Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns, whose biogenesis is regulated by specific cis-acting elements and trans-acting factors. Some circRNAs are abundant and evolutionarily conserved, and many circRNAs exert important biological functions by acting as microRNA or protein inhibitors ('sponges'), by regulating protein function or by being translated themselves. Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases and cancer. Although the circular nature of these transcripts makes their detection, quantification and functional characterization challenging, recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging.

The biogenesis, biology and characterization of circular RNAs.

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis

Translation of CircRNAs

Circular RNAs, a novel class of endogenous noncoding RNAs, are characterized by their covalently closed loop structures without a 5′ cap or a 3′ Poly A tail. Although the mechanisms of circular RNAs’ generation and function are not fully clear, recent research has shown that circular RNAs may function as potential molecular markers for disease diagnosis and treatment and play an important role in the initiation and progression of human diseases, especially in tumours. This review summarizes some information about categories, biogenesis, functions at the molecular level, properties of circular RNAs and the possibility of circular RNAs as biomarkers in cancers.

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CircRNA: functions and properties of a novel potential biomarker for cancer

Circular RNA and its mechanisms in disease: From the bench to the clinic.

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

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Rolling Circle Translation of Circular RNA in Living Human Cells

Rolling Circle Amplification in a Prokaryotic Translation System Using Small Circular RNA

2′-Deoxy-4′-thioribonucleic acid (4′-thioDNA) having a sulfur atom instead of an oxygen atom in the furanose ring has a nuclease resistance and hybridization ability higher than that of natural DNA. Despite its great potential for various biological applications, a long 4′-thioDNA having all four kinds of 2′-deoxy-4′-thionucleosides has not been reported. In this study, we describe systematic analysis of the incorporation of 2′-deoxy-4′-thionucleoside 5′-triphosphates (dSNTPs) using various DNA polymerases. We found that family B DNA polymerases, which do not have 3′→5′ exonuclease activity, could efficiently incorporate dSNTPs via single nucleotide insertion and primer extension. Moreover, 104-mer PCR product was obtained even under the conditions in the presence of all four kinds of dSNTPs when KOD Dash DNA polymerase was used. The resulting PCR product was converted into a natural dsDNA by using PCR with dNTPs, and sequencing of the natural dsDNA revealed that the PCR cycle successfully proceeded witho...

PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside 5'-triphosphates.

https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/60827/1/manuscript.pdf

Transcription of 4'-thioDNA templates to natural RNA in vitro and in mammalian cells

We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene) EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 70-80 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39-85% yield This method has potential uses in biotechnology and chemical biology

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Hideto Maruyama

Papers

Rolling Circle Translation of Circular RNA in Living Human Cells

Rolling Circle Amplification in a Prokaryotic Translation System Using Small Circular RNA

PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside 5'-triphosphates.

Transcription of 4'-thioDNA templates to natural RNA in vitro and in mammalian cells

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester