Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity

Abstract

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and posttranscriptional modifications were directly characterized by mass spectrometric analyses.

MicroRNA Pharmacoepigenetics: Posttranscriptional Regulation Mechanisms behind Variable Drug Disposition and Strategy to Develop More Effective Therapy

“…However, research on miRNA pharmacoepigenetics and development of miRNA-based therapy may be limited by the utilization of synthetic miRNA agents consisting of excessive artificial modifications on the phosphate linkages and/or ribose rings and thus exhibit different physicochemical and biologic properties or toxicities. By contrast, recombinant RNA technology has been successfully employed to costeffectively produce large quantities of miRNA agents Chen et al, 2015;Li et al, 2015;Wang et al, 2015), which are comprised of no or posttranscriptional modifications on the nucleobases and may better capture the structures, functions, and safety properties of natural RNAs. These natural ncRNA agents are produced in cells and should represent a novel class of miRNA agents for research and development.…”

“…3). MicroRNAs are able to suppress the expression of some efflux transporters and enzymes and lead to a greater degree of intracellular drug accumulation, resulting in higher efficacy (Zhu et al, 2008;Pan et al, 2009g;Liang et al, 2010;Pan et al, 2013;Shang et al, 2014;Li et al, 2015). Meanwhile, some miRNAs may directly reduce the protein outcome of pharmacological targets [e.g., (proto)oncogenes] and thus control disease progression (e.g., tumor growth and metastasis) Zhao et al, 2015).…”

“…There is growing evidence supporting the feasibility of combination therapy. MicroRNAs downregulate the expression of efflux transporters in tumor cells, resulting in a higher level of intracellular drug accumulation and thus an improved efficacy (Zhu et al, 2008;Pan et al, 2009g, 2013Liang et al, 2010;Shang et al, 2014;Li et al, 2015). In addition, miRNAs may directly target (proto-) oncogenes and thus additively or synergistically suppress tumor growth Zhao et al, 2015).…”

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

“…HPLC was conducted using a 1 ϫ 150-mm XBridge C 18 column (Waters) using mobile phase A and mobile phase B (200 mM hexafluoroisopropanol, 8.15 mM triethylamine in 50% methanol (Burdick and Jackson), pH 7.0) at a flow rate of 30 l/min. The chromatographic gradient and mass spectrometric conditions are identical to the conditions described before (56).…”

“…The fractionated oligomer was digested with snake venom phosphodiesterase and Antarctic phosphatase before subjecting it to subsequent LC-MS (56,58) with detection in positive polarity.…”