Abstract
Single-stranded (ss) circular oligodeoxynucleotides were previously found to undergo rolling circle transcription (RCT) by phage and bacterial RNA polymerases (RNAPs) into tandemly repetitive RNA multimers. Here, we redesign them to encode minimal primary miRNA mimics, with the long term aim of intracellular transcription followed by RNA processing and maturation via endogenous pathways. We describe an improved method for circularizing ss synthetic DNA for RCT by using a recently described thermostable RNA ligase, which does not require a splint oligonucleotide to juxtapose the ligating ends. In vitro transcription of four templates demonstrates that the secondary structure inherent in miRNA-encoding vectors does not impair their RCT by RNAPs previously shown to carry out RCT. A typical primary-miRNA rolling circle transcript was accurately processed by a human Drosha immunoprecipitate, indicating that if human RNAPs prove to be capable of RCT, the resulting transcripts should enter the endogenous miRNA processing pathway in human cells. Circular oligonucleotides are therefore candidate vectors for small RNA delivery in human cells, which express RNAPs related to those tested here.
Highlights
Small RNAs such as miRNA and siRNA are well established as sequence-specific regulators of gene expression at the mRNA level [1]
In addition to describing an improved method for circularizing the linear rolling circle transcription (RCT) template oligonucleotides, we show here that miRNA-encoding circular oligodeoxynucleotides (COLIGOs) are substrates for RCT by bacterial and bacteriophage RNA polymerases (RNAPs)
We find that the secondary structure necessarily present in a COLIGO encoding pri-miRNAs does not impair RCT by bacterial and bacteriophage RNAP
Summary
Small RNAs such as miRNA and siRNA are well established as sequence-specific regulators of gene expression at the mRNA level [1]. Plasmid and viral vectors encoding short hairpin transcripts (shRNA) carry the same information in relatively stable form, but typically waste more than 99% of their nucleotide (nt) mass. They must be biosynthesized in cells or by microorganisms, risking contamination in therapeutic applications, and can undergo genomic integration, potentially causing cancer [7]. These shortcomings in small RNA delivery methods have prompted us to investigate single stranded circular oligodeoxynucleotides (COLIGOs) as minimized relay molecules for shuttling small RNA sequence information into cells
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