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WO2017082517A1 - Method for real-time detection of rna splicing using light scattering in plasmon nanostructures - Google Patents

Method for real-time detection of rna splicing using light scattering in plasmon nanostructures Download PDF

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WO2017082517A1
WO2017082517A1 PCT/KR2016/008647 KR2016008647W WO2017082517A1 WO 2017082517 A1 WO2017082517 A1 WO 2017082517A1 KR 2016008647 W KR2016008647 W KR 2016008647W WO 2017082517 A1 WO2017082517 A1 WO 2017082517A1
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splicing
probe
rna
scattering
messenger rna
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심상준
원훙안
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Korea University Research and Business Foundation
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals

Definitions

  • the present invention relates to a method for detecting RNA splicing using plasmon nanostructures and light scattering, and more particularly, to detect RNA splicing in a single molecule in real time through an optical microscope using light scattering in plasmon nanostructures. It is about a method.
  • RNA splicing is a molecular mechanism that removes introns from the transcripts of early messenger RNA precursors (pre-mRNAs) by cleavage of conserved sequences called splice sites. ribozyme) and contribute to the diversity of mature RNA for protein translation (Fica et al., Nature 503: 229, 2013). This mechanism plays an important role in the onset of cancer, genetic diseases and biological evolution (Herbert et al., Nat. Genet. 21: 265, 1999). Mutations at the intron / exon boundary generally impair RNA splicing and cause loss of mRNA diversity resulting in diseases such as beta-thalassemia (Srebrow et al., J. Cell Sci.
  • RNA splicing can be analyzed by gel-based methods (e.g. Northern blots) and primer extensions, but has limitations such as time, labor and consumable costs and cannot be used for monitoring messenger RNA precursors. (Vandenbroucke et al., Nucl . Acids Res . 29: e68, 2001).
  • FRET fluorescence resonance energy transfer
  • AuNPs gold nanoparticles
  • the wavelength combined with the collective vibration of free electrons on the surface of gold nanoparticles (AuNPs) produces plasmon resonance frequencies (Hutter et al., Adv . Mater . 16: 1685, 2004).
  • Gold nanoparticles (AuNPs) have excellent plasmon properties, including scattering, absorption and resonance based on their geometric and relative positions (Sharma et al., Mater. Today 15:16, 2012).
  • LSPR local surface plasmon resonance
  • SERS electromagnetic surface enhanced Raman scattering
  • SERS Surface-enhanced Raman scattering
  • RNA splicing in a single particle can be detected, and the present invention was completed.
  • An object of the present invention is a method for real-time detection of RNA splicing using light scattering in plasmon nanostructures, and detection of RNA splicing through optical microscopy using scattering intensity, local surface plasmon resonance and surface-enhanced Raman scattering in a single molecule And a method of monitoring.
  • the present invention comprises the steps of (a) cloning the target gene into the expression vector and transforming the vector in vivo except cells, tissue or human, to generate a transcript of the recombinant target messenger RNA precursor; (b) a plasmon probe or surface enhanced Raman scattering (SERS) -tag probe is reacted with a transcript of said recombinant target messenger RNA precursor or introduced directly into vivo, excluding cells, tissues or humans to enhance said plasmon probe or surface Attaching a Raman scattering (SERS) -tag probe to the boundaries of exons / introns and introns / exons of the messenger RNA precursor; And (c) measuring scattering intensity, local surface plasmon resonance of Rayleigh scattering, and surface enhanced Raman scattering through an optical microscope to detect RNA splicing. .
  • SERS surface enhanced Raman scattering
  • FIG. 1 is a schematic diagram of an optical platform for messenger RNA precursor splicing monitoring.
  • (a) Represents a cell-free system comprising a recombinant human beta-globin on the left and a spliceosome complex for RNA splicing analysis on the right.
  • (b) Shown the position of the plasmon probe (left) and the surface enhanced Raman scattering (SERS) probe (right) at exon-intron boundaries before and after RNA splicing.
  • SERS surface enhanced Raman scattering
  • RNA splicing process shows the RNA splicing process.
  • c TEM images of individual binding probes after RNA splicing.
  • FIG. 3 shows an optical based analysis of messenger RNA precursor splicing.
  • SERS surface enhanced Raman scattering
  • RNA splicing inhibition is the result for RNA splicing inhibition.
  • Scattering intensity correlates with the presence of inhibitors and arrows indicate the addition of RNA splicing buffer (left) and HeLa cell extract (right).
  • SERS surface enhanced Raman scattering
  • c shows real time plasmon migration during RNA splicing.
  • d Plasmon migration over time during RNA splicing.
  • RNA splicing was confirmed in single messenger RNA precursor molecules through Rayleigh scattering and surface-enhanced Raman scattering with a plasmon and surface-enhanced Raman scattering (SERS) -tag based optical microscope in which gold nanoparticles are bound.
  • SERS plasmon and surface-enhanced Raman scattering
  • SERS surface enhanced Raman scattering
  • RNA splicing is inhibited by 33 ⁇ M of biflavonoid isoginkgetin, a common inhibitor of RNA splicing.
  • the detection process of the plasmon binding and surface enhanced Raman scattering (SERS) of the present invention is shown in Figure 1, specifically (i) the messenger RNA precursor of the beta-globin molecule is cloned and transcribed in E. coli, (ii) Derived messenger RNA precursor molecules are purified by RNA purification kit (Qiagen) and injected into chambers coated with RNA specific probes, and (iii) two plasmons for plasmon binding, specific DNA probes that bind to exon / intron boundaries. Two surface enhanced Raman scattering (S) -probes for) -probe and surface enhanced Raman scattering (SERS) were added to the chamber.
  • S surface enhanced Raman scattering
  • SERS surface enhanced Raman scattering
  • Plasmon (P) -probes and surface enhanced Raman scattering (S) -probes were hybridized with messenger RNA precursors at the exon2 / intron3 interface, capping with gold nanoparticles and the Raman molecule cyanine-3 (Cy3), respectively.
  • RNA splicing platforms such as RNA splicing inhibition and cancer inhibitory effect by isoginkgetin.
  • the present invention can account for scattering intensity, local surface plasmon resonance and surface enhanced Raman scattering using optically based methods, thereby monitoring and detecting RNA splicing.
  • optical-based methods have several advantages, such as the study of the dynamics of splicing, splice suppression, cis-trans replacement splicing, and rapid measurement of RNA splices.
  • the effects of isogingzetin on messenger RNA precursor splicing and splicing kinetics at the single molecule level were analyzed.
  • the ability to perform splicing of individual messenger RNA precursor molecules, high resolution and optically based methods and the like can demonstrate the relative stability to weakly stabilized RNA molecules and their lifetimes.
  • RNA splicing is important for gene regulation at the post-translational level, promoting or inhibiting RNA splicing using this platform is an essential element of modern molecular biology useful for drug screening.
  • the present invention provides a method for producing a transcript of a recombinant target messenger RNA precursor by (a) cloning a target gene into an expression vector and transforming the vector into cells other than cells, tissues, or humans; (b) a plasmon probe or surface enhanced Raman scattering (SERS) -tag probe is reacted with a transcript of said recombinant target messenger RNA precursor or introduced directly into vivo, excluding cells, tissues or humans to enhance said plasmon probe or surface Attaching a Raman scattering (SERS) -tag probe to the boundaries of exons / introns and introns / exons of the messenger RNA precursor; And (c) measuring scattering intensity, local surface plasmon resonance of Rayleigh scattering, and surface enhanced Raman scattering through an optical microscope to detect RNA splicing. .
  • SERS surface enhanced Raman scattering
  • the step (b) may be characterized in that performed in-vitro (in vitro) or in-vivo (in vivo).
  • the RNA splicing analysis is preferably measured by Rayleigh scattering method including a nanoparticle binding pattern, it is measured by the method of scattering intensity including a one-dimensional binding pattern with at least one nanoparticle.
  • the surface enhanced Raman scattering (SERS) -tag including the formation of an electromagnetic surface enhancement pattern is preferably performed by the surface enhanced Raman scattering (SERS) method of nanoparticle bonding, but is not limited thereto.
