Revealing the potential of genetic circuits on single DNA molecules

In a new Natural communications In this study, researchers explored the construction of genetic circuits on single DNA molecules, demonstrating localized protein synthesis as a guiding principle for dissipative nanodevices, providing insight into artificial cell design and applications of the nanobiotechnology.

The term “genetic circuit” is a metaphorical description of the complex network of genetic elements (such as genes, promoters, and regulatory proteins) within a cell that interact to control gene expression and cellular functions.

In the field of artificial cell design, scientists aim to replicate and engineer these genetic circuits to create functional, autonomous units. These circuits act as molecular machinery responsible for orchestrating cellular processes by precisely regulating the production of proteins and other molecules.

By understanding and manipulating these circuits, researchers can design artificial cells with programmable behaviors, mimicking the functionality of natural cells.

Within the framework of the mentioned study, the focus is on building genetic circuits on single DNA molecules. This is a novel approach because it moves away from the traditional cellular context and explores the possibility of creating genetic circuits in acellular conditions.

First author, Dr. Ferdinand Greiss of the Weizmann Institute of Science in Israel, explained the researchers’ motivation to Phys.org: “We are trying to reconstruct biological processes outside of the complex circuits of living cells, hopefully improving -the, our understanding of the guiding principles of nature. research is geared toward building future artificial cells, and unique DNA molecules could be the genetic foundation. »

Gene regulation

Gene regulation is the process by which cells control gene expression, determining when and to what extent information from a gene is used in the synthesis of functional molecules like proteins or RNA. It plays a crucial role in maintaining cellular functions, responding to environmental changes and proper development.

Regulation of gene expression involves transcription and translation. During transcription, a specific segment of DNA serves as a template for the synthesis of complementary mRNA molecules by RNA polymerase. This mRNA transports the genetic code from the nucleus to the cytoplasm, where translation takes place.

Translation involves the conversion of mRNA into proteins. Ribosomes read the mRNA sequence, facilitating the assembly of amino acids into a polypeptide chain, forming the protein encoded by the gene.

“In prokaryotic systems, the processes of transcription and translation are coupled. This means that once RNA polymerase produces mRNA from DNA, the ribosome can find the ribosomal binding site on the nascent mRNA to begin synthesizing the protein. The nascent protein can fold and function while still attached to the DNA by the RNA polymerase-mRNA-ribosome complex. After transcription or translation is complete, the protein “The nascent cell detaches from the DNA and disperses into the overall solution,” explained co-author Shirley Shulman Daube of the Weizmann Institute of Science in Israel.

The importance lies in the increase in the local concentration of nascent proteins, which is approximately 1000 times that of the surrounding bulk solution. This spatial organization and increase in concentration could have implications for cellular functions and potentially play a role in the construction of artificial cells using single DNA molecules.

Building a genetic circuit on a single DNA molecule

“Genetic circuits are based on genetically encoded molecules, such as transcription factors, that are produced from DNA and bind to DNA to regulate their own production and that of other molecules,” said co-author Dr. Vincent Noireaux of the University of Minnesota. .

To build the genetic circuit on a single DNA molecule, the researchers designed specific sequences with the genes of bacteriophage lambda (E. coli).

The genetic circuit involved a negative cascade, driven by the CI repressor gene and its operator binding site, complexly controlling the HT gene. This HT gene encoded the HaloTag (HT) protein, a crucial element for visualizing nascent proteins on individual DNA molecules.

The study implemented strict conditions, including low DNA surface density, to ensure precise localized protein synthesis.

Simultaneously, a positive cascade unfolded with the fusion of bacteriophage T7 RNA polymerase (HT-T7 RNAP) and HT protein, enabling real-time monitoring of gene expression via a downstream reporter gene , GFP.

A far-red fluorogenic dye (MaP655-Halo) enhanced the detection of nascent proteins, providing a comprehensive view of genetic circuit dynamics.

The negative cascade, or suppression, regulates and inhibits the production of specific proteins under certain conditions. On the other hand, positive cascades contribute to the activation and expression of specific genes within the genetic circuit.

The research went beyond simple observation, integrating a feedback circuit with a synthetic repressor dCro. This component was crucial in regulating gene expression through a meticulously designed synthetic promoter.

Without cellular confinement

Researchers have discovered that localized protein synthesis on a single DNA molecule can drive genetic circuits in cell-free conditions, without confinement of cellular compartments. The dynamics of genetic circuits were meticulously observed under very dilute conditions.

Lead author Dr. Roy Bar-Ziv of the Weizmann Institute of Science in Israel highlighted the importance of their findings: “The regulation of gene expression depends on the binding of proteins to DNA, blocking or increasing the activity of a gene. concentrations of proteins to find and bind specific sequences on the DNA molecule. Unexpectedly, we find that localized protein synthesis can transiently increase concentration long enough for proteins to do the same without cellular confinement.

Essentially, this finding challenges the conventional notion that high concentrations are essential for gene regulation, introducing a new aspect of localized protein synthesis as a means of influencing genetic circuitry in cell-free conditions.

For future work, the researchers plan to exploit localized protein synthesis as a guiding principle to improve the functionality of artificial cells constructed from single DNA molecules, addressing challenges at low concentrations. They also foresee potential applications in self-encoded nanodevices and plan to explore correlations between DNA structure, gene expression dynamics and protein synthesis.

The research also involved contributions from MPI Medical Research’s Nicolas Lardon and Professor Kai Johnsson, who developed the fluorogenic dye (MaP655-Halo); Yoav Barak, who contributed to the optimization of DNA preparation; and Leonie Schütz with Professor Elmar Weinhold, a pioneer in the development of methyltransferases for site-specific DNA modifications with biotins.

© 2024 Science X Network