Nucleic acid molecules for pseudouridylation
A nucleic acid molecule, pseudouridine technology, applied in the field of snoRNA, can solve problems such as instability
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Embodiment 1
[0115] Example 1: Design of pseudouridylated guide oligonucleotides derived from the structure of small nucleolar RNAs (snoRNAs) for converting specific uridines to ψ in ACA19 target RNAs.
[0116] The present inventors questioned whether it is possible to induce pseudourylation using a shortened box H / ACA snoRNA. To this end, a pseudouridylated guide RNA was designed in which most of the 5′ hairpin and H-box of the full-length ACA19 snoRNA were removed ( Figure 4 ). Shortened guide RNAs and full-length snoRNAs were generated by in vitro transcription using T7 RNA polymerase. For testing in cell lysates, by using T7 RNA polymerase (in [α- 32 p]UTP) to generate short substrate RNAs for these guides by in vitro transcription or by two-part ligation. For the latter, first, the 5′ hydroxyl of a synthetic RNA oligonucleotide that is pseudouridylated at the 5′ end of the uridine terminated with [γ- 32 P] phosphate for radiolabelling. The 5' end of the radiolabeled RNA oligonuc...
Embodiment 2
[0120] Example 2: Design of psEON for conversion of specific uridines in premature stop codons of human CFTR.
[0121] It was investigated whether uridine in the premature stop codon (PTC) in the human CFTR gene could be converted to ψ. For example, the CFTR-G542X mutation was selected. The testing process was as described in Example 1.
[0122] Substrate RNA (two-part connection) is constructed similarly to Example 1, and the final target sequence is 5'-GACAAUAUAGUUCUU U GAGAAGGUGGAAUC-3' (target uridine underlined; SEQ ID NO: 2).
[0123] Figure 8 Four CFTR-G542X psEONs are shown with their introduced chemical modifications: black dots indicate the 2'-OMe modification on the ribose moiety of the nucleotide, open dots indicate the bonded PS modification between the two ribose sugars. Figure 9Results are shown for pseudouridylation using four CFTR-G542X psEONs with the corresponding guide RNA (lack of chemical modification) as a positive control and no guide RNA as a neg...
Embodiment 3
[0124] Example 3: Design of snoRNAs for conversion of specific uridines in premature stop codons in mouse Idua RNA.
[0125] investigated whether uridine in PTC in mouse Idua RNA could be converted to ψ. As an example, the Idua-W392X mutation in mouse RNA was chosen, which corresponds to the human IDUAW402X mutation known to cause Hurler Syndrome. For this, a substrate RNA was constructed similar to Example 1 (two-part ligation) with the final sequence 5'-GAUGGAGAACAACUC U AGGCAGAGGUCUCA-3' (target uridine underlined; SEQ ID NO: 3).
[0126] Figure 10 The positions of the four Idua-W392X psEONs and the introduced chemical modifications are shown: black dots indicate the 2′-OMe modification on the ribose moiety of this nucleotide, open dots indicate the bonded PS modification between the two ribose sugars. Figure 11 Results are shown for pseudouridylation using four Idua-W392X psEONs with the corresponding guide RNA (lack of chemical modification) as a positive control and...
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