  • the local surface plasmon resonance, scattering intensity and surface enhanced Raman scattering (SERS) of the Rayleigh scattering may be characterized in that it occurs in the solid phase or solution phase of the optical microscope chamber.
  • the binding pattern may be characterized in that the binding pattern of the probe including the nanoparticles according to the number of introns of the messenger RNA precursor molecule.
  • the surface enhanced Raman scattering (SERS) -tag probe may be characterized in that the Raman dye (Taman dye), the Raman dye (Cyanine-3), Cyanine-5, Cyanine-5.5, Cyanine 7, 4-aminothiophenol, 4-methylbenzenethiol, 4-naphthalene Thiol (2-naphthalenethiol), rhodamine-5- (and-6) -isothiocyanate, tetramethylrhodamine-5-isothiocyanate (tetramethylrhodamine- 5-isothiocyanate), Rhodamine B Rhodamine 6G nile blue (rhodamine B Rhodamine 6G nile blue), preferably selected from the group consisting of, but not limited to TAMRA.
  • the Raman dye Timan dye
  • the Ramanine-3 Cyanine-5, Cyanine-5.5, Cyanine 7, 4-aminothiophenol, 4-methylbenzenethiol, 4-na
  • the plasmon probe may be characterized by binding in parallel to the RNA molecule.
  • the plasmon probe preferably has a homology of 50 to 100% with the target gene messenger RNA precursor sequence, the plasmon probe comprises 2 to 100 or more nanoparticles according to the number of messenger RNA precursor molecules Preferably, but not limited thereto.
  • the plasmon probe may be characterized by having a spatial separation of 1 ⁇ 80nm after RNA splicing.
  • the messenger RNA precursor is preferably selected from the group consisting of messenger RNA precursor, micro RNA precursor, hnRNA, rRNA, tRNA and viral RNA, but is not limited thereto.
  • the RNA splicing may be characterized in that introns are removed from messenger RNA precursor molecules or pre-micro RNAs containing at least one intron next to the exon, wherein the removal of the introns is spliced. It may be characterized in that it is carried out by the cleavage mechanism of the spliceosom.
  • the RNA splicing may be performed by kinetic monitoring of messenger RNA precursor using nanoparticles
  • the nanoparticles are gold, silver, platinum and copper having a diameter of 10 ⁇ 100nm. It is preferably any one selected from the group consisting of nanoparticles, but is not limited thereto.
  • the nanoparticles may be characterized in that it further comprises a stabilizer, the nanoparticles and stabilizer may be characterized in that the thiol group is directly bonded.
  • the stabilizer is preferably selected from the group consisting of thiol polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, thiol oligoethylene glycol, cetyltrimethylammonium bromide and polystyrene sulfonic acid, but is not limited thereto.
  • the cell is preferably selected from the group consisting of prokaryotic cells, eukaryotic cells, normal cells and cancer cells, but is not limited thereto.
  • the optical microscope comprises a dark field condenser; Spectrograph for spectral graph; CCD camera for signal processing; White light sources of halogen or xenon for Rayleigh scattering; Laser sources for surface enhanced Raman scattering (SERS) signal detection; And it may be characterized in that it comprises a reaction chamber for RNA splicing analysis.
  • the optical microscope of the present invention can be used without limitation as long as it can measure scattering intensity, local surface plasmon resonance of Rayleigh scattering and surface enhanced Raman scattering.
  • the chamber may be characterized in that the biotin-DNA probe for messenger RNA precursor capture is coated with a streptavidin molecule fixed in the vertical direction, the chamber is surface enhanced Raman scattering (SERS) signal It may be characterized in that it further comprises a plasmon nanoparticle-probe mixture for detection.
  • SERS surface enhanced Raman scattering
  • HBB human beta-globin gene
  • GenBank: GQ370762.1 human beta-globin gene
  • a messenger RNA precursor was prepared from the gene.
  • HBB human beta-globin gene
  • GenBank: GQ370762.1 human beta-globin gene
  • a messenger RNA precursor was prepared from the gene.
  • Messenger RNA Precursor Splicing (Exon-2 / Intron-2 / Exon-3) is a series of catalytic reactions by spliceosome of HeLa nuclear extract of the Messenger RNA Precursor Splicing Kit (Proteinone, USA). Was performed.
  • RNA splicing was explored by monitoring RNA splicing using integrated optical construction for surface enhanced Raman scattering (SERS), scattering intensity and local surface plasmon resonance (LSPR).
  • SERS surface enhanced Raman scattering
  • LSPR local surface plasmon resonance
  • Plasmon binding probe-1 (P1) and probe-2 (P2) correspond to the border site between exon-2 / intron-2 and intron-2 / exon-3.
  • the thiol-modified probe was reacted with 10 ⁇ l of 10N DTT at room temperature for 10 minutes, and then excess DTT was removed three times with 50 ⁇ l of ethyl acetate to obtain a thiol-fragment from the thiol-modified probe.
  • the resulting probes were immediately reacted in 50 nm (0.05 mg / mL) citric acid-gold nanoparticles (AuNPs) solution to make plasmon probes (Kimling et al., J. Phys. Chem . B 110: 15700, 2006).
  • Ligand exchange was performed by adding 0.1 M BSPP (bis (p-sulfonatophenyl) phenylphosphine dipotassium salt, Sigma-Aldrich) to 1 mL citric acid-gold nanoparticle solution. Excess BSPP was removed by centrifugation, and particles were separated into two portions by gradual washing with 10 mM Tris (pH 8.0), 40 mM NaCl buffer. One part was reacted with P1 and P2 probes for 10 hours, and then poly (ethylene glycol) methyl ether thiol CH 3 O (CH 2 CH 2 O) n was obtained so that the ratio of poly (ethylene glycol): gold nanoparticles was 1: 1,000. CH 2 CH 2 SH (Sigma Aldrich, USA) was added.
  • BSPP bis (p-sulfonatophenyl) phenylphosphine dipotassium salt, Sigma-Aldrich
  • probe-1 S1 with modified gold nanoparticles bound at a ratio of 1:50 (AuNP / probe) and probe-2 (S2 with Cy3 binding at the 3 'end) ) (SEQ ID NO: 5, IDT DNA, USA) was used.
  • the plasmon probe corresponding to the boundary region between exon-2 / intron-2 and intron-2 / exon-3 was capped with 50 nm gold nanoparticles to have a binding ratio of 1:50 (AuNP: probe), and the surface
  • An enhanced Raman scattering (SERS) probe was constructed as a probe for RNA splicing through Raman fingerprinting for surface enhanced Raman scattering (SERS) by binding Cy3 to the 3'-end.
  • the bound probe was then hybridized with thiol-PEG, HS (CH 2 ) 11 (OCH 2 CH 2 ) 3 OH (OEG3) and 1 ⁇ M BSA (10 mM Tris. HCl, pH 8.0, and 160 mM NaCl). It was inactivated in RNA splicing buffer (provided in the RNA splicing kit).
  • the reaction chamber was made of straptavidin-coated glass (SMS, Arrayit), and 100 ⁇ M biotin capture probe (5'-GCTGCTGGTGGTCTACCCT-biotinylated-3 ': SEQ ID NO: 6) was injected for 2 hours to streptavidin- After the biotin binding reaction, the surface was treated with 2 mL superblock buffer (37516, Thermo Scientific) to minimize nonspecific reactions.
  • the beta-globin messenger RNA precursor 200 ⁇ L, 1 ng / ⁇ L
  • 10 mM Tris-HCl pH 7.5
  • 0.1 mM EDTA 0.1 mM EDTA
  • RNA splicing of messenger RNA precursor monomolecules was monitored by scattering intensity, local surface plasmon resonance and integrated optical configuration of Raman scattering.
  • the optical configuration was set on an upright Eclipse Nickel U microscope (Nikon) with a darkfield capacitor, then integrated Monora500i for Raman scattering and Monora320i shooting port (Dongwoo Optron, South Korea) for scattering intensity, iXon EM + CCD camera (512 ⁇ 512 active) pixel chip) (Andor, Oxford Instruments).
  • the light source used a white light source of halogen or xenon, and the laser used sources of 785 nm and 632.5 nm.
  • the reaction chamber reacted with a 1 mg / ml biotin capture probe (Integrated DNA Technology) for the first 15 minutes, and 300 ⁇ l of T50 (50 mM NaCl / 10 reaction chamber had a 1 mg / ml biotin capture probe (Integrated DNA) for the first 15 minutes. Technology) and washed with 300 ⁇ l of T50 (50 mM NaCl / 10 mM Tris, pH 8.0) Next, the chamber was reacted with SuperBlock (37516, Thermo Scientific) for 45 minutes, followed by 300 ⁇ l of T50.
  • SuperBlock 37516, Thermo Scientific
  • the chamber was allowed to react with 200 ⁇ l of messenger RNA precursor molecule solution (5 ng / ⁇ l), which resulted in messenger RNA precursor molecules bound only to a glass surface coated with a specific capture probe and other mRNA molecules in solution. Due to the very high surface area of the modified glass surface, it is possible to concentrate low concentrations of beta-globin messenger RNA precursors at high rates.
  • the beta-globin messenger RNA precursor capture device was adjusted to an optimum annealing temperature of 40 ° C. using a temperature controller (TC-324B, Warner Instruments) to plasmon in the hybridization solution (10 mM Tris. HCl, pH 8.0 and 160 mM NaCl).
