F IG UR E 3 Representative force-extension curves obtained with our methodology in the PBS 34 mM buffer, for different DAPI concentrations. [2,20] Such result was also obtained by authors that use non-stretching (zero force) techniques, [21,[23][24][25][26][27][28] although experiments performed in a higher force regime can lead to different results.…”

“…These results show that: (a) the difference in the two ionic strengths used here do not affect the bare DNA persistence length, a result also verified by other authors [31] ; and (b) when DAPI binds to DNA, the electrostatic character of the interaction is evidenced for lower ionic strengths, which is accompanied by higher changes on the mechanical properties of the DNA-dye complexes. We have also included in the figure the results obtained from the data of Japaridze et al [9] and Wang et al, [22] which have used ionic strengths different from the two ones used in this work. Such parameter can be estimated directly from the ionic strength I of the electrolyte solution by the approximate formula 0.304/ ffiffi I p , which gives the Debye length in nanometers if the ionic strength is expressed in molar units.…”

Intercalative DNA binding governs fluorescence enhancement of SYBR Gold

“…(iii) and (iv) Broadening of the rotation curves and a slope in the pre-buckling regime (Figure 2A and Supplementary Figure S1C) are similar to what has been observed for other intercalators, notably ethidium bromide (24,47), PicoGreen (43), and the bis-intercalator YOYO-1 (27). The two effects can be understood from the properties of the DNA tethers and how they are changed upon SYBR Gold intercalation.…”

“…Conversely, small-molecule binding to DNA can alter its structure and mechanical properties. Specifically, intercalation lengthens and unwinds the DNA helix (7,8), and the changes upon intercalation into DNA have been investigated at the single-molecule level using optical tweezers (3,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21), AFM force spectroscopy (22,23), and magnetic tweezers (MT) (24)(25)(26)(27). In contrast, minor groove binding has only much smaller effects, if any, on DNA length and winding angle (24).…”

Binding mechanism of anti-cancer chemotherapeutic drug mitoxantrone to DNA characterized by magnetic tweezers

“…In brief, the surface of the flow cell was covalently coated with sigmacote (Sigma-Aldrich, Hamburg, Germany) for a homogeneous hydrophobic surface and subsequently functionalized with anti-digoxigenin (200 μg/ml, Roche, Penzberg, Germany). For MT experiments, we prepared λ-dsDNA fragments which were functionalized at one end with several biotins (Biotin-14-dCTP, Metabion, Steinkirchen, Germany) and with several digoxigenins (Dig-11-dUTP, Roche, Penzberg, Germany) at the other end according to a published protocol [ 29 , 32 , 33 ]. The 11.8 kbp fragments, corresponding to a contour length of about 4 µm, were separated by gel electrophoresis.…”