Determining the Elasticity of Short DNA Fragments using Optical Tweezers and Protein-Mediated DNA Loop Formation Assays

Abstract

In the light of the recent controversy over whether DNA on short length scales becomes more elastic or not, we contribute two additional measurements that take sequence effects into account: We have directly stretched short DNA molecules of varying AT / CG content with optical tweezers to determine their persistence length. Scaling the result for known end-point entropic effects, we find a persistence length of 44±2nm and 59±3nm for the AT and CG rich constructs, respectively, but no appreciable overall softening, in line with our previous measurements that found no softening in DNA molecules as short as 250 bp. Secondly, we have used tethered particle motion (TPM) microscopy to observe protein-mediated DNA loop formation in the lactose repressor system with the same DNA constructs between the operators. While an absolute determination of the persistence length of the DNA from such kinetic measurements is notoriously difficult, as ongoing discussions of ring cyclization experiments by various groups suggest, we can unequivocally state that any sequence effect in these measurements is much smaller than what we found in the stretching experiments with the optical tweezers. This suggests two distinct response regimes of DNA to force: one for low curvatures commensurate with thermal forces, in which elastic-rod descriptions for the DNA hold and sequence effects are observed, and a second non-linear high curvature region in which elasticity is not sequence-dependent and likely dominated by the conformation of the phosphate backbone.