RNA Synthetic Biology

What is Synthetic Biology?

Synthetic biology has gained considerable attention in recent years. It refers to the genetic design and modification of cells or living organisms by constructing biological functional components, devices, and systems to endow them with desired biological functions that meet human needs, and even the creation of entirely new biological systems. At its core, synthetic biology aims to make cells work for humans, producing the substances we desire. It centers around the "artificial design and writing of genomes" and can be applied to address specific needs and purposes. It leverages the diversity of substances found in the natural world and, from an engineering perspective, involves designing and constructing components or modules that allow for the reengineering or optimization of existing biological systems, the integrated analysis of life processes, or the design and synthesis of entirely new, controllable biological systems. This serves to help humans achieve goals such as disease treatment, product manufacturing, environmental management.

Figure 1. Synthetic biology-inspired cell engineering can be employed for various medical applications. ( N, L, Zhao.; et al, 2023)

Key technologies involved in synthetic biology include:

• Excavation/synthesis of crucial components (DNA/RNA sequences). The key foundational technologies in synthetic biology are used in the cell construction phase, mainly applying the "central dogma," which involves the transmission of genetic information from DNA to RNA to proteins.
• Optimization of host cells (selection and modification of strains).
• Construction of metabolic pathways/gene expression pathways and product identification.
• Methods for purification, separation, and scale-up production.

What is RNA in Synthetic Biology?

RNA is an attractive molecule for synthetic biology for several reasons. Firstly, RNA plays a role in controlling gene expression at both the transcription and translation levels, and it is an essential component of the CRISPR/Cas gene editing system. Secondly, the relationship between RNA sequences and their secondary or tertiary structures is well-characterized, allowing for the programming of interactions between single or multiple RNA chains to achieve specific structures or desired binding event sequences. Thirdly, RNA molecules can be designed to bind to various ligands (small molecules, proteins, and other RNAs), making them useful as sensors and actuators. Lastly, because RNA naturally exists in all living organisms, synthetic RNA molecules can be easily transplanted into different hosts.

Figure 2. Synthetic RNA molecules expressed within cells. (J, M, Kim.; et al, 2020)

What are the Applications of Synthetic RNA?

• Synthetic RNA for Transcription Regulation

Synthetic RNA molecules expressed within cells can be optimized for various tasks, including controlling gene expression and organizing small molecules and proteins. While protein regulators played a critical role in the early development of synthetic biological circuits, significant progress has been made in creating libraries of synthetic RNA genetic regulators. RNA-mediated gene expression control typically involves specific structural motifs within mRNA, such as intrinsic terminator structures in bacteria that prevent transcription elongation. Small RNA-based transcription activators have been developed to control the expression of multi-gene metabolic pathways.

• Engineering RNA to Direct the CRISPR/Cas System

Clustered regularly interspaced short palindromic repeats (CRISPR) motifs occur naturally in prokaryotes.CRISPR and CRISPR-associated proteins (Cas) function as microbial analogs of the acquired immune system in higher organisms.The versatility, modularity, and efficacy of the CRISPR-Cas system have a wide range of applications, including genome editing, post-transcriptional engineering, imaging, and diagnostics.The CRISPR interference (CRISPRi) system efficiently represses the expression of target genes in E. coli with no detectable off-target effects. Engineering the structure of a guide RNA (gRNA) to conditionally control Cas9 activity is an effective alternative to engineering the Cas9 protein itself. The activity of Cas9 is tolerant to significant modifications to the structure of standard gRNAs involving small molecules and nuclease-controlled auxiliary structural domains for gRNA activity. Antisense rna was shown to be effective in inhibiting the active form of gRNA. Switching of programmable RNA structures can further extend the regulatory functions of gRNAs. the potential of the CRISPR-Cas gRNA engineering strategy as a tool of choice for the control of synthetic gene circuits in bacterial and mammalian cells.

• Synthetic RNA for Control of Translation

Synthetic RNA translation activators known as ribose regulators were among the first synthetic RNA regulatory devices . Like natural sRNA regulators, the molecular structure of synthetic ribonucleic acid regulators prevents the ribosome from binding to the RBS, thereby inhibiting translation initiation. A trans-acting RNA could be designed to unwind the hairpin structure, releasing the RBS and allowing the ribosome to enter and continue translation.

• Organizing Molecular Components with RNA

Due to the ability to predict RNA base pairing through various algorithms, RNA has become a versatile polymer for constructing self-assembled structures at the nanoscale. These nanostructures include both natural and engineered structural patterns, where sequences are locally folded into known secondary or tertiary structures. These structural motifs can be modularly combined to obtain molecular scaffolds with desired shapes, including ligands that can spatially organize target ligands on the scaffold.

References

1. N, L, Zhao.; et al. Synthetic Biology-inspired Cell Engineering in Diagnosis, Treatment, and Drug Development. Signal Transduction and Targeted Therapy.2023,8: 112.
2. J, M, Kim.; et al. Synthetic RNA Molecules Expressed within Cells. Current Opinion in Biotechnology. 2020, 63: 135–141.