Applying 6-Methylisoxanthopterin-Enhanced Fluorescence To Examine Protein-DNA Interactions in the Picomolar Range

作者：Moreno Andrew; Knee Joseph; Mukerji Ishita^*

来源：Biochemistry, 2012, 51(34): 6847-6859.

DOI：10.1021/bi300466d

Incorporation of fluorescent nucleoside analogues, into duplex DNA usually leads to a reduction in quantum yield, which Significantly limits their potential use and application. We have identified two pentamer DNA sequences containing 6-methylisoxanthopterin (6-MI) (ATFAA and AAFTA, where F is 6-MI) that exhibit significant enhancement of fluorescence upon formation of duplex DNA with quantum yields close to that of monomeric 6-MI. The enhanced fluorescence dramatically increases the utility and sensitivity of the probe and is used to study protein DNA interactions of nanomolar specificity in this work. The increased sensitivity Of 6-MI allows anisotropy binding measurements to be performed at DNA concentrations of 1 nM, and fluorescence intensity. measurements at 50 pM DNA. The ATFAA sequence was incorporated into DNA constructs to measure the binding affinity of four different protein DNA interactions that exhibit sequence specific and non-sequence-specific recognition. In all cases, the K-d values obtained were consistent with previously reported values measured by other methods. Time resolved and steady state fluorescence measurements demonstrate that 6-MI fluorescence is very sensitive to local distortion and reports on different degrees of protein-induced perturbations with single base resolution, where the largest changes occur at the site of protein binding.

• 出版日期2012-8-28