Quantifying length-dependent DNA end-binding by nucleoproteins.

The ends of nucleic acids oligomers alter the statistics of interior nonspecific ligand binding and act as binding sites with altered properties. While the former aspect of "end effects" has received much theoretical attention in the literature, the physical nature of end-binding, and hence its potential impact on a wide range of studies with oligomers, remains poorly known. Here, we report for the first time end-binding to DNA using a model helix-turn-helix motif, the DNA-binding domain of ETV6, as a function of DNA sequence length. Spectral analysis of ETV6 intrinsic tryptophan fluorescence by singular value decomposition showed that end-binding to nonspecific fragments was negligible at >0.2 kbp and accumulated to 8% of total binding to 23-bp oligomers. The affinity for end-binding was insensitive to salt but tracked the affinity of interior binding, suggesting translocation from interior sites rather than free solution as its mechanism. As the presence of a cognate site in the 23-bp oligomer suppressed end-binding, neglect of end-binding to the short cognate DNA does not introduce significant error. However, the same applies to nonspecific DNA only if longer fragments (>0.2 kbp) are used.