  • the probe was reacted with a surface enhanced Raman scattering (SERS) probe.
  • SERS surface enhanced Raman scattering
  • the chamber was equilibrated with RNA splicing buffer (20 mM HEPES-Na (pH 7.9), 20% Glycerol, 42 mM (NH 4 ) 2 SO 4 , 0.5 mM DTT, 0.2 mM EDTA), followed by in vitro messenger RNA precursor.
  • RNA splicing 190 ⁇ l of RNA splicing buffer containing HeLa cell nuclear extract (P002-1, Protein One, 10 ⁇ L, 5 ng / ⁇ L) was injected into the chamber at time point 0 seconds.
  • RNA splicing was detected by scattering intensity, LSPR and Raman scattering in plasmon binding mode.
  • RNA splicing includes 20 nM messenger RNA precursor, 30% nuclear extract, 20 mM potassium chloride, 2.5 mM magnesium chloride, 10 mM creatine phosphate, 0.5 mM DTT, 0.4 units / l RNasin, 40 mM Tris-HCl, pH 8, 0.5 mM ATP and 1% (v / v) DMSO were performed by reacting at 40 ° C for 10 minutes. mRNA splicing products, ie binding particles of messenger RNA precursor and DNA probe, were separated by gel electrophoresis.
  • the 1% agarose gel on the right side of FIG. Post splicing reaction (lane 3).
  • the splicing mixture contains an intermediate band showing dimer formation by removal of intron-2, and the splicing mRNA, in which two probes are plasmon-bound, is shorter than the messenger RNA precursor due to the removal of 850 bp of intron-2. lost. This is slower than gold nanoparticle functionalized DNA probes and faster than gold nanoparticle functionalized DNA probes hybridized with messenger RNA precursors.
  • Dimer bands were separated by an electroelution system (Elutrap electroelution system, Whatman) and observed with a transmission electron microscope (TEM) (Fig. 2c).
  • Plasmon probes are generally capable of producing plasmon bonds and reproducing optical recordings when the distance is reduced due to RNA splicing and the composition of the gold nanoparticles of the messenger RNA precursor molecules is completely irreversible.
  • Exon-2 (222 bp) and exon-3 (126 bp) are isolated from intron-2 (851 bp), exon-2 and exon-3 are linked, so that the two probes bind plasmons with enhancement of the plasmon sites of LSPR and SERS Reach the site.
  • RNA splicing were confirmed by real-time PCR with 29 nm generation (FIG. 3B) and SERS spectra (FIG. 3C) of plasmon shift in local surface plasmon resonance (LSRP) (FIG. 3D).
  • Gold nanoparticles bind dimers in monomers to increase scattering intensity and are linked to splicing of messenger RNA precursors.
  • the orientation of bound gold nanoparticles was indicated on the reaction chamber surface based on the movement of messenger RNA precursor splicing, and real-time PCR was performed when the intron fragment diffused into solution.
  • Nonspecific sites of gold nanoparticles were blocked with 5% HS (CH 2 ) 11 (OCH 2 CH 2 ) 3 OH (OEG3) and 1 ⁇ M BSA.
  • Gold nanoparticles can reproduce optical recordings of plasmon surfaces and plasmon bonds for SERS.
  • Two or more binders are removed by nonspecific binding blocking on the surface of the gold nanoparticles, and only one probe specifically binds to the exon / intron boundary.
  • the reaction time and the concentration of the intramolecular DNA probe were adjusted until capped with 5% or less gold nanoparticles. After RNA splicing, the binding of the two gold nanoparticles is stabilized and continuously monitored in units of time.
  • Gold nanoparticles in solution are optionally oriented with polarized light of scattered light, and the increased concentration of gold nanoparticles indicates RNA splicing yield in the chamber.
  • the platform is due to the high sensitivity and specificity of splice site recognition of spliceosomes.
  • RNA splicing was performed by injecting E. coli extract into the chamber to observe scattering intensity.
  • RNA splicing time is the total time of total splicing, including spliceosome assembly, from splicing initiation to splicing of introns.
  • the 2.5 minute delay in messenger RNA precursor splicing is the time to correct errors in RNA splicing.
  • the time of various splicing depends on the length of the 5 'splicing site or intron, or on the affinity of the enhancer and the silence (Wang et al., RNA 14: 802, 2008).
  • the varying rates of intron removal can be attributed to differences in rates at the stage of spliceosome assembly, such as control of insertion rate, prior to progression of initial splicing sites, pairing of splicing sites, proper splice of exons, etc. Because. Another reason is the binding of serine / arginine (SR) proteins in introns to inhibit splicing progression (Wang et al., RNA 14: 802, 2008).
  • SR serine / arginine
  • SERS surface enhanced Raman scattering
  • SERS intensity is proportional to the number of Raman dies of the P1 probe and P2-Cy3 probe reaching the surface of the gold nanoparticles, thus reflecting the relative activity of the spliceosome and the efficiency of messenger RNA precursor splicing Indicates.
  • Normalized surface enhanced Raman scattering (SERS) intensity change is a direct indicator of messenger RNA precursor splicing efficiency (FIG. 4B). As shown in FIG. 4, the intensity of all peaks showed a normal curve for 3 minutes before the injection of HeLa extract containing the spliceosome complex.
  • SERS Raman scattering
  • the rate of increase of the Raman signal is proportional to the concentration of the messenger RNA precursor because the higher the concentration of the messenger RNA precursor increases the splicing rate of the messenger RNA precursor.
  • the detection range was set from 0.1 ⁇ g / ⁇ l to 5 ⁇ g / ⁇ l, and messenger RNA precursor splicing lower than 0.1 ⁇ g / ⁇ l did not change within a given detection time, and 10 minutes recording of the negative control E. coli extract was also performed. No change in intensity was observed. This result demonstrated an increase in Raman signals in messenger RNA precursor splicing assays based on spliceosome activity rather than self-assembly of RNA formation or magnetic binding of Raman die with gold nanoparticles in the reaction solution.
  • RNA splicing inhibition of 33 ⁇ M isogingzetin was measured using plasmon binding and scattering intensity of the SERS signal at 37 ° C. for 0-10 minutes. The approach of high time resolution messenger RNA precursor splicing at the single molecule level has been shown by quantifying the rate of RNA splicing through probes and isogingzetin.
  • RNA splicing results are reasonable to measure scattering intensity.
  • Two representative scattering phenomena, scattering intensity and Raman scattering, are shown in FIGS. 5A and 5B.
  • Raman scattering and scattering intensity which can reflect changes in RNA splicing with two very close plasmon probes, include scattering trajectory intensity changes.
  • Comparison of scattering intensity (FIG. 5A) and Raman scattering (FIG. 5B) inhibitors with and without RNA splicing showed an increase in the average intensity of plasmon binding.
  • scattering intensity for RNA splicing activity is explained by the affinity of the inhibitor in the action of splicing time (FIG. 5C) and the inhibitory activity against the spliocesome complex.
  • the inhibition (%) on the action of reaction time from the inhibition progress curve was determined by continually measuring the accumulation of mRNA product at all times with or without inhibitor (FIG. 5D). This indicates instability of the self-assembly of the spliceosome by isogingzetin. Thus, the increase in growth inhibitor concentration observed in t1 / 2 splicing is explained by the low binding force of spliceosome to the exon-intron boundary region.
  • RNA splicing monitoring For scattering intensity, plasmon binding and Raman scattering based RNA splicing monitoring, data was recorded with the Andor CCD detector using software Solis T. All binding pairs are shown to increase slowly or radically, indicating plasmon shift and Raman shift. All recordings following the spliceosome complex injection included a gradual drop in intensity set to zero seconds. Only binding pairs include assays that show the binding of dimer's plasmon migration and plasmon intensity after RNA splicing. Scattering and spectral data of Rayleigh scattering and Raman scattering are plotted with the OriginPro program (version 8.6). This process was further analyzed by the OriginPro program for splicing time determinations defined by the time difference between the addition of spliceosome complexes and the increase in observed intensity.
  • RNA splicing Based on scattering intensity, localized surface plasmon resonance and surface enhanced Raman scattering according to the present invention, a method for detecting and monitoring RNA splicing in a single particle via optical microscopy has been found to provide high sensitivity, time savings and low cost. It is useful as a platform for in-depth study of splicing such as splicing, screening of splicing inhibitors and RNA nanotechnology.

