RNA-based biosensors win a race for metabolite-sensing in cells

Dr. Sudeshna Manna’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Manna is currently a postdoctoral scholar at the group of Prof. Ming Hammond in the University of Utah, USA, where she works on RNA engineering to develop genetically encodable tools for synthetic biology, cell biology, and biotechnology applications. She pursued her Ph.D. in 2019 in the Indian Institute of Science Education and Research (IISER) Pune, India, under the supervision of Prof. S. G. Srivatsan. Her Ph.D. was focused on the chemical biology of nucleic acids, particularly on the development of nucleoside probes to understand the structure and recognition of nucleic acids in the cell-free and cellular environment. Before joining her Ph.D., she obtained her master’s and bachelor’s degrees in chemistry from the Indian Institute of Technology (IIT) Guwahati and Jadavpur University, Kolkata, India, respectively. Her research interest revolves around synthetic biology, nucleic acids, and protein-based tools for diagnostics and therapeutics and exploring new antimicrobial targets. Here, Sudeshna talks about her first author work on “Guanidine Biosensors Enable Comparison of Cellular Turn-on Kinetics of Riboswitch-Based Biosensor and Reporter” published in ACS Synthetic Biology (2021).

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How would you explain your paper’s key results to the non-scientific community?

It is necessary to sense dynamic biological processes in the cells in various biotechnology and synthetic biology applications. An important criterion in such sensing processes is that the time scale of the target biological event should match with the time scale of the activation of the in vivo sensors. Over the years, considerable efforts have been laid to develop novel biosensors for the important target molecules. However, optimization and analysis of the activation kinetics of the cell-based biosensors remain unexplored.


Figure 1. Riboswitch-based guanidine biosensor with fast in vivo activation kinetics. (a) Proposed activation mechanism of guanidine biosensors. (b) Comparison of in vivo turn-on kinetics shows 15 times faster kinetics of the biosensor than the reporter derived from the same riboswitch.

In recent years, RNA-based biosensor has emerged as an attractive tool for real-time in vivo sensing. In this article, we studied how fast an RNA-based biosensor gets activated in the cells and how efficient it is compared to other RNA-based tools. Using flow cytometry, we compared the in-cell turn-on kinetics of two genetically programmable riboswitch-based sensing tools, a riboswitch-based reporter and a riboswitch-based biosensor, in response to a bacterial metabolite, guanidine. Riboswitch is a cis-regulatory RNA element that, after binding to a particular target, undergoes conformational changes and regulates gene expressions in bacteria. In guanidine reporter, guanidine riboswitch is fused to the upstream of a reporter gene so that the guanidine binding induces the reporter gene expression. On the other hand, in guanidine biosensors, guanidine riboswitch is fused to a fluorogenic dye (DFHBI) binding aptamer in such a way that the guanidine binding facilitates the fluorescence turn-on (Fig 1a). We chose the guanidine as a target molecule for our study because it is cell-permeable and nontoxic to bacterial cells up to millimolar concentration. This allowed us to compare the activation kinetics of the biosensor and the reporter by direct addition of guanidine to the cells.

The key finding of our study is that the guanidine biosensor responds as quickly as in 4 min of the addition of guanidine in the cells, which is ~ 15 times faster than the reporter derived from the same riboswitch (Fig 1b). To our knowledge, this is the first study that compares the activation kinetics of a biosensor and a reporter for a particular target. Another exciting finding of this study is that after being expressed in bacteria, the biosensor maintains its fast-sensing activity for up to 11 days. These key results highlight the efficacy of an RNA-based biosensor in detecting dynamic processes for various applications. (The work is featured on the front cover of the March issue of ACS Synthetic Biology. https://pubs.acs.org/toc/asbcd6/10/3)

“The study showcases the response time of an RNA-based tool that involves RNA folding and its cognate ligand binding.”

What are the possible consequences of these findings for your research area?

The genetically encodable label-free RNA-based fluorescent biosensor is an emerging tool in the field of cell-based sensors. It is crucial to ameliorate the biosensors ‘ activation kinetics to develop a good biosensor that is suitable for real-time sensing of dynamic biological processes. Our study shows the fast activation of the programmable RNA-based biosensors. The study showcases the response time of an RNA-based tool that involves RNA folding and its cognate ligand binding. Hence, this study will help scientific community interested in designing RNA-based genetic devices for various applications.

What was the exciting moment (eureka moment) during your research?

The eureka moment for us was when we observed our biosensors were able to detect guanidine within 4 min of its addition in the cells. We were also amazed by the observation that these RNA-based biosensors, once expressed in bacteria, are stable and maintain their activity for up to 11 days.

What do you hope to do next?

My next research goal is to develop a high-throughput method to study the interactions of the antimicrobial agents with therapeutically relevant riboswitches and the effects of antimicrobial agents on riboswitches’ regulatory action.

Where do you seek scientific inspiration?

The excitement of discovering the unknown and having opportunities to learn something new every day always drives me to do science. At a very young age, I came across the biography of great scientists Marie Curie, Rosalind Franklin, and Stephen Hawking. I realized the joy of discovery and the happiness of untangling a mystery. The exemplary work done by scientists worldwide have always remained my scientific inspiration. Throughout my academic carrier, I have been fortunate to have excellent mentors who inspired me in many ways to achieve my scientific goal and made me confident to choose a career in science.

How do you intend to help Indian science improve?

The current pandemic situation worldwide has reminded us the importance of scientific research in saving lives. From the very early stage of my Ph.D., I have been interested in research that is beneficial for on human health. So, I would be happy to help Indian science by developing new techniques in disease diagnostics and therapeutics, which will benefit the people of our country. Besides, I am interested in scientific communication with the general public to help build logical minds in society.

Reference

Sudeshna Manna, Johnny Truong, and Ming C. Hammond. Guanidine Biosensors Enable Comparison of Cellular Turn-on Kinetics of Riboswitch-Based Biosensor and Reporter. ACS Synthetic Biology 2021 10 (3), 566-578. DOI: 10.1021/acssynbio.0c0058.3

Email: sudeshna.manna@utah.edu

Google Scholar: https://scholar.google.com/citations?user=ZSXdo5UAAAAJ&hl=en

Hammond Lab Link: https://www.mchgrp.chem.utah.edu/

Edited by: Manveen K Sethi (Volunteer, Bio Patrika)

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