CN112816395A - RNA marker, and living cell marking method and application containing same - Google Patents
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Abstract
本发明涉及活细胞检测技术领域，尤其涉及一种RNA标记物及包含该标记物的活细胞标记方法和应用。所述方法包括标记待测基因，具体为使用RNA标记物一对一地标记待检测基因，所述RNA标记物包含多个茎环结构；其中，每个茎环结构独立地选自两种不同的茎环结构；任意两个不同的RNA标记物之间，所述两种不同的茎环结构中的任一种占自身茎环结构总数比例的差值为15～50％。本发明利用不同的RNA茎环结构标记基因后相对荧光强度和绝对荧光强度不同的原理实现了同时对更多种mRNA的标记和观测，拓展了可用的mRNA标记物数量，节约了所用荧光蛋白种类，对于复杂的基因网络的成像和生物学过程研究具有重要意义。
The present invention relates to the technical field of live cell detection, in particular to an RNA marker and a live cell labeling method and application comprising the marker. The method includes labeling the gene to be detected, specifically, using an RNA marker to label the gene to be detected one-to-one, the RNA marker comprising a plurality of stem-loop structures; wherein, each stem-loop structure is independently selected from two different The stem-loop structure; between any two different RNA markers, the difference between the proportion of any of the two different stem-loop structures in the total number of stem-loop structures is 15-50%. The invention utilizes the principle of different relative fluorescence intensity and absolute fluorescence intensity after different RNA stem-loop structures are used to mark genes, thereby simultaneously marking and observing more mRNAs, expanding the number of available mRNA markers, and saving the types of fluorescent proteins used , has important implications for the imaging of complex gene networks and the study of biological processes.
Description
Technical Field
The invention relates to the technical field of living cell detection, in particular to an RNA marker, a living cell marking method containing the RNA marker and application of the RNA marker.
Background
Imaging systems based on RNA Stem-loop structures (Stem-loops) and RNA binding proteins are common methods for imaging living cells. The RNA stem-loop structures currently used for live cell imaging are mainly MS2, PP7, lambda boxB and U1A. In general, in order to label a specific RNA, it is necessary to insert multiple tandem stem-loop structural sequences into an untranslated region of a target gene, such as an intron, 5'UTR, 3' UTR, etc. The transcribed target gene carries multiply RNA stem-loop structures that recruit RNA-binding proteins to which typically 1-3 copies of a fluorescent protein are fused, thereby forming fluorescent spots within the cell.
Among the many live cell imaging methods, the MS2 system and the PP7 system have now become standard methods for mRNA imaging in live cells, since the functional and kinetic studies of mRNA currently available ensure that the use of these two systems does not affect normal mRNA function. At present, the resolution of single mRNA molecule can be achieved by utilizing stem-loop system imaging, and quantitative analysis of the whole process from RNA generation to degradation in the gene transcription process can be realized.
Although living cells can achieve single molecule resolution and high precision at present, the imaging method based on the combination of stem-loop structure and RNA binding protein still has shortcomings. The biggest problem is that the throughput (the gene factors that can be observed in one cell at the same time) of live cell RNA imaging is not high, and the live cell RNA imaging is difficult to be used for researching the gene network level. Specific reasons for this problem include: (1) currently for live cell imaging, as many markers are available at the transcriptional level as at the protein level. The protein layer utilizes fluorescent proteins with various colors and properties (such as maturation time and degradation rate) to label target proteins, so that the technology is mature at present, and the available fluorescent proteins are various; for live cell imaging, the most widely used stem-loop labeling methods have few available stem-loop types. (2) Since the RNA stem-loop structures currently used for labeling live-cell mRNA are mainly derived from bacteriophage, it is relatively time-consuming to develop completely new RNA stem-loop structures. (3) As each RNA stem-loop structure needs a fluorescent protein to be fused with corresponding RNA binding protein for imaging, part of the fluorescent proteins are overlapped on a fluorescence spectrum, and the fluorescent proteins are simultaneously observed by using a fluorescence microscope to cause cross lines among channels, thereby reducing the types of the fluorescent proteins which can be actually and simultaneously observed.
Disclosure of Invention
In order to solve at least one problem in the prior art, the invention provides an RNA marker, a living cell marking method comprising the RNA marker and application of the RNA marker. The invention carries out fluorescence detection after marking a plurality of genes through any two different RNA stem-loop structures, and can measure the real-time expression level and other related information of the gene to be detected through the difference of relative fluorescence intensity, absolute fluorescence intensity and cell positioning.
In a first aspect, the present invention provides an RNA marker comprising 24 to 48 stem-loop structures; wherein each stem-loop structure is independently selected from two different stem-loop structures; at least 12 of each stem-loop structure are present in the RNA marker.
Further, the two different stem-loop structures are two stem-loop structures recruiting different RNA binding proteins, preferably any two of MS2, PP7, λ boxB, and U1A; more preferably MS2 and PP 7.
