RNA molecules comprising non-canonical base pairs
A base-pairing and base-pairing technology, used in DNA/RNA fragments, recombinant DNA technology, genetic engineering, etc., can solve the problems of transcriptional self-silencing, damage to the stability and efficacy of target gene silencing, etc.
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Embodiment 1
[0550] Example 1: Materials and Methods
[0551] Synthesis of Genetic Constructs
[0552] To design a typical ledRNA construct, target RNA regions of about 100-1000 nucleotides in length, typically 400-600 nucleotides in length, were identified. In one embodiment, the 5' half of the sequence and about 130 nt of the flanking region and similarly the 3' half and 130 nt of the flanking region are oriented in antisense orientation relative to the promoter. These sequences are interrupted by 400-600 nucleotides of the sense target sequence ( figure 1 A). The 5' end of the resulting construct is preceded by a promoter, such as a T7 or SP6 RNA polymerase promoter, and the 3' end is engineered to include restriction enzyme cleavage sites to allow in vitro transcription termination.
[0553] For transcription in cells such as bacterial cells, inducible promoters are used, for example, to introduce promoter and terminator sequences to facilitate expression as a transgene. The do...
Embodiment 2
[0559] Example 2: Design of ledRNA
[0560] like figure 1 Schematically shown in A, a typical ledRNA molecule comprises a sense sequence that can be thought of as two adjacent sense sequences covalently linked and identical to the target RNA, complementary to the sense sequence and divided into two The antisense sequence of the region, and two loops separating the sense sequence from the antisense sequence. Thus, a DNA construct encoding this form of ledRNA contains, in 5' to 3' order, a promoter for transcription of the coding region of the ledRNA, a first antisense region complementary to the region towards the 5' end of the target RNA, a first loop sequence, a sense sequence, a second loop sequence, then a second antisense region complementary to the 3' end region of the target RNA, and finally a means to terminate transcription. In this arrangement, two antisense sequences flank the sense and loop sequences. When transcribed, the two regions of the antisense sequence ...
Embodiment 3
[0566] Example 3: Stability of ledRNA
[0567] The ability of ledRNA to form dsRNA structures was compared to dsRNAs that are open-ended (ie, without loops, formed by the annealing of separate single-stranded sense and antisense RNAs) and long hpRNAs. The mixture of ledRNA, long hpRNA, and sense and antisense RNA was denatured by boiling and annealed in annealing buffer (250 mM Tris-HCl, pH 8.0 and 100 mM MgCl) 2 / buffer), followed by electrophoresis in a 1.0% agarose gel under native conditions.
[0568] like figure 2 As shown, both GUS ledRNA and GFP ledRNA gave dominant RNA bands of expected mobility for double-stranded molecules, indicating the formation of predicted ledRNA structures. This is in contrast to the mixture of sense and antisense RNAs, which show only weak bands of dsRNA, indicating that most sense and antisense RNAs do not readily anneal to each other to form dsRNAs. Hairpin RNA samples gave two prominent bands, indicating that only a portion of the tra...
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