Methods for RNA analysis
a technology of rna and rna molecules, applied in the field of rna analysis, can solve the problems of reducing the efficacy of rna therapeutics, complicated interpretation of the resulting band pattern, and limited methods for analyzing short rna molecules, so as to facilitate translation or localization and prevent degradation of rna molecules
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example 1
on of Hammerhead Ribozymes
[0322]1. A hammerhead ribozyme can be directed to cleave 3′ of any NUH sequence as shown in FIG. 5 (N=G,A,C,U; H=A,C,U) (Haseloff and Gerlach, 1988. Nature 334: 585-591; McCall et al., 2000. Molecular Biotechnology 14: 5-17). The schematic diagram of FIG. 5 shows how helix I and helix III anneal to the target RNA sequence.
[0323]2. The trans-acting hammerhead ribozyme HHNUH2d was designed to target the 5′ region of the RNA sequences shown in FIGS. 1 to 3, forming helix III with mRNA positions 1-12, and helix I with mRNA positions 14-18 (FIG. 6). The 5′ region of the target RNA sequence contains two possible recognition sites, NUH1 (positions 10-12) and NUH2 (positions 11-13), of which NUH2 is the preferred target site.
[0324]Sequence of the trans-acting hammerhead ribozyme HHNUH2d (SEQ ID NO: 2):
5′-GCAUGGCUGAUGAGGCCUCGACCGAUAGGUCGAGGCCGAAAAGCUUUCUCCC-3′
[0325]3. The ribozyme HHNUH2d was synthesized and HPLC purified by Biomers.net GmbH (Ulm, Germany), and 200 ...
example 2
on of the mRNA
[0326]1. Preparation of DNA and mRNA Constructs
[0327]For the present example DNA sequences encoding HsNY-ESO-1 mRNA according to SEQ ID NO: 3 (FIG. 1), PpLuc mRNA according to SEQ ID NO: 4 (FIG. 2) and HsPSCA RNA according to SEQ ID NO: 5 (FIG. 3) were prepared and used for subsequent in vitro transcription reactions.
[0328]According to a first preparation, the DNA sequences coding for the above mentioned mRNAs were prepared. The constructs were prepared by modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)), a stretch of 64 adenosines (poly-A-sequence), a stretch of 30 cytosines (poly-C-sequence), and a histone stem loop. In FIGS. 1 to 3 the sequences of the corresponding mRNAs are shown.
[0329]The 5′ region of the target RNA sequence contains two possible recognition sites, NUH1 (positions 10-12) and NUH2 (positions ...
example 3
sis Assay
[0339]1. Principle of the Assay
[0340]The hammerhead ribozyme HHNUH2d of example 1 was incubated with the in vitro transcribed RNAs of example 2 (Table 2) and the cleavage products were separated by denaturing polyacrylamide gel electrophoresis (PAGE) or high performance liquid chromatography (HPLC).
[0341]2. Ribozyme Cleavage Reaction
[0342]Reaction scales for gel analysis were usually 1× (10 pmol RNA). For HPLC analysis, 15× reaction (150 pmol RNA) were set up, allowing a more sensitive detection and thus a more precise determination of the respective mRNA populations. Per reaction, 10 pmol of HHNUH2d and 10 pmol of the respective substrate RNA were annealed in 0.625 mM EDTA in a total volume of 6 μl (2 min at 95° C., 0.1° C. / sec to 25° C., 10 min at 25° C.). After addition of 4 μl of 100 mM MgCl2, 125 mM Tris / HCl, pH 7.5 (final concentration 40 mM MgCl2, 50 mM Tris / HCl), the reaction was incubated at 25° C. for 1 hour. For analysis via polyacrylamide gel electrophoresis (PA...
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