In a second aspect, the present invention provides an RNA labeling system comprising the RNA label and RNA binding proteins corresponding to two different stem-loop structures in the RNA label, respectively; different RNA binding proteins are linked to different types of fluorescent proteins, respectively.
Further, the RNA binding proteins corresponding to the two different stem-loop structures in the RNA marker are RNA binding proteins capable of being recruited by the corresponding stem-loop structures, such as MS2 stem-loop structure corresponding to MCP protein and PP7 stem-loop structure corresponding to PCP protein.
Further, the different types of fluorescent proteins are spectrally non-overlapping fluorescent proteins.
In a third aspect, the present invention provides a method for simultaneously detecting multiple RNAs in a living cell, comprising: marking a gene to be detected, expressing RNA binding protein and carrying out fluorescence detection;
the marked genes to be detected comprise:
marking a gene to be detected one by using a plurality of RNA markers, wherein each RNA marker comprises 24-48 stem-loop structures, and each stem-loop structure is independently selected from two different stem-loop structures; and simultaneously comprises at least 12 stem-loop structures in the RNA markers of two different stem-loop structures;
and between any two different RNA markers, the difference of the proportion of any one of the two different stem-loop structures in the total number of the stem-loop structures is 15-50%.
Further, the two different stem-loop structures are any two of MS2, PP7, λ boxB and U1A; preferably MS2 and PP 7. Taking MS2 and PP7 as an example, assuming that the RNA markers contain N stem-loop structures, the difference between the ratio N1/N of MS2 between the RNA markers corresponding to different genes to be detected and the ratio N2/N of another RNA marker MS2 should be not less than 15-50% of the value set by the present invention (for example, if the ratio of MS2 in adjacent RNA markers is set to 0, 1/2, 1/1, 2/1, 1, there are 5 RNA markers that can detect 5 RNAs simultaneously), and if the difference is less than 15%, it is difficult to distinguish the fluorescence detection results of two RNA markers adjacent to each other.
Further, the expressed RNA binding protein is: simultaneously expressing RNA binding proteins corresponding to the two different stem-loop structures in the living cells, wherein the two RNA binding proteins are respectively connected with different types of fluorescent proteins; the fluorescence detection is to perform fluorescence imaging on the cells after the RNA binding protein is expressed to obtain the fluorescence intensity of different types of fluorescent proteins, and the detection of multiple RNAs is realized through the relative fluorescence intensity and the absolute fluorescence intensity of the different types of fluorescent proteins.
Furthermore, different types of fluorescent proteins are fluorescent proteins with different spectra, and the fluorescence intensities of the fluorescent proteins with different spectra can be accurately distinguished under the precision of the current fluorescence detection instrument.
Further, when the two different stem-loop structures are MS2 and PP7, the RNA binding protein corresponding to MS2 is MCP protein connected with CFP fluorescent protein; the RNA binding protein corresponding to PP7 is PCP protein and is connected with mCherry fluorescent protein.
Further, the detection of multiple RNAs by the relative fluorescence intensity and absolute fluorescence intensity of different types of fluorescent proteins is:
and determining the RNA type corresponding to each fluorescence point according to the relative intensity of different fluorescence in each fluorescence point in the fluorescence imaging result, and determining the expression difference of different types of RNA according to the absolute fluorescence intensity.
Furthermore, the location of the corresponding RNA can be determined based on the localization of the fluorescent protein within the cell.
After the genes are labeled by different kinds of RNA markers, the RNA markers are also transcribed along with gene transcription, so that binding protein (the binding protein is fused with fluorescent protein) is recruited at a transcription site, fluorescent spots are formed at the transcription site, each fluorescent spot has two kinds of fluorescence, the relative intensity of the two kinds of fluorescence indicates the proportion of the markers, so that the RNA is the RNA of which gene, the fluorescence intensity of the fluorescent spots is positively correlated with the transcription level, and the stronger the transcription is, the stronger the fluorescence intensity of the fluorescent spots is. The method can be used for realizing time sequence imaging of gene transcription by combining a time sequence fluorescence imaging method.
The invention further provides the application of the RNA marker, the RNA marking system and the method in living cell imaging.
The invention has the following beneficial effects:
1. the invention realizes the marking and observation of more mRNA under the condition of limited stem-loop structure types by carrying out combined marking on different stem-loop structures in proportion, expands the quantity of available mRNA markers and ensures that 2 mRNA markers can achieve the technical effect of detecting 5 or even more RNAs.
2. The method saves the types of the used fluorescent proteins, and is beneficial to introducing new RNA stem-loop structures and fluorescent proteins to carry out imaging and biological process research of more complex gene networks in the later period.
3. The invention realizes the marking of each element of the simple network (five-gene synthesis network) and real-time live cell imaging for the first time, and has potential to mark the endogenous biological network and image the live cells in real time in the follow-up process.
