In α-proteobacteria, strict regulation of cell cycle progression is necessary for the specific cellular differentiation required for adaptation to diverse environmental niches. The symbiotic lifestyle of Sinorhizobium meliloti requires a drastic cellular differentiation that includes genome amplification. To achieve polyploidy, the S. meliloti cell cycle program must be altered to uncouple DNA replication from cell division. In the α-proteobacterium Caulobacter crescentus, cell cycle-regulated transcription plays an important role in the control of cell cycle progression but this has not been demonstrated in other α-proteobacteria. Here we describe a robust method for synchronizing cell growth that enabled global analysis of S. meliloti cell cycle-regulated gene expression. This analysis identified 462 genes with cell cycle-regulated transcripts, including several key cell cycle regulators, and genes involved in motility, attachment, and cell division. Only 28% of the 462 S. meliloti cell cycle-regulated genes were also transcriptionally cell cycle-regulated in C. crescentus. Furthermore, CtrA-and DnaA-binding motif analysis revealed little overlap between the cell cycle-dependent regulons of CtrA and DnaA in S. meliloti and C. crescentus. The predicted S. meliloti cell cycle regulon of CtrA, but not that of DnaA, was strongly conserved in more closely related α-proteobacteria with similar ecological niches as S. meliloti, suggesting that the CtrA cell cycle regulatory network may control functions of central importance to the specific lifestyles of α-proteobacteria.cell cycle regulation | symbiosis | alpha-proteobacteria
The delivery of small interfering RNA (siRNA) remains a major hurdle for the clinical translation of RNA interference (RNAi) therapeutics. Due to its low valency and rigid nature, siRNA typically requires high excesses of cationic delivery materials to package it stably and deliver it to the cytoplasm of target cells, resulting in high toxicities and inefficient gene silencing in vivo. To address these challenges, we pair a polymeric form of siRNA, p-shRNA, with optimized biodegradable polycations to form stable complexes that induce far more potent gene silencing than with siRNA complexes. Furthermore, we unveil a set of design rules governing p-shRNA delivery, using degradable polycations containing hydrophobic and stabilizing polyethylene glycol domains that enable both stable condensation and efficient release inside cells. We demonstrate the therapeutic potential of this approach by silencing the oncogene STAT3 in a well-established B16F10 mouse melanoma model to significantly prolong survival. By blending nucleic acid engineering and polymer design, our system provides a potentially translatable platform for RNAi-based therapies.
Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.
Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3′ terminal poly adenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complex synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilize the ribonucleoproteins (RNPs) via a family of polypeptides bearing different arrangements of cationic side groups. We find that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and ~4-fold increase in mice. These findings suggest a set of new design principles for gene delivery via synergistic co-assembly of mRNA with helper proteins.
Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3′ terminal poly adenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complex synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilize the ribonucleoproteins (RNPs) via a family of polypeptides bearing different arrangements of cationic side groups. We find that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and ~4-fold increase in mice. These findings suggest a set of new design principles for gene delivery via synergistic co-assembly of mRNA with helper proteins. Graphical AbstractCo-assembly of mRNA/PABP nanoplexes with polyamines enhances mRNA translation via improvement of both mRNA stability and protein translation. [1][2][3][4] Unlike protein-based biologics, mRNA co-opts natural biological processes to express proteins and thereby achieve a desired therapeutic effect. [5][6] In contrast to DNA, however, mRNA does not need to enter the nucleus to be functional, which allows for transfection of non-dividing cells with potentially high efficiency. [7] Additionally, mRNA does not integrate into the genome and hence has little risk of insertional mutagenesis. [8][9] These advantages enable the potential treatment of a broad spectrum of diseases, many of which cannot be addressed with current technologies. [1][2] Nevertheless, inefficient transfection of exogenous mRNA remains a key barrier to broad applications of mRNA-based drugs. [10] Endogenous mRNA associates with specific proteins at different stages when trafficking from the nucleus to cytoplasmic ribosomes for maximal protein production. In contrast, the existing approach towards transfecting exogenous mRNA remains direct complexation of mRNA with cationic carriers. [11][12][13] These carriers are designed to protect mRNA from degradation, enable uptake by cells, and facilitate endosomal escape. [10] However, it was recently reported that certain polycation-based delivery vehicles can partially block mRNA from effectively recognizing the complementary protein responsible for mRNA translation. [14] To circumvent this problem, we recently demonstrated that preloading the 5′ end of mRNA with recombinant cap-binding protein, eIF4E, increases the formation of the mRNA translation initiation complex and leads to enhanced mRNA transfection via improved mRNA stability and expression inside cells. [15] Nevertheless, the 3′ poly A tail of mRNA is susceptible to de-adenylation (an enzyme-mediated hydrolysis of poly A), which can trigger RNA degradation by 3′-5′ RNA exonucleases in the cytoplasm. [16] Additionally, fundamental biology studies have shown that the poly A tail of endogenous mRNA i...
Large dsRNA molecules can cause potent cytotoxic and immunostimulatory effects through the activation of pattern recognition receptors; however, synthetic versions of these molecules are mostly limited to simple sequences like poly-I:C and poly-A:U. Here we show that large RNA molecules generated by rolling circle transcription fold into periodic-shRNA (p-shRNA) structures and cause potent cytotoxicity and gene silencing when delivered to cancer cells. We determined structural requirements for the dumbbell templates used to synthesize p-shRNA, and showed that these molecules likely adopt a co-transcriptionally folded structure. The cytotoxicity of p-shRNA was robustly observed across four different cancer cell lines using two different delivery systems. Despite having a considerably different folded structure than conventional dsRNA, the cytotoxicity of p-shRNA was either equal to or substantially greater than that of poly-I:C depending on the delivery vehicle. Furthermore, p-shRNA caused greater NF-κB activation in SKOV3 cells compared to poly-I:C, indicating that it is a powerful activator of innate immunity. The tuneable sequence and combined gene silencing, immunostimulatory and cytotoxic capacity of p-shRNA make it an attractive platform for cancer immunotherapy.
RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.