Biology subjects

Glasscock, C. J.

Publications and source records attributed to Glasscock, C. J..

4 recordsLinked to original sources

bioRxiv2026

Generative design of sequence specific DNA binding proteins

De novo protein design has advanced rapidly in recent years, yet the programmable recognition of specific DNA sequences remains a longstanding challenge. Here we describe a deep learning based approach for designing sequence selective DNA binding proteins. Our method combines structure generation using RFdiffusion3 with explicit screening against off-target interactions using AlphaFold3. We test this approach by generating 96 designs for each of 15 diverse DNA targets and identify specific binders for 7 targets, representing a ~100-fold improvement in success rates over previous approaches. We further characterize the binding landscape using variant competition assays and randomized library screening, revealing robust sequence discrimination across diverse targets. Together, these results represent a significant step forward in de novo sequence specific DNA binder design.

synthetic biology↗

bioRxiv2023

Computational design of sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing.

synthetic biology↗

bioRxiv2019

Dynamic control of pathway expression with riboregulated switchable feedback promoters

Dynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the hosts natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.

synthetic biology↗

bioRxiv2018

De novo Design of Translational RNA Repressors

A central goal of synthetic biology is the development of methods for the predictable control of gene expression. RNA is an attractive substrate by which to achieve this goal because the relationship between its sequence, structure, and function is being uncovered with increasing depth. In addition, design approaches that use this relationship are becoming increasingly effective, as evidenced by significant progress in the de novo design of RNA-based gene regulatory mechanisms that activate transcription and translation in bacterial cells. However, the design of synthetic RNA mechanisms that are efficient and versatile repressors of gene expression has lagged, despite their importance for gene regulation and genetic circuit construction. We address this gap by developing two new classes of RNA regulators, toehold repressors and looped antisense oligonucleotides (LASOs), that repress translation of a downstream gene in response to an arbitrary input RNA sequence. Characterization studies show that these designed RNAs robustly repress translation, are highly orthogonal, and can be multiplexed with translational activators. We show that our LASO design can repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity and predictability. These results demonstrate significant yet easy-to-implement improvements in the design of synthetic RNA repressors for synthetic biology, and point more broadly to design principles for repressive RNA interactions relevant to modern drug design.

synthetic biology↗