Development of microRNA (miRNA or miR) based treatments such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. In recent work published by a team of scientists led by researchers from UC Davis, investigators presented a new approach to high-yield and large-scale biosynthesis of chimeric miR-34a agent in Escherichia coli using tRNA scaffold, which may act as a prodrug for cancer therapy. In their study, investigators quickly purified the recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) to a high degree of homogeneity (> 98%) using anion-exchange fast protein liquid chromatography (FPLC), whose primary sequence and posttranscriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed favorable cellular stability while it was degradable by several ribonucleases. Through LC Sciences’ small RNA sequencing service and qPCR studies, it was revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, whereas the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of varoius types of human carcinoma cells in a dose dependent manner and to much greater degrees than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, it was shown that tRNA/mir-34a significantly suppressed the growth of human non-small cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared to the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effects on blood chemistries and IL-6 levels in mouse models, suggesting that recombinant RNAs were well tolerated. Their findings provoke a conversation on producing biological miRNAs to perform miRNA actions, and point towards a new direction to develop miRNA-based therapies.
The tRNA-carried pre-miR-34a was selectively processed to mature miR-34a in human carcinoma cells while tRNA scaffold was degraded to tRFs, as revealed by unbiased deep sequencing studies.
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Reference
W. P. Wang, P. Y. Ho, Q. X. Chen, B. Addepalli, P. A. Limbach, M. M. Li, W. J. Wu, J. L. Jilek, J. X. Qiu, H. J. Zhang et al. (2015) Bioengineering novel chimeric microRNA-34a for prodrug cancer therapy: High-yield expression and purification, and structural and functional characterization JPET doi: 10.1124/jpet.115.225631 [article]