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Abstract

The present invention relates to a method for detecting RNA splicing using plasmon nanostructures and light scattering and, more particularly, to a method for real-time detection of RNA splicing in a single molecule through an optical microscope using light scattering in a plasmon nanostructure. The method for detecting and monitoring RNA splicing in a single particle through an optical microscope on the basis of the scattering intensity, local surface plasmon resonance and surface enhancement Raman scattering according to the present invention is useful as a platform for studying multiple splicing, such as binding of microRNA to drug for RNA splicing screening, due to the high sensitivity, time reduction and low cost.

Description

플라즈몬 나노구조에서 광산란을 이용한 RNA 스플라이싱을 실시간 검지하는 방법Real-time Detection of RNA Splicing Using Light Scattering in Plasmon Nanostructures

본 발명은 플라즈몬 나노구조 및 광산란을 이용하여 RNA 스플라이싱을 검지하는 방법에 관한 것으로, 보다 상세하게는 플라즈몬 나노구조에서 광산란을 이용한 광학 현미경을 통해 단일 분자에서의 RNA 스플라이싱을 실시간 검지하는 방법에 관한 것이다.The present invention relates to a method for detecting RNA splicing using plasmon nanostructures and light scattering, and more particularly, to detect RNA splicing in a single molecule in real time through an optical microscope using light scattering in plasmon nanostructures. It is about a method.

RNA 스플라이싱(splicing)은 스플라이스(splice) 부위라 불리는 보존 서열의 분열(cleavage)에 의해 초기 전령 RNA 전구체(pre-mRNA)의 전사체에서 인트론을 제거하는 분자 메커니즘으로, 라이보자임(ribozyme) 구조 및 단백질 번역을 위한 성숙 RNA의 다양성에 기여한다(Fica et al., Nature 503:229, 2013). 이런 메커니즘은 암의 발병, 유전 질환 및 생물학적 진화에서 중요한 역할을 한다(Herbert et al., Nat. Genet. 21:265, 1999). 인트론/엑손의 경계에서의 돌연변이는 일반적으로 RNA 스플라이싱을 손상시키고, mRNA의 다양성을 상실시켜 베타-탈라세미아(β-thalassemia)와 같은 질병의 원인이 된다(Srebrowet al., J. Cell Sci.119:2635, 2006; Faial et al., Nat. Genet. 47:105, 2015). 현재 RNA 스플라이싱은 젤-기반 방법(예: 노던 블롯) 및 프라이머 익스텐션(primer extendion)에 의해 분석될 수 있으나, 시간, 노동력 및 소모품 비용 등의 한계가 있고 전령 RNA 전구체의 모니터링에는 사용할 수 없다(Vandenbroucke et al., Nucl . Acids Res. 29:e68, 2001). RNA splicing is a molecular mechanism that removes introns from the transcripts of early messenger RNA precursors (pre-mRNAs) by cleavage of conserved sequences called splice sites. ribozyme) and contribute to the diversity of mature RNA for protein translation (Fica et al., Nature 503: 229, 2013). This mechanism plays an important role in the onset of cancer, genetic diseases and biological evolution (Herbert et al., Nat. Genet. 21: 265, 1999). Mutations at the intron / exon boundary generally impair RNA splicing and cause loss of mRNA diversity resulting in diseases such as beta-thalassemia (Srebrow et al., J. Cell Sci. 119: 2635, 2006; Faial et al., Nat. Genet . 47: 105, 2015). Currently RNA splicing can be analyzed by gel-based methods (e.g. Northern blots) and primer extensions, but has limitations such as time, labor and consumable costs and cannot be used for monitoring messenger RNA precursors. (Vandenbroucke et al., Nucl . Acids Res . 29: e68, 2001).

최근, 형광 프로브가 인트론 래리어트(lariat) RNA를 검출하기 위해 사용되고 있으나(Furukawa et al., Angew . Chem . Int Ed Engl. 50:12020, 2011), 짧은 수명, 광-표백제 및 형광 공명 에너지 전달(FRET)에서 공명을 위한 좁은 거리범위 등의 한계를 가진다. DNA 나노기술 및 분자생물학에서는 동력학적 연구의 한계를 극복하기 위해 플라즈몬 물질의 플라즈몬 결합에서의 산란 강도를 연구하고 있다(Reinhard et al., Nano Lett.5:2246, 2005; Ross et al., Nat. Nanotechnol . 10:453, 2015; Reinhard et al., Proc . Natl . Acad . Sci . U.S.A. 104:2667, 2007; Anker et al., Nat. Mater. 7:442, 2008; Nguyen et al., Biosens . Bioelectron. 66:497, 2015). 금나노 입자(AuNPs) 표면의 자유전자들의 집단적인 진동과 결합된 파장은 플라즈몬 공명 주파수를 생성한다(Hutter et al., Adv . Mater. 16:1685, 2004). 금나노 입자(AuNPs)는 자신의 기하학적 및 상대적 위치를 기반으로 한 산란, 흡수, 공명을 포함한 뛰어난 플라즈몬 특성을 갖는다(Sharma et al., Mater. Today 15:16, 2012). 플라즈몬 결합은 산란 강도, 단일 입자가 결합 입자로 이동시 진동수를 연구하는 국소 표면 플라즈몬 공명(LSPR) 및 전자기 향상을 연구하는 전자기 표면 증강 라만 산란(SERS)으로 탐색할 수 있다(Sharma et al., Mater. Today 15:16, 2012). Recently, fluorescent probes have been used to detect intron lariat RNA (Furukawa et al., Angew . Chem . Int Ed Engl . 50: 12020, 2011), but short lifetime, photo-bleach and fluorescence resonance energy transfer. (FRET) has limitations such as narrow range for resonance. In order to overcome the limitations of kinetic studies, DNA nanotechnology and molecular biology are studying the scattering intensity at the plasmon binding of plasmon materials (Reinhard et al., Nano Lett . 5: 2246, 2005; Ross et al., Nat. Nanotechnol . 10: 453, 2015; Reinhard et al., Proc . Natl . Acad . Sci . USA . 104: 2667, 2007; Anker et al., Nat. Mater . 7: 442, 2008; Nguyen et al., Biosens . Bioelectron . 66: 497, 2015). The wavelength combined with the collective vibration of free electrons on the surface of gold nanoparticles (AuNPs) produces plasmon resonance frequencies (Hutter et al., Adv . Mater . 16: 1685, 2004). Gold nanoparticles (AuNPs) have excellent plasmon properties, including scattering, absorption and resonance based on their geometric and relative positions (Sharma et al., Mater. Today 15:16, 2012). Plasmon binding can be explored with scattering intensity, local surface plasmon resonance (LSPR), which studies the frequency of movement of a single particle to the binding particle, and electromagnetic surface enhanced Raman scattering (SERS), which studies electromagnetic enhancement (Sharma et al., Mater). Today 15:16, 2012).

