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Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.

We seek to build an optical pooled screening platform with single-molecule readouts to quantify transcription factor(TF)-DNA binding kinetics across thousands of TF variants in live cells. As a step toward this goal, this pilot study tackles the frontline challenge of scaling optical pooled screening (OPS) with chromosomal barcodes, previously demonstrated for ~10² chromosomal genotypes in Escherichia coli (E. coli) (Soares et al., 2025), to 10³–10⁴ genotypes. We implemented a dual-barcode in situ genotyping and pooled λ-Red recombineering workflow to enable high-throughput single-molecule phenotyping of LacI–mVenus variants. In a six-genotype pilot library (WT, Q18M, V52A, Q55N, G58A and a negative control lacking a specific genomic Lac operator), in situ genotyping correctly identified 5/6 strains, and live-cell imaging recovered expected phenotypes for identified strains. This pilot establishes OPS as a practical foundation for single-molecule phenotyping with large TF variants library and identifies below design constraints to address before scaling : dual-barcode decoding efficiency, rare inter-donor recombination, and instability of long LacO arrays.

Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For E. coli lac repressor (LacI), the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both in vitro on DNA microarrays with 2,479 different Lac operators and in vivo via single-molecule experiments. We conclude that a hinge region mutation causing less helix propensity enhances the specificity but reduces binding strength globally, while a hinge region mutation causing higher helix propensity has opposite effects. However, altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to IPTG induction for the strongly binding mutant. Together with earlier reports of affinity–inducibility trade-offs in LacI, our data support the model in which the hinge region governs a trade-off between binding stability and inducibility rather than between speed and binding stability.