Stabilization and Translation of Immobilized mRNA on Latex Beads for Cell-Free Protein Synthesis System

Kobatake, Eiry; Ebisawa, Akira; Asaka, Orie; Yanagida, Yasuko; Ikariyama, Yoshihito; Aizawa, Masuo

doi:10.1385/abab:76:3:217

Cited by 5 publications

(3 citation statements)

References 19 publications

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“…The above results demonstrate that mRNA tethering to poly dT media can effectively protect the mRNA from nuclease attack. Kobatake et al (1999) immobilized mRNA on the nonporous oligo(dT) latex beads and investigated the degradation of the immobilized mRNA encoding protein A against three different types of nucleases, including 3′‐OH exonuclease, 5′‐OH exonuclease, and endonuclease. Compared with the solution‐based mRNA, the stability of the immobilized mRNA against all these three types of nucleases was improved, and the immobilized mRNA could be used for cell‐free protein synthesis.…”

Section: Resultsmentioning

confidence: 99%

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

Che,

Feng,

et al. 2023

Biotech & Bioengineering

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The messenger RNA (mRNA) 5′‐cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large‐scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre‐and‐post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2′‐O‐methyltransferase can efficiently catalyze the capping of poly dT media‐tethered mRNA to produce mRNA with cap‐1 structure under an optimized condition. We have therefore designed an integrated purification and solid‐based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3′‐end poly‐A in mRNA, in situ capping of mRNA 5′‐end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution‐based capping, and post‐separation and recovering steps. Specifically, the new process accomplished a 1.76‐fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid‐based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution‐based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on‐column continuous mode. The results presented in this work demonstrated that the new on‐column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost‐effective platform technology suitable for large‐scale production of capped mRNA.

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Section: Resultsmentioning

confidence: 99%

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

Che,

Feng,

et al. 2023

Biotech & Bioengineering

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“…Hence, we demonstrate the utility of our newly constructed linker-DNA that may prove to be a highly effective tool for generating arrays harboring larger proteins. Furthermore, the utility of our linker-DNA in immobilizing the end of an mRNA-template onto solid surfaces for solid-phase translation improves the half-life of the mRNA molecule, which is very short in cell-free systems, by protecting its 3′ terminus against contaminating nucleases ( 27 ). In this context, to confirm the stability of our immobilized mRNA, magnetic bead-bound GFP-mRNAs were used for multiple sequential solid-phase cell-free translation reactions.…”

Section: Discussionmentioning

confidence: 99%

Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA

Biyani

Husimi

Nemoto

2006

Nucleic Acids Research

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A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA–protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA–protein fusion is ligated to the 3′ ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA–protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of ‘RNA chip-to-protein chip’ using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution.

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“…Translation of immobilized mRNA was previously performed to stabilize the labile RNA against nucleases, but this method resulted in much lower protein yields compared to TL of free mRNA. 33,34 Novel automated modular microfluidic systems with integrated sensors for online monitoring and regulation of relevant process parameters could allow a continuous, controlled and resource-conserving CFPS. The presented microfluidic TRITT platform fulfills the precondition for such a modular micro-reactor: the active transfer of the product of transcription (RNA) from the TK to a separate TL compartment, where the RNA serves as template for protein synthesis, is realized in an automatable manner.…”

Section: Introductionmentioning

confidence: 99%

On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode

Georgi

Blechert

et al. 2016

Lab Chip

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Many pharmaceuticals are proteins or their development is based on proteins. Cell-free protein synthesis (CFPS) is an innovative alternative to conventional cell based systems which enables the production of proteins with complex and even new characteristics. However, the short lifetime, low protein production and expensive reagent costs are still limitations of CFPS. Novel automated microfluidic systems might allow continuous, controllable and resource conserving CFPS. The presented microfluidic TRITT platform (TRITT for Transcription - RNA Immobilization & Transfer - Translation) addresses the individual biochemical requirements of the transcription and the translation step of CFPS in separate compartments, and combines the reaction steps by quasi-continuous transfer of RNA templates to enable automated CFPS. In detail, specific RNA templates with 5' and 3' hairpin structures for stabilization against nucleases were immobilized during in vitro transcription by newly designed and optimized hybridization oligonucleotides coupled to magnetizable particles. Transcription compatibility and reusability for immobilization of these functionalized particles was successfully proven. mRNA transfer was realized on-chip by magnetic actuated particle transfer, RNA elution and fluid flow to the in vitro translation compartment. The applicability of the microfluidic TRITT platform for the production of the cytotoxic protein Pierisin with simultaneous incorporation of a non-canonical amino acid for fluorescence labeling was demonstrated. The new reaction mode (TRITT mode) is a modified linked mode that fulfills the precondition for an automated modular reactor system. By continual transfer of new mRNA, the novel procedure overcomes problems caused by nuclease digestion and hydrolysis of mRNA during TL in standard CFPS reactions.

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Stabilization and Translation of Immobilized mRNA on Latex Beads for Cell-Free Protein Synthesis System

Cited by 5 publications

References 19 publications

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA

On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode

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