표면 증강 라만 산란(SERS) 기술은 복잡한 생물 검체에서 구분되는 "지문"인 라만 스펙트럼을 검출하고 모니터링 할 수 있으므로, 단일분자 수준에서 생물학적 모니터링 및 진단을 위한 매우 민감하고 선택적인 기술이다(Lee et al., Adv . Funct . Mater. 24:2079-2084, 2014; Fabris et al., Adv . Funct . Mater. 18:2518, 2008). 생물학적 모니터링에서 표면 증강 라만 산란(SERS) 기반 플랫폼은 텔로미어 신장, 메틸화 및 알츠하이머 병의 펩티드 집적 등의 진단 및 모니터링에 이용할 수 있다(Wang et al., Chem . Commun. 51:10953, 2015; Chou et al., Nano Lett.8:1729, 2008).Surface-enhanced Raman scattering (SERS) technology is a very sensitive and selective technology for biological monitoring and diagnostics at the single-molecule level because it can detect and monitor the Raman spectrum, the "fingerprint" that is distinguished from complex biological samples (Lee et al. , Adv . Funct . Mater . 24: 2079-2084, 2014; Fabris et al., Adv . Funct . Mater . 18: 2518, 2008). In biological monitoring, surface-enhanced Raman scattering (SERS) based platforms can be used for diagnostics and monitoring of telomeres, methylation and peptide accumulation in Alzheimer's disease (Wang et al., Chem . Commun . 51: 10953, 2015; Chou et. al., Nano Lett. 8: 1729, 2008).

이에, 본 발명자들은 종래의 문제점을 개선하고 RNA 스플라이싱(splicing)을 모니터링할 수 있는 방법을 개발하고자 예의 노력한 결과, 산란 강도, 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 기반으로 광학 현미경을 통해 단일 입자에서의 RNA 스플라이싱을 검지할 수 있음을 확인하고, 본 발명을 완성하였다.Accordingly, the present inventors have made diligent efforts to improve the conventional problems and develop a method for monitoring RNA splicing. Based on scattering intensity, local surface plasmon resonance and surface enhanced Raman scattering, It was confirmed that RNA splicing in a single particle can be detected, and the present invention was completed.

발명의 요약Summary of the Invention

본 발명의 목적은 플라즈몬 나노구조에서 광산란을 이용한 RNA 스플라이싱을 실시간 검지하는 방법으로, 단일 분자에서 산란 강도, 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 이용하여 광학 현미경을 통한 RNA 스플라이싱 검출 및 모니터링하는 방법을 제공하는데 있다.An object of the present invention is a method for real-time detection of RNA splicing using light scattering in plasmon nanostructures, and detection of RNA splicing through optical microscopy using scattering intensity, local surface plasmon resonance and surface-enhanced Raman scattering in a single molecule And a method of monitoring.

상기 목적을 달성하기 위하여, 본 발명은 (a) 표적 유전자를 발현 벡터에 클로닝하고 세포, 조직 또는 인간을 제외한 생체 내로 벡터를 형질전환시켜, 재조합 표적 전령 RNA 전구체의 전사체를 생성하는 단계; (b) 플라즈몬 프로브 또는 표면 증강 라만 산란(SERS)-태그 프로브를 상기 재조합 표적 전령 RNA 전구체의 전사체와 반응시키거나, 직접 세포, 조직 또는 인간을 제외한 생체 내로 도입시켜, 상기 플라즈몬 프로브 또는 표면 증강 라만 산란(SERS)-태그 프로브를 전령 RNA 전구체의 엑손/인트론 및 인트론/엑손의 경계에 부착시키는 단계; 및 (c) 광학 현미경을 통해 산란 강도, 레일리 산란의 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 측정하여, RNA 스플라이싱을 검출하는 단계를 포함하는 RNA 스플라이싱의 검출 및 모니터링 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of (a) cloning the target gene into the expression vector and transforming the vector in vivo except cells, tissue or human, to generate a transcript of the recombinant target messenger RNA precursor; (b) a plasmon probe or surface enhanced Raman scattering (SERS) -tag probe is reacted with a transcript of said recombinant target messenger RNA precursor or introduced directly into vivo, excluding cells, tissues or humans to enhance said plasmon probe or surface Attaching a Raman scattering (SERS) -tag probe to the boundaries of exons / introns and introns / exons of the messenger RNA precursor; And (c) measuring scattering intensity, local surface plasmon resonance of Rayleigh scattering, and surface enhanced Raman scattering through an optical microscope to detect RNA splicing. .

도 1은 전령 RNA 전구체 스플라이싱 모니터링을 위한 광학 플랫폼에 대한 도식도이다. (a) 좌측의 재조합 인간 베타-글로빈과 우측의 RNA 스플라이싱 분석을 위한 스플라이세오솜(spliceosome) 복합체를 포함하는 세포-프리 시스템을 나타낸 것이다. (b) RNA 스플라이싱 전, 후의 엑손-인트론 경계에서 플라즈몬 프로브의 위치(좌측) 및 표면 증강 라만 산란(SERS) 프로브의 위치(우측)를 나타낸 것이다. (c) 플라즈몬 이동 및 라만 이동에서 단량체와 결합체의 스펙트럼을 나타낸 것이다.1 is a schematic diagram of an optical platform for messenger RNA precursor splicing monitoring. (a) Represents a cell-free system comprising a recombinant human beta-globin on the left and a spliceosome complex for RNA splicing analysis on the right. (b) Shown the position of the plasmon probe (left) and the surface enhanced Raman scattering (SERS) probe (right) at exon-intron boundaries before and after RNA splicing. (c) Spectrum of monomer and conjugate in plasmon shift and Raman shift.

도 2는 RNA 스플라이싱 과정을 나타낸 것이다. (a) 프로브 결합, 인트론 루프 형성 및 인트론 제거로 인한 광 신호(좌에서 우로) 생성 과정을 나타낸 것이다. (b) 유도 정제한 베타-글로빈 RNA(좌), 금 나노입자들이 코팅된 프로브(1), 전령 RNA 전구체와 금 나노입자들이 코팅된 프로브의 복합체(2), mRNA와 금 나노입자들이 코팅된 프로브를 포함하는 RNA 스플라이싱(3). (c) RNA 스플라이싱 후 개별 결합 프로브의 TEM 이미지. (d) RNA 스플라이싱 전 전령 RNA 전구체와 금 나노입자들이 코팅된 프로브의 단일 복합체(좌) 및 RNA 스플라이싱 후 개별 결합 프로브(우)의 암시야 이미지. 2 shows the RNA splicing process. (a) The process of generating optical signals (from left to right) due to probe coupling, intron loop formation and intron removal. (b) Inductively purified beta-globin RNA (left), probe coated with gold nanoparticles (1), complex of messenger RNA precursor and probe coated with gold nanoparticles (2), coated with mRNA and gold nanoparticles RNA splicing (3) comprising a probe. (c) TEM images of individual binding probes after RNA splicing. (D) RNA splicing single field of left messenger RNA precursor and probe coated with gold nanoparticles (left) and dark field image of individual binding probes (right) after RNA splicing.

도 3은 전령 RNA 전구체 스플라이싱의 광학 기반 분석을 나타낸 것이다. (a) RNA 스플라이싱 반응에 의한 플라즈몬 결합의 통합 강도를 85Hz에서 기록하였다. (b) 전령 RNA 전구체 스플라이싱에 의해 LSPR 청색편이가 발생하였으며, 스펙트럼 이동의 피크는 로렌츠 함수에 의해 계산되었다. (c) HeLa 세포 추출물(레드) 및 대장균 세포 추출물(블랙)의 mRNA 형성에 의한 Cy3의 표면 증강 라만 스펙트럼. (d) 스플라이싱 시간에 따른 스플라이싱된 인트론-2 농도를 실시간 PCR로 확인한 결과이다.3 shows an optical based analysis of messenger RNA precursor splicing. (a) The integration intensity of plasmon binding by RNA splicing reaction was recorded at 85 Hz. (b) LSPR blue shift occurred by messenger RNA precursor splicing, and the peak of spectral shift was calculated by Lorentz function. (c) Surface enhanced Raman spectra of Cy3 by mRNA formation of HeLa cell extract (red) and E. coli cell extract (black). (d) Spliced intron-2 concentration according to the splicing time was confirmed by real-time PCR.

도 4는 표면 증강 라만 산란(SERS) 방법을 이용한 전령 RNA 전구체 스플라이싱의 실시간 동역학을 나타낸 것이다. (a) 전령 RNA 전구체 스플라이싱 측정을 위해 표면 증강 라만 산란(SERS) 스펙트럼은 10분간 15초 노출을 실시. (b) HeLa와 대장균의 세포 추출물에서 1120 cm-1, 1383 cm-1 및 1589 cm-1의 라만 피크 강도의 상대적 변화에 대해 시간별 측정.4 shows real-time kinetics of messenger RNA precursor splicing using surface enhanced Raman scattering (SERS) method. (a) Surface enhanced Raman scattering (SERS) spectra were subjected to 15 second exposure for 10 minutes for messenger RNA precursor splicing measurements. (b) Hourly measurements for relative changes in Raman peak intensities of 1120 cm -1 , 1383 cm -1 and 1589 cm -1 in HeLa and E. coli cell extracts.

