Zueva, E. | Search bioRxiv

bioRxiv2025

Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis

Computational protein design can create binders against targets of interest, but identifying binders with sufficient affinity still requires biochemical screening of many designs. In this work, we test flow-induced dispersion analysis (FIDA) as a method for screening binders in a time and cost-effective manner. FIDA uses Taylor dispersion analysis to determine the hydrodynamic radius of fluorescently labelled biomolecules and their complexes. Here, we use FIDA to assess the binding of RFdiffusion-designed protein binders against the small helical peptide ALFA-tag and the GK domain of PSD-95. Successful binders can be identified in a single measurement using heat-treated bacterial lysates allowing rapid identification of binders with high affinity, and thermostability. Subsequent titration experiments show micromolar affinities for ALFA-tag binders and nanomolar affinities for GK domain binders. The lack of immobilization, the minimal sample volume, and the compatibility with complex biological samples, positions FIDA as a valuable tool for the screening and characterization of computationally generated protein binders.

biophysics↗

bioRxiv2025

Transposon invasion of primate genomes shaped human inflammatory enhancers and susceptibility to inflammatory diseases

Human immune inflammatory response reflects the evolutionary adaptation of immune-cell regulatory elements1, where recent mutations can control both pathogen defence and susceptibility to chronic inflammatory and autoimmune diseases2-4. The impact of the deeper evolutionary history of these elements within primate genomes on human inflammatory responses remains poorly understood. Evolutionary young transposons have uniquely reshaped primate genomes5 and spread novel cis-regulatory sequences6. To understand how these events influenced human inflammation, we traced sequence changes in annotated human immune-cell enhancers back to macaque. We show that Alu elements and endogenous retroviruses dispersed motifs for the inflammation-related NF-{kappa}B and IRF1, redefining their binding patterns and contributing most prominently to great ape-specific binding sites. After the human-macaque split, many of these motifs shifted toward higher predicted binding affinity. In humans, population genetics analyses reveal that positive selection favors alleles, often Alu-derived, that increase enhancer affinity toward NF-{kappa}B. Enhancers containing Alu elements are more likely to undergo positive selection at the locus level, particularly when associated with chronic inflammatory diseases. As the most mutable enhancer sequences, Alus harbor disproportionately high numbers of single nucleotide polymorphisms and significantly contribute to selected alleles, some associated with chronic inflammatory diseases. We propose that the invasion of primate-specific transposons has created unique opportunities to adapt inflammatory responses in rapidly evolving great apes, with ancestral Alus continuing to influence evolutionary potential in humans.

genomics↗