Biology subjects

Chacko, A. N.

Publications and source records attributed to Chacko, A. N..

3 recordsLinked to original sources

bioRxiv2025

A programmable genetic platform for engineering noninvasive biosensors

Creating genetic sensors for noninvasive visualization of biological activities in deep, optically opaque tissues holds immense potential for basic research and the development of genetic and cell-based therapies. MRI stands out among deep-tissue imaging methods for its ability to generate high-resolution images without ionizing radiation. However, the adoption of MRI as a mainstream biomolecular technology has been hindered by the lack of adaptable methods to link molecular events with genetically encodable MRI contrast. To address this challenge, we introduce universal reporter circuit-based activatable sensors (URCAS), a highly programmable platform for the systematic creation of genetic sensors for MRI. In developing URCAS, we engineered protease-activatable MRI reporters using two distinct approaches: protein stabilization and subcellular trafficking. We established the applicability of URCAS in five diverse mammalian cell types and showcased its versatility by assembling a toolkit of genetic sensors for viral proteins, small-molecule drugs, logic gates, protein-protein interactions, and calcium, without requiring new customization for each target. Our findings suggest that URCAS provides a modular, programmable platform for streamlining the development of noninvasive, nonionizing, and genetically encoded sensors for biomedical research and in vivo diagnostics.

bioengineering↗

bioRxiv2024

Exploring the potential of water channels for developing MRI reporters and sensors without the need for exogenous contrast agents

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells.

bioengineering↗

bioRxiv2023

Engineering ligand stabilized aquaporin reporters for magnetic resonance imaging

Imaging transgene expression in live tissues requires reporters that are detectable with deeply penetrant modalities, such as magnetic resonance imaging (MRI). Here, we show that LSAqp1, a water channel engineered from aquaporin-1, can be used to create background-free, drug-gated, and multiplex images of gene expression using MRI. LSAqp1 is a fusion protein composed of aquaporin-1 and a degradation tag that is sensitive to a cell-permeable ligand, which allows for dynamic small molecule modulation of MRI signals. LSAqp1 improves specificity for imaging gene expression by allowing reporter signals to be conditionally activated and distinguished from the tissue background by difference imaging. In addition, by engineering destabilized aquaporin-1 variants with different ligand requirements, it is possible to image distinct cell types simultaneously. Finally, we expressed LSAqp1 in a tumor model and showed successful in vivo imaging of gene expression without background activity. LSAqp1 provides a conceptually unique approach to accurately measure gene expression in living organisms by combining the physics of water diffusion and biotechnology tools to control protein stability.

bioengineering↗