Drawings
FIG. 1 illustrates the principle of selection and action of the RNA markers provided by the present invention;
FIG. 2 is an example of a specific experimental procedure provided by the present invention;
FIG. 3 is a graph showing the variation of the characteristic difference between different marker ratios according to the present invention with the increase of the fluorescence intensity of the markers, i.e., the stronger the transcription, the more obvious the ratio differentiation between the markers;
FIG. 4 shows the time-series live cell imaging effect of gene transcription with different RNA markers under the Dox induction system provided by the present invention;
FIG. 5 shows the principle of construction of the five-gene line cell line according to the present invention;
FIG. 6 shows the time sequence variation of fluorescence intensity information of five RNA marker genes in a single cell initiated by the Dox induction system provided by the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
This example provides a living cell RNA labeling system, which is as follows:
1. selection of RNA markers
Two most widely used live cell RNA marker systems, PP7 and MS2, were selected, and the principle is shown in FIG. 1, and by the difference of the relative number of the stem-loop structures of the two, new markers with different ratios were composed, and based on the original 24 XP 7(1:0) and 24 XPS 2(0:1), three new marker systems of 24 XP 7:24 XPS 2(1:1), 32 XP 7:16 XPS 2(2:1) and 16 XP 7:32 XPS 2(1:2) were further adopted, because MS2 and PP7 capture different RNA-binding proteins, respectively, the different marker systems showed different characteristics in fluorescence imaging (FIG. 1).
2. Example of a flow Process for labeling and time-series imaging of Living cells Using the labeling System of the present invention
Transfecting plasmids encoding target genes and RNA markers in certain proportion into cells to construct a cell line, carrying out single cell imaging on the cell line, wherein each single cell forms a fluorescent spot when the target genes and the RNA markers are transcribed, the fluorescent spot has different fluorescent intensity outputs in two fluorescent channels, RNA types corresponding to the markers can be judged according to the relative fluorescent intensity of the two channels, the transcription intensity can be judged according to the actual fluorescent intensity, and the real-time transcription intensity of a plurality of single cells in a period of time can be obtained by combining a time sequence fluorescent microscope (figure 2).
3. Construction of five Gene line cell lines
Induced expression of PP7/MS2 marker was achieved using the Tet on induction system: the marker is uniformly positioned at the 3' UTR position behind a gene stop codon, and the transcription of a reporter fluorescent protein Citrine (YFP) and an RNA marker is induced by Doxycylin (hereinafter referred to as Dox); RNA-binding proteins PCP-3 × mCherry-NLS and NLS-MCP-3 × CFP-NLS were used and a human U2OS cell line (lentivirus-infected construction cell line) was constructed that constantly expressed both proteins (FIG. 1).
Further, in this embodiment, the RNA marker is integrated into the U2OS cell line to obtain a five-gene transcription regulation circuit, which is an AND gate system (AND gate), AND the specific process is as follows: upstream Dox starts transcription expression, Dox induces 1:0 marker and LacI-VP64, starts downstream LacO-minor CMV promoter and expresses 2:1 marker and VP 64-Frb; dox induced both the 0:1 marker and ZFN-VP64 at the same time, and late initiated expression of the downstream 1:2 marker and Gal 4-FKBP. Finally, under the action of Rapamycin (Rapamycin, hereinafter referred to as RAPA), Gal4 and VP64 jointly initiate the transcription of downstream 1:1 marker and YFP reporter fluorescent protein, and the principle is shown in FIG. 5.
4. Fluorescence signal detection
The statistics of the imaging data of a plurality of single cells respectively transfected with one marker show that the same marker is stably expressed in different cells, and the proportion information of the marker is not significantly fluctuated due to the change of fluorescence intensity and the heterogeneity of the cells. Meanwhile, since PP7 and MS2 are proportional in the labeling, the higher the fluorescence intensity of barcode, the more distinct the characteristic difference between different ratios (FIG. 3). The ratio of labels varied throughout the imaging process, although the fluorescent spot transcriptional intensity varied, the ratio of RNA labels remained stable (FIG. 4).
In this example, the above five gene transcription regulation circuits were imaged, and the results are shown in fig. 6: the fluorescent spots generated by transcription and the transcription signals changing along with time can be seen after Dox induction is added, and the proportion of the RNA markers is kept stable in spite of the change of the transcription intensity of the fluorescent spots in the whole imaging process.
And when Dox induction is added, the expression is carried out from the upstream to the downstream in a time sequence manner, and the fluorescence intensity information of five RNA markers in a single cell can be distinguished; recording the time-series signals of the fluorescent spots in the cells can see that the markers at the same level are different in the time for starting transcription and the transcription fluctuation, and the downstream genes are expressed after the upstream transcription (FIG. 6).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
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| CN116643036A (en) * | 2023-05-25 | 2023-08-25 | 深圳湾实验室 | RNA modified living cell imaging system and composition and application thereof |
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