Technique for Expressing any RNA in Curcular Form
Antisense mRNA and ribozymes (catalytic RNA) have great potential for controlling gene expression. A major road block remains: many linear anti-sense or ribozyme RNAs are unstable when expressed in living cells. Due to this instability, these RNAs do not accumulate enough to provide an adequate level of inhibition for practical control of gene expression.
The most promising method to stabilize RNA transcripts involves making them circular. Existing methods of circularizing RNA either have low yields, or are limited in the size and type of RNA sequence that may be made circular. Additionally, none of these existing methods have sufficient flexibility to engineer circular RNAs in vivo.
Scientists at the University of California have developed a method for making virtually any RNA in a covalent, circular form, both in vitro and in vivo. Their method uses a specially designed restriction fragment in which the coding sequence for the desired RNA is inserted. When this restriction fragment is cloned into an expression vector and transcribed, the resulting RNA strand will spontaneously splice out the inserted sequence as a circular RNA, free of any intron sequences or undesired catalytic activity. These circles may be as small as 50 bases and larger than 500 bases.
Tests in yeast and E. coli demonstrate that the simple, two step method produces covalent circular RNA in vivo, with excellent yield and accuracy. Furthermore, the circles can be made from transcripts synthesized by RNA polymerase II. Thus, inserting the restriction fragment into an expression vector with a carefully chosen promoter will allow temporal and tissue specific control of circle production. This method will be useful in the design of stable forms of RNA to regulate gene expression, such as ribozyme or antisense regulators. Additional applications include the design of circular mRNAs to make long protein chains with repeating segments. Hence, this new technique would be the method of choice for anyone constructing circular RNA, or wanting to express a circular RNA in vivo.
US 5,773,244 [MORE INFO
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