도 5는 RNA 스플라이싱 억제에 대한 결과이다. (a) 산란 강도는 억제제의 존재 여부와 상관관계가 있으며, 화살표는 RNA 스플라이싱 버퍼(좌) 및 HeLa 세포 추출물(우)의 첨가를 나타낸다. (b) RNA 스플라이싱에 의해 생성되는 표면 증강 라만 산란(SERS) 스펙트럼의 종점을 나타낸 것이다. (c) RNA 스플라이싱 동안 실시간 플라즈몬 이동을 나타낸 것이다. (d) RNA 스플라이싱 동안 플라즈몬 이동을 시간에 따라 나타낸 것이다.5 is the result for RNA splicing inhibition. (a) Scattering intensity correlates with the presence of inhibitors and arrows indicate the addition of RNA splicing buffer (left) and HeLa cell extract (right). (b) shows the endpoint of the surface enhanced Raman scattering (SERS) spectrum produced by RNA splicing. (c) shows real time plasmon migration during RNA splicing. (d) Plasmon migration over time during RNA splicing.

발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred 구현예Embodiment

다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

본 발명에서는 금나노 입자가 결합된 플라즈몬 및 표면 증강 라만 산란(SERS)-태그 기반의 광학 현미경으로 레일리(Rayleigh) 산란 및 표면 증강 라만 산란을 통하여 단일 전령 RNA 전구체 분자에서 RNA 스플라이싱을 확인하였다. 베타-글로빈 유전자의 전령 RNA 전구체의 엑손-2/인트론-2 및 인트론 -2/엑손-3의 경계 부위에 결합하는 플라즈몬 프로브를 이용하여, RNA 스플라이싱으로 인트론이 제거될 경우에 성숙 전령 RNA에 결합하는 플라즈몬 프로브의 재배열로 인한 표면 증강 라만 산란(SERS) 스펙트럼 및 산란 강도의 증폭을 플라즈몬 밴드로 확인하였다. 플라즈몬 프로브는 엑손 2 및 엑손 3 연결에 대응하는 29nm 부근에서 플라즈몬 이동이 나타났다. RNA 스플라이싱에서 표면 증강 라만 산란(SERS) 지문 및 산란 강도의 강한 증가가 나타나므로 명확한 전령 RNA 전구체 스플라이싱을 알 수 있다. RNA 스플라이싱의 일반적인 억제제인 바이플라보노이드 이소깅제틴(isoginkgetin) 33μM에 의해 RNA 스플라이싱은 억제된다. In the present invention, RNA splicing was confirmed in single messenger RNA precursor molecules through Rayleigh scattering and surface-enhanced Raman scattering with a plasmon and surface-enhanced Raman scattering (SERS) -tag based optical microscope in which gold nanoparticles are bound. . Mature messenger RNA when introns are removed by RNA splicing using a plasmon probe that binds to the boundaries of exon-2 / intron-2 and intron-2 / exon-3 of the messenger RNA precursor of the beta-globin gene Amplification of surface enhanced Raman scattering (SERS) spectra and scattering intensity due to rearrangement of plasmon probes binding to the plasmon band was confirmed. The plasmon probe showed plasmon shift around 29 nm corresponding to exon 2 and exon 3 linkages. A clear increase in surface enhanced Raman scattering (SERS) fingerprint and scattering intensity in RNA splicing reveals a clear messenger RNA precursor splicing. RNA splicing is inhibited by 33 μM of biflavonoid isoginkgetin, a common inhibitor of RNA splicing.

본 발명의 플라즈몬 결합 및 표면 증강 라만 산란(SERS)의 검출 과정은 [도 1]에 나타냈으며, 구체적으로는 (i) 베타-글로빈 분자의 전령 RNA 전구체를 클로닝하여 대장균에서 전사시키고, (ii) 유도된 전령 RNA 전구체 분자는 RNA 정제 키트(Qiagen)로 정제하여 RNA 특이 프로브가 코팅된 챔버에 주입하고, (iii) 엑손/인트론 경계에 결합하는 특정 DNA 프로브인 플라즈몬 결합을 위한 2개의 플라즈몬(P)-프로브 및 표면 증강 라만 산란(SERS)를 위한 2개의 표면 증강 라만 산란(S)-프로브를 챔버에 추가하였다. 플라즈몬(P)-프로브 및 표면 증강 라만 산란(S)-프로브는 각각 금 나노입자들 및 라만 분자 시아닌-3(Cy3)로 캡핑하여, 엑손2/인트론3 경계에서 전령 RNA 전구체와 혼성화 하였다. (iv) 전령 RNA 전구체 분자와 플라즈몬(P)-프로브 및 표면 증강 라만 산란(S)-프로브의 혼성화 후, 과량의 프로브를 제거하기 위해 챔버를 세척하고, (v) 플라즈몬(P)-프로브 및 표면 증강 라만 산란(S)-프로브와 혼성화된 전령 RNA 전구체는 RNA 스플라이싱을 위해 HeLa 핵 추출물과 함께 반응시키고, (vi) 플라즈몬 이동, 산란 강도 및 표면 증강 라만 산란(SERS) 스펙트럼을 실시간으로 종점에서 수집하여, RNA 스플라이싱 모니터링을 위해 시간별로 측정하였다. 또한, 본 발명에서는 이소깅제틴(isoginkgetin)에 의한 RNA 스플라이싱 억제 및 암 억제 효과 등의 RNA 스플라이싱 플랫폼을 조사하였다.The detection process of the plasmon binding and surface enhanced Raman scattering (SERS) of the present invention is shown in Figure 1, specifically (i) the messenger RNA precursor of the beta-globin molecule is cloned and transcribed in E. coli, (ii) Derived messenger RNA precursor molecules are purified by RNA purification kit (Qiagen) and injected into chambers coated with RNA specific probes, and (iii) two plasmons for plasmon binding, specific DNA probes that bind to exon / intron boundaries. Two surface enhanced Raman scattering (S) -probes for) -probe and surface enhanced Raman scattering (SERS) were added to the chamber. Plasmon (P) -probes and surface enhanced Raman scattering (S) -probes were hybridized with messenger RNA precursors at the exon2 / intron3 interface, capping with gold nanoparticles and the Raman molecule cyanine-3 (Cy3), respectively. (iv) hybridization of the messenger RNA precursor molecule with plasmon (P) -probe and surface enhanced Raman scattering (S) -probe, followed by washing the chamber to remove excess probes, (v) plasmon (P) -probe and Messenger RNA precursors hybridized with surface enhanced Raman scattering (S) -probe react with HeLa nuclear extract for RNA splicing and (vi) plasmon migration, scattering intensity and surface enhanced Raman scattering (SERS) spectra in real time Collected at endpoints, measured over time for RNA splicing monitoring. In addition, the present invention investigated RNA splicing platforms such as RNA splicing inhibition and cancer inhibitory effect by isoginkgetin.

