Fluorogenic nucleic acid molecule and target-rna fluorescent labeling method
A technology of fluorescent molecules and nucleic acid molecules, applied in the field of fluorescent nucleic acid molecules, can solve the problems of fluorescent proteins that are difficult to explain the results
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[0135] Next, although an Example etc. demonstrate this invention in detail, this invention is not limited to these examples.
[0136]
[0137] Plasmids used in the following experiments were prepared as follows.
[0138] Fusion to the NheI / NotI site of pEGFP-N1 (manufactured by Clontech) and subcloning 3×NLS (LVPKKKRKVVPKKKKRKVVPKKKKVFEGPDPPV: SEQ ID NO: 21) and iRFP713 (hereinafter referred to as "iRFP", kindly presented by Mr. Michiyuki Matsuda, non-patent literature 11) coding sequence to obtain pNLS-iRFP. In order to screen candidates for paired fluorescence-generating RNAs (candidate RNAs) of DFHBI family molecules, F30-2×dBroccoli was obtained by PCR amplification from pAV5S-F30-2×dBroccoli (#66845, manufactured by Addgene). Other RNAs are constructed by synthesizing oligonucleotide fragments. These oligonucleotide fragments were subcloned into the NotI site of pNLS-iRFP using In-Fusion (manufactured by Clontech Corporation). Primers for In-Fusion were designed in s...
reference example 1
[0144] F30-2×dBroccoli (made by inserting the nucleotide sequence of dBroccoli at two places on the ring structure in the F30 scaffold sequence, SEQ ID NO: 22) is considered to have the highest fluorescence intensity among Broccoli mutants. Therefore, first, in order to investigate whether this F30-2×dBroccoli can visualize RNA polymerase II (Pol II)-dependent mRNA in human cells, downstream of the RNA polymerase II-dependent CMV promoter, DFHBI- F30-2×dBroccoli, which emits green fluorescence in the presence of 1T, was expressed in HEK293T cells together with NLS-iRFP (nucleus-localized infrared fluorescent protein iRFP). Specifically, the plasmid vector (pNLS-iRFP-#0) was transfected into HEK293T cells, and DFHBI-1T was added to the culture medium for culture, wherein the plasmid vector (pNLS-iRFP-#0) was introduced into pNLS-iRFP expressing NLS-iRFP is formed by integrating F30-2×dBroccoli into the 3' untranslated region of pNLS-iRFP.
[0145] As a result, the NLS-iRFP-pos...
Embodiment 1
[0148] Screening the fluorescent RNA paired with the DFHBI family molecule is the fluorescent RNA capable of improving the intensity of the generated fluorescence by combining with the DFHBI family molecule.
[0149] Such as figure 1 As shown, the sequences of well-known fluorescent molecule binding aptamers such as Spinach and Broccoli are similar, and are composed of a terminal stem (Terminal stem), a G-quadruplex, a stem-loop (Stem-loop), and a connector (Connector). These segments are determined by the crystal structure of Spinach and the base sequence alignment with other fluorescent molecule binding aptamers.
[0150] A:G purine-purine mismatches and C-G Watson-Click base pairs in the double-strand-four-strand transition region contained in the Spinach mutant were shown in the terminal stem to greatly affect the brightness of the aptamer in the cellular environment (non-patent literature 9). Thus, GAGAC-GGCUC was used for the terminal stem sequence.
[0151] Looking a...
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