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. 2023 Mar 23;51(11):5364–5376. doi: 10.1093/nar/gkad207

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Quantitative analysis of ZFY reveals that its downstream recognition depends on the presence of perfect core. (A) Workflow of two rounds of HT-SELEX with previously identified binding site as the anchor position. (B) Tested constructs, Spec-seq library design and the variants distribution of observed binding energy under human ZFY (F7-F13) construct. (C) Position energy logo constructed by regression of all single variants of reference site. (D) Dependent recognition model for Zinc Finger Y (ZFY); Without intact core, there is no observed specificity towards downstream region. (E) Energy levels for representative binding sites under different constructs; S.S.N. is short name for Specific-Specific-Non-Specific, and so on. (F) Workflow of florescence anisotropy assay to measure the intrinsic dissociation rates of various protein-DNA complexes; >200X unlabeled competitor probe was added to the pre-equilibrated binding reaction right before the kinetics monitoring processes. (G) Upper panel shows dissociation curves for full-length ZFY over various DNA probes; Lower panel shows dissociation curves for full-length and various truncated ZFY constructs over S.S.S. (long) DNA probe; Single phase exponential curve was used to fit all observed data, except the ZFY-Full over S.S.S. (long) in lower panel, which shows two-phase exponential curve; Residual Standard Error (RSE) were listed on he upper right panels. (H) Summary of observed dissociation rates of different constructs over various probes; Each sample was measured for at least three times; Sample means and standard deviations were derived from fitted single exponential curves.