본 발명은 광학 기반의 방법을 이용하여 산란 강도, 국소 표면 플라즈몬 공명과 표면 증강 라만 산란을 설명할 수 있으므로, RNA 스플라이싱을 모니터링 및 검출할 수 있다. 분자적 방법과 비교하여 광학 기반의 방법은 스플라이싱의 동역학 연구, 스플라이싱 억제, 시스-트랜스 대체 스플라이싱 및 RNA 스플라이스의 빠른 측정 등의 여러 가지 장점이 있다. 본 발명에서는 단일분자 수준에서 전령 RNA 전구체 스플라이싱 및 스플라이싱 동력학에 대한 이소깅제틴의 영향 등을 분석하였다. 따라서, 개별 전령 RNA 전구체 분자의 스플라이싱을 수행하는 능력, 높은 해상도 및 광학 기반의 방법 등은 약하게 안정화된 RNA 분자 및 그 수명에 대한 상대적 안정성을 입증할 수 있다. 종래의 방법으로는 모니터링 할 수 없었던, 전령 RNA 전구체 스플라이싱의 동력학을 직접 분석으로 10분에서 모니터링 하였다. 산란 강도 및 표면 증강 라만 산란(SERS) 스펙트럼은 RNA 스플라이싱 모니터링의 성공을 나타내며, RNA 스플라이싱 억제제로 광학 기반 방법은 RNA 스플라이싱 억제에 사용할 수 있음을 확인하였다. 또한, 암, 분자면역학 및 분자 진화 연구에 중요한 선택적 스플라이싱(alternative splicing) 검출, 다중 엑손-인트론 전령 RNA 전구체의 RNA 스플라이싱 검출, RNA의 인트론으로부터 마이크로-RNA의 생성(마이크로 RNA의 성숙 ) 및 숙주 mRNA의 처리로부터 siRNA의 모니터링 등을 광학 기반 RNA 스플라이싱 모니터링을 통해 수행할 수 있다. RNA 스플라이싱은 번역 후 수준에서 유전자 조절에 중요하므로, 이 플랫폼을 이용한 RNA 스플라이싱 촉진 또는 억제는 약물 스크리닝에 유용한 현대분자생물학의 필수 요소이다.The present invention can account for scattering intensity, local surface plasmon resonance and surface enhanced Raman scattering using optically based methods, thereby monitoring and detecting RNA splicing. Compared to molecular methods, optical-based methods have several advantages, such as the study of the dynamics of splicing, splice suppression, cis-trans replacement splicing, and rapid measurement of RNA splices. In the present invention, the effects of isogingzetin on messenger RNA precursor splicing and splicing kinetics at the single molecule level were analyzed. Thus, the ability to perform splicing of individual messenger RNA precursor molecules, high resolution and optically based methods and the like can demonstrate the relative stability to weakly stabilized RNA molecules and their lifetimes. The kinetics of messenger RNA precursor splicing, which could not be monitored by conventional methods, was monitored at 10 minutes by direct analysis. Scattering Intensity and Surface Enhancement Raman scattering (SERS) spectra indicate the success of RNA splicing monitoring, and it has been confirmed that optically based methods as RNA splicing inhibitors can be used to inhibit RNA splicing. In addition, selective splicing detection important for cancer, molecular immunology and molecular evolution studies, RNA splicing detection of multiple exon-intron messenger RNA precursors, generation of micro-RNA from introns of RNA (micro RNA maturation) And siRNA monitoring from the treatment of host mRNA and the like can be performed via optical based RNA splicing monitoring. Since RNA splicing is important for gene regulation at the post-translational level, promoting or inhibiting RNA splicing using this platform is an essential element of modern molecular biology useful for drug screening.

따라서, 본 발명은 일 관점에서 (a) 표적 유전자를 발현 벡터에 클로닝하고 세포, 조직 또는 인간을 제외한 생체 내로 벡터를 형질전환시켜, 재조합 표적 전령 RNA 전구체의 전사체를 생성하는 단계; (b) 플라즈몬 프로브 또는 표면 증강 라만 산란(SERS)-태그 프로브를 상기 재조합 표적 전령 RNA 전구체의 전사체와 반응시키거나, 직접 세포, 조직 또는 인간을 제외한 생체 내로 도입시켜, 상기 플라즈몬 프로브 또는 표면 증강 라만 산란(SERS)-태그 프로브를 전령 RNA 전구체의 엑손/인트론 및 인트론/엑손의 경계에 부착시키는 단계; 및 (c) 광학 현미경을 통해 산란 강도, 레일리 산란의 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 측정하여, RNA 스플라이싱을 검출하는 단계를 포함하는 RNA 스플라이싱의 검출 및 모니터링 방법에 관한 것이다.Thus, in one aspect, the present invention provides a method for producing a transcript of a recombinant target messenger RNA precursor by (a) cloning a target gene into an expression vector and transforming the vector into cells other than cells, tissues, or humans; (b) a plasmon probe or surface enhanced Raman scattering (SERS) -tag probe is reacted with a transcript of said recombinant target messenger RNA precursor or introduced directly into vivo, excluding cells, tissues or humans to enhance said plasmon probe or surface Attaching a Raman scattering (SERS) -tag probe to the boundaries of exons / introns and introns / exons of the messenger RNA precursor; And (c) measuring scattering intensity, local surface plasmon resonance of Rayleigh scattering, and surface enhanced Raman scattering through an optical microscope to detect RNA splicing. .

본 발명에 있어서, 상기 (b) 단계는 인-비트로(in vitro) 또는 인-비보(in vivo)에서 수행하는 것을 특징으로 할 수 있다. In the present invention, the step (b) may be characterized in that performed in-vitro (in vitro) or in-vivo (in vivo).

본 발명에 있어서, 상기 RNA 스플라이싱 분석은 나노입자 결합 패턴을 포함하는 레일리 산란 방법으로 측정하는 것이 바람직하며, 적어도 하나 이상의 나노입자와의 일차원 결합 패턴을 포함하는 산란 강도의 방법으로 측정하는 것이 바람직하고, 전자기 표면 증강 패턴 형성을 포함하는 표면 증강 라만 산란(SERS)-태그와 나노입자 결합의 표면 증강 라만 산란(SERS) 방법으로 수행하는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the RNA splicing analysis is preferably measured by Rayleigh scattering method including a nanoparticle binding pattern, it is measured by the method of scattering intensity including a one-dimensional binding pattern with at least one nanoparticle. Preferably, the surface enhanced Raman scattering (SERS) -tag including the formation of an electromagnetic surface enhancement pattern is preferably performed by the surface enhanced Raman scattering (SERS) method of nanoparticle bonding, but is not limited thereto.

본 발명에 있어서, 상기 레일리 산란의 국소 표면 플라즈몬 공명, 산란 강도 및 표면 증강 라만 산란(SERS)은 광학 현미경 챔버의 고체상 또는 용액상에서 발생하는 것을 특징으로 할 수 있다.In the present invention, the local surface plasmon resonance, scattering intensity and surface enhanced Raman scattering (SERS) of the Rayleigh scattering may be characterized in that it occurs in the solid phase or solution phase of the optical microscope chamber.

또한, 본 발명에 있어서, 상기 결합 패턴은 전령 RNA 전구체 분자의 인트론 수에 따른 나노입자를 포함하는 프로브의 결합 패턴인 것을 특징으로 할 수 있다.In addition, in the present invention, the binding pattern may be characterized in that the binding pattern of the probe including the nanoparticles according to the number of introns of the messenger RNA precursor molecule.

본 발명에 있어서, 상기 표면 증강 라만 산란(SERS)-태그 프로브는 라만 다이(Raman dye)가 태그된 것을 특징으로 할 수 있으며, 상기 라만 다이(Raman dye)는 시아닌-3(Cyanine-3), 시아닌-5(Cyanine-5), 시아닌-5.5(Cyanine-5.5), 시아닌 7(Cyanine 7), 4-아미노티오페놀(4-aminothiophenol), 4-메틸벤젠티올(4-methylbenzenethiol), 2-나프탈렌티올(2-naphthalenethiol), 로다민-5-(and-6)-이소티오시아네이트(rhodamine-5-(and-6)-isothiocyanate), 테트라메틸로다민-5-이소티오시아네이트(tetramethylrhodamine-5-isothiocyanate), 로다민 B 로다민 6G 닐 블루(rhodamine B Rhodamine 6G nile blue), FAM 및 TAMRA으로 구성된 군에서 선택되는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the surface enhanced Raman scattering (SERS) -tag probe may be characterized in that the Raman dye (Taman dye), the Raman dye (Cyanine-3), Cyanine-5, Cyanine-5.5, Cyanine 7, 4-aminothiophenol, 4-methylbenzenethiol, 4-naphthalene Thiol (2-naphthalenethiol), rhodamine-5- (and-6) -isothiocyanate, tetramethylrhodamine-5-isothiocyanate (tetramethylrhodamine- 5-isothiocyanate), Rhodamine B Rhodamine 6G nile blue (rhodamine B Rhodamine 6G nile blue), preferably selected from the group consisting of, but not limited to TAMRA.

본 발명에 있어서, 상기 플라즈몬 프로브는 RNA 분자에 병렬로 결합하는 것을 특징으로 할 수 있다.In the present invention, the plasmon probe may be characterized by binding in parallel to the RNA molecule.

본 발명에 있어서, 상기 플라즈몬 프로브는 표적 유전자 전령 RNA 전구체 서열과 50~100%의 상동성을 갖는 것이 바람직하며, 상기 플라즈몬 프로브는 전령 RNA 전구체 분자의 수에 따라 2~100개 이상의 나노입자를 포함하는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the plasmon probe preferably has a homology of 50 to 100% with the target gene messenger RNA precursor sequence, the plasmon probe comprises 2 to 100 or more nanoparticles according to the number of messenger RNA precursor molecules Preferably, but not limited thereto.

