Mechanical Properties of Base-Modified DNA Are Not Strictly Determined by Base Stacking or Electrostatic Interactions

Abstract

This work probes the mystery of what balance of forces creates the extraordinary mechanical stiffness of DNA to bending and twisting. Here we explore the relationship between base stacking, functional group occupancy of the DNA minor and major grooves, and DNA mechanical properties. We study double-helical DNA molecules substituting either inosine for guanosine or 2,6-diaminopurine for adenine. These DNA variants, respectively, remove or add an amino group from the DNA minor groove, with corresponding changes in hydrogen-bonding and base stacking energy. Using the techniques of ligase-catalyzed cyclization kinetics, atomic force microscopy, and force spectroscopy with optical tweezers, we show that these DNA variants have bending persistence lengths within the range of values reported for sequence-dependent variation of the natural DNA bases. Comparison with seven additional DNA variants that modify the DNA major groove reveals that DNA bending stiffness is not correlated with base stacking energy or groove occupancy. Data from circular dichroism spectroscopy indicate that base analog substitution can alter DNA helical geometry, suggesting a complex relationship among base stacking, groove occupancy, helical structure, and DNA bend stiffness.

Nanomechanics of Diaminopurine-Substituted DNA

“…This result is consistent with what has been reported previously for experiments using AFM and optical tweezing (14,19). Similarly, previously reported MT experiments measuring DNA under supercoiling found that DAP substitution increased the size of plectonemic writhes, a finding that is consistent with DAP-DNA being harder to bend (6).…”

“…To further investigate the effect of DAP substitution upon DNA conformational stability in the low-tension (entropic) and high-tension (overstretching) regimes, we performed a series of atomic force microscopy (AFM) imaging, magnetic tweezer (MT)-based stretching, circular dichroism (CD) spectroscopy, and thermal melting experiments on equivalent WT and DAP-substituted DNA sequences. The AFM and MT experiments confirm the findings of Virstedt et al and Peters et al that under low tension, DAP-DNA is flexurally stiffer (14,19). Interestingly, the AFM and MT assays yielded conflicting measurements of molecular contour length.…”

“…The spectra of all four WT molecules show the expected behavior of B-DNA, specifically a local maximum in the 270-280 nm range and a local minimum around 250 nm (41,42). The spectrum of 4.6 kb DAP molecules is very similar to the (14,19). Interestingly, the 40, 54, and 65% GC sequences show a progressive shift from non-WT behavior toward a WT-like spectrum.…”

“…2) by DDE ¼ À0.12 5 0.03 kcal/mol. Interestingly, this is smaller than one might expect, given reported DAPstacking energies measured by the ''dangling-bond'' assay (18,19,36). Peters et al report that stacking interactions of a single DAP base with a neighboring GC basepair contributes approximately À5 kcal/mol to the enthalpy of hybridization, compared with À4 kcal/mol for adenine (18,19).…”

“…This additional bond strengthens the effective interaction between the paired backbones and, like sequences with high GC content, raises the melting temperature (1,14). Second, the additional amino group extends into the minor groove of the B-form DNA helix (B-DNA), decreasing the compressibility of the groove through steric hindrance, reducing bending flexibility, and biasing the molecule to deviate from the typical B-form conformation at low tension (15)(16)(17)(18)(19). The decrease in bending flexibility results in DAP-containing molecules behaving as stiffer entropic springs, characterized by longer persistence lengths, and forming larger plectonemic loops upon supercoiling (6).…”