Biology subjects

Siddiquee, R.

Publications and source records attributed to Siddiquee, R..

4 recordsLinked to original sources

bioRxiv2026

Directed evolution of multimeric proteins is enabled by dual-compensatory gene duplication

Gene duplication has played a critical role in the evolutionary history of proteins, enabling complex multimers to emerge from simpler precursors. Yet in protein engineering, current methods for directed evolution do not exploit gene duplication, hampering access to the vast array of diverse variants that are only enriched in the presence of a wild-type copy. We establish a directed evolution strategy for multimeric proteins that harnesses gene duplication to compensate for metabolic burden and self-assembly fitness, allowing previously inaccessible variants to be enriched. Starting from a homomeric 240-mer capsid, gene duplication enables selection of both extreme homomeric variants and obligate heteromers. This strategy significantly expands engineering access to diverse high-performing variants, while also supporting a plausible model for evolutionary diversification of higher-order multimers in nature.

evolutionary biology↗

bioRxiv2024

High-fidelity in vitro packaging of diverse synthetic cargo into encapsulin protein cages

Cargo-filled protein cages are powerful tools in biotechnology with demonstrated potential as catalytic nanoreactors and vehicles for targeted drug delivery. While endogenous biomolecules can be packaged into protein cages during their expression and self-assembly inside cells, synthetic cargo molecules are typically incompatible with live cells and must be packaged in vitro. Here we report a fusion-based in vitro assembly method for packaging diverse synthetic cargo into encapsulin protein cages that outperforms standard in cellulo assembly, producing cages with superior uniformity and thermal stability. Fluorescent dyes, proteins and cytotoxic drug molecules can all be selectively packaged with high efficiency via a peptide-mediated targeting process. The exceptional fidelity and broad compatibility of our in vitro assembly platform enables generalisable access to cargo-filled protein cages that host novel synthetic functionality for diverse biotechnological applications.

biochemistry↗

bioRxiv2024

A programmable seeker RNA guides target selection by IS1111 and IS110 type insertion sequences.

IS1111 and IS110 insertion sequence (IS) family members encode an unusual DEDD transposase type and exhibit specific target site selection. The IS1111 group include identifiable subterminal inverted repeats (sTIR) not found in the IS110 type [1]. IS in both families include a noncoding region (NCR) of significant length and, as each individual IS or group of closely related IS selects a different site, we had previously proposed that an NCR-derived RNA was involved in target selection [2]. Here, we found that the NCR is usually downstream of the transposase gene in IS1111 family IS and upstream in the IS110 type. Four IS1111 and one IS110 family members that target different sequences were used to demonstrate that the NCR determines a short seeker RNA (seekRNA) that co-purified with the transposase. The seekRNA was essential for transposition of the IS or a cargo flanked by IS ends from and to the preferred target. Short sequences matching both top and bottom strands of the target were identified in the seekRNA but their order in IS1111 and IS110 family IS was reversed. Reprogramming the seekRNA and donor flank to target a different site was demonstrated, indicating future biotechnological potential for these systems.

microbiology↗

bioRxiv2020

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a {beta}-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.

biochemistry↗