Developing New Tools to Fight Human Pathogens: A Journey through the Advances in RNA Technologies

Abstract

A long scientific journey has led to prominent technological advances in the RNA field, and several new types of molecules have been discovered, from non-coding RNAs (ncRNAs) to riboswitches, small interfering RNAs (siRNAs) and CRISPR systems. Such findings, together with the recognition of the advantages of RNA in terms of its functional performance, have attracted the attention of synthetic biologists to create potent RNA-based tools for biotechnological and medical applications. In this review, we have gathered the knowledge on the connection between RNA metabolism and pathogenesis in Gram-positive and Gram-negative bacteria. We further discuss how RNA techniques have contributed to the building of this knowledge and the development of new tools in synthetic biology for the diagnosis and treatment of diseases caused by pathogenic microorganisms. Infectious diseases are still a world-leading cause of death and morbidity, and RNA-based therapeutics have arisen as an alternative way to achieve success. There are still obstacles to overcome in its application, but much progress has been made in a fast and effective manner, paving the way for the solid establishment of RNA-based therapies in the future.

Keywords: CRISPR; RNA chaperones; RNA metabolism; RNA regulators; RNA tool; ribonucleases; small non-coding RNAs (ncRNAs); synthetic biology; virulence.

Figure 1

Landmarks on RNA technologies. An overview of the most relevant achievements and pioneer experiments around the RNA molecule. The timeline is color-coded for each field (green for synthetic biology; orange for siRNAs; purple for ncRNAs; turquoise for CRISPR/Cas systems; salmon for mRNA vaccines; yellow for RNA-seq technologies; blue for ribonucleases) [7,33,34,35,36,37,38,39,40,41,42,43].

Figure 2

(A) Targeting fundamental viral proteins to inhibit SARS-CoV-2. The use of nsp14 and nsp15 ribonucleases as druggable targets may impair SARS-CoV-2 viral replication cycle, and therefore, it can be a good way to tackle infection. (B) Synthetic sRNA expression system. Vector containing the E. coli MicC scaffold, in which a customized seed sequence complementary to the endogenous target transcript is inserted [61]. (C) RNase E mediated thermoregulation. When temperature is low, the RNase E cleavage site (RC) is hybridized with the anti-RNase cleavage site (ARC) forming a hairpin, thus blocking the cleavage by RNase E and allowing gene expression to occur. When temperature increases, the RC is exposed, the mRNA is cleaved by RNase E, and the expression of the gene is impaired (RBS stands for ribosomal binding site and AUG for the initiation codon). This is adapted from [62]. (D) Regulation of the FMN riboswitch by Ribocil. FMN riboswitch in the absence of any compound; it presents a conformation that allows gene expression to occur. Upon binding to the riboswitch, Ribocil induces a rearrangement of its structure that sequesters the RBS, thus preventing translation; this is adapted from [63]. Figure created using BioRender.com (accessed on 11 November 2022).

Figure 4

(A) Simplified workflow of a dual RNA-seq protocol. Host cells are infected in vitro with pathogen cells, lysed and total RNA is extracted. The sequencing library is prepared, and sequencing is performed in a NGS platform, obtaining simultaneously the results for both species. During bioinformatic data analysis, after quality control and data cleaning, the reads from the host and the pathogen are separated in silico in the mapping step. Annotation and quantification are carried out independently for each species, allowing to analyze host and pathogen differential gene expression in parallel, as well as to predict functional correlations between species [230]. (B) Main categories of third-generation sequencing (TGS). (Left panel) Single-molecule real-time (SMRT) sequencing—Sequence is determined through emission of fluorescence due to the incorporation of a fluorescently labelled deoxyribonucleotide (dNTP) by the DNA polymerase in the nascent complementary strand of the cDNA template molecule. The DNA polymerase is anchored to the bottom of a nanowell. (Right panel) Nanopore sequencing—Sequence is obtained without imaging. The template nucleic acid is bound to a motor protein which takes the molecule to a protein nanopore. When the template molecule is translocated through the pore, each nucleotide with its own modifications produces a characteristic current shift that is recorded. Unlike the other methods, direct RNA-seq uses an RNA molecule as template [236]. (C) Antisense oligonucleotides (ASOs) mechanism. (Left panel) General mechanism of ASOs activity. The oligonucleotide binds to the complementary RNA, impairing ribosome progression and/or causing transcript cleavage of a target duplex of mRNA/ASO by RNase H. (Right panel) Targeting of ncRNA–mRNA interaction. In this case, the ASO can be designed to mimic the ncRNA and block its binding to the mRNA (anti-mRNA ASO) or mimic the mRNA sequence to sequester the ncRNA (anti-ncRNA ASO) [247]. (D) mRNA vaccines mechanism. The nucleoside-modified mRNA containing the coding sequence of the protein of interest (SARS-CoV-2 Spike protein) is encapsulated in a lipid nanoparticle (LNP). Upon human vaccination, the LNP is internalized, and the mRNA coding sequence is recognized by the host translation machinery, leading to the production of Spike proteins. This will induce the production of specific antibodies by the host immune system, inducing an immune response cascade [248]. Figure created using BioRender.com (accessed on 11 November 2022).

Figure 3

CRISPR technologies. (A) CRISPR-based antimicrobials. The system has been successfully tested through the directed degradation of the antibiotic resistance gene located in a plasmid (left side) leading to the recovery of the bacterial antibiotic sensitivity or the directed degradation of chromosomal genes, and consequently, cell death (bactericidal) [186]. (B) CRISPR-based diagnostics. When CRISPR effector proteins (Cas) recognize the specific target site, their collateral cleavage capability is triggered (this indiscriminate nucleic acid cleavage only happens when the crRNA finds its match). The addition of a reporter, that only releases the signal upon cleavage, enables the emission of a signal that can be easily detected [187]. Figure created using BioRender.com (accessed on 11 November 2022).