본 발명에 있어서, 상기 플라즈몬 프로브는 RNA 스플라이싱 후, 1~80nm의 공간 분리를 갖는 것을 특징으로 할 수 있다.In the present invention, the plasmon probe may be characterized by having a spatial separation of 1 ~ 80nm after RNA splicing.

본 발명에 있어서, 상기 전령 RNA 전구체는 전령 RNA 전구체, 마이크로 RNA 전구체, hnRNA, rRNA, tRNA 및 viral RNA로 구성된 군에서 선택되는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the messenger RNA precursor is preferably selected from the group consisting of messenger RNA precursor, micro RNA precursor, hnRNA, rRNA, tRNA and viral RNA, but is not limited thereto.

본 발명에 있어서, 상기 RNA 스플라이싱은 엑손 옆에 적어도 하나 이상의 인트론을 함유하고 있는 전령 RNA 전구체 분자 또는 프리-마이크로 RNA에서 인트론이 제거되는 것을 특징으로 할 수 있으며, 상기 인트론의 제거는 스플라이세오솜(spliceosom)의 절단 메커니즘에 의해 수행되는 것을 특징으로 할 수 있다.In the present invention, the RNA splicing may be characterized in that introns are removed from messenger RNA precursor molecules or pre-micro RNAs containing at least one intron next to the exon, wherein the removal of the introns is spliced. It may be characterized in that it is carried out by the cleavage mechanism of the spliceosom.

또한, 본 발명에 있어서, 상기 RNA 스플라이싱은 나노입자를 이용한 전령 RNA 전구체의 동력학 모니터링에 의해 수행되는 것을 특징으로 할 수 있으며, 상기 나노입자는 직경 10~100nm의 금, 은, 백금 및 구리 나노입자로 구성된 군에서 선택되는 어느 하나인 것이 바람직하나, 이에 한정되는 것은 아니다.In addition, in the present invention, the RNA splicing may be performed by kinetic monitoring of messenger RNA precursor using nanoparticles, the nanoparticles are gold, silver, platinum and copper having a diameter of 10 ~ 100nm. It is preferably any one selected from the group consisting of nanoparticles, but is not limited thereto.

본 발명에 있어서, 상기 나노입자는 안정제를 추가로 함유하는 것을 특징으로 할 수 있으며, 상기 나노입자와 안정제는 티올기가 직접 결합하는 것을 특징으로 할 수 있다. 또한, 상기 안정제는 티올폴리에틸렌글리콜(PEG), 폴리에틸렌글리콜(PEG) 유도체, 티올올리고에틸렌글리콜, 세틸트리메틸암모늄브로마이드 및 폴리스티렌술폰산으로 구성된 군에서 선택되는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the nanoparticles may be characterized in that it further comprises a stabilizer, the nanoparticles and stabilizer may be characterized in that the thiol group is directly bonded. In addition, the stabilizer is preferably selected from the group consisting of thiol polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, thiol oligoethylene glycol, cetyltrimethylammonium bromide and polystyrene sulfonic acid, but is not limited thereto.

본 발명에 있어서, 상기 세포는 원핵세포, 진핵세포, 정상세포 및 암세포로 구성된 군에서 선택되는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the cell is preferably selected from the group consisting of prokaryotic cells, eukaryotic cells, normal cells and cancer cells, but is not limited thereto.

본 발명에 있어서, 상기 광학 현미경은 암시야 콘덴서; 스펙트럼 그래프용 분광기; 신호처리용 CCD 카메라; 레일리 산란용 할로겐 또는 제논의 백색 광원; 표면 증강 라만 산란(SERS) 신호 검출용 레이저 소스; 및 RNA 스플라이싱 분석용 반응 챔버를 포함하는 것을 특징으로 할 수 있다.In the present invention, the optical microscope comprises a dark field condenser; Spectrograph for spectral graph; CCD camera for signal processing; White light sources of halogen or xenon for Rayleigh scattering; Laser sources for surface enhanced Raman scattering (SERS) signal detection; And it may be characterized in that it comprises a reaction chamber for RNA splicing analysis.

본 발명의 광학 현미경은 산란 강도, 레일리 산란의 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 측정할 수 있는 것이라면, 제한 없이 사용이 가능하다.The optical microscope of the present invention can be used without limitation as long as it can measure scattering intensity, local surface plasmon resonance of Rayleigh scattering and surface enhanced Raman scattering.

본 발명에 있어서, 상기 챔버는 전령 RNA 전구체 포획을 위한 비오틴-DNA 프로브가 수직 방향으로 고정된 스트렙타비딘 분자가 코팅되어 있는 것을 특징으로 할 수 있으며, 상기 챔버는 표면 증강 라만 산란(SERS) 신호 검출용 플라즈몬 나노입자-프로브 혼합물을 추가로 포함하는 것을 특징으로 할 수 있다.In the present invention, the chamber may be characterized in that the biotin-DNA probe for messenger RNA precursor capture is coated with a streptavidin molecule fixed in the vertical direction, the chamber is surface enhanced Raman scattering (SERS) signal It may be characterized in that it further comprises a plasmon nanoparticle-probe mixture for detection.

실시예Example

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

실시예 1: 베타-글로빈(β-globin) 프리-mRNA의 클로닝 및 전사 Example 1 Cloning and Transcription of Beta-globin Pre-mRNA

RNA 스플라이싱 연구를 위한 베타-글로빈 전령 RNA 전구체 생산을 위해, 인간 베타-글로빈(HBB) 유전자(GenBank: GQ370762.1)를 사용하였다. 3개의 엑손(1,142bp 엑손-1, 223bp 엑손 2, 129bp 엑손-3) 및 2개의 인트론(130bp 인트론-1, 2,850bp 인트론-2)을 포함하는 1,474bp의 인간 베타-글로빈(HBB, NCBI GQ370762.1)유전자로부터 전령 RNA 전구체를 제조하였다. For the production of beta-globin messenger RNA precursors for RNA splicing studies, the human beta-globin (HBB) gene (GenBank: GQ370762.1) was used. 1,474 bp human beta-globin (HBB, NCBI GQ370762) containing three exons (1,142 bp exon-1, 223 bp exon 2, 129 bp exon-3) and two introns (130 bp intron-1, 2,850 bp intron-2) .1) A messenger RNA precursor was prepared from the gene.

RNA 스플라이싱 연구를 위한 베타-글로빈 전령 RNA 전구체 생산을 위해, 인간 베타-글로빈(HBB) 유전자(GenBank: GQ370762.1)를 사용하였다. 3개의 엑손(1,142bp 엑손-1, 223bp 엑손 2, 129bp 엑손-3) 및 2개의 인트론(130bp 인트론-1, 2,850bp 인트론-2)을 포함하는 1,474bp의 인간 베타-글로빈(HBB, NCBI GQ370762.1)유전자로부터 전령 RNA 전구체를 제조하였다.For the production of beta-globin messenger RNA precursors for RNA splicing studies, the human beta-globin (HBB) gene (GenBank: GQ370762.1) was used. 1,474 bp human beta-globin (HBB, NCBI GQ370762) containing three exons (1,142 bp exon-1, 223 bp exon 2, 129 bp exon-3) and two introns (130 bp intron-1, 2,850 bp intron-2) .1) A messenger RNA precursor was prepared from the gene.

5'-AAAGTCGACGCTGCTGGTGGTCTACCC-3'(GTCGAC: SalI site) : 서열번호 15'-AAA GTCGAC GCTGCTGGTGGTCTACCC-3 '(GTCGAC: SalI site): SEQ ID NO: 1

5'-AAACTCGAGTTAGTGATACTTGTGGGCCAGG-3'(CTCGAG: XhoI site) : 서열번호 25'-AAA CTCGAG TTAGTGATACTTGTGGGCCAGG-3 '(CTCGAG: XhoI site): SEQ ID NO: 2

엑손-2/인트론-2/엑손-3을 포함하는 인간 베타-글로빈 전령 RNA 전구체의 유전자 절편을 플라스미드 pET32b (+)(Novagen) 시스템을 이용하여 클로닝하고, E. coli BL21 (DE3) (Real Biotech)에서 1mM IPTG로 유도하여 전사시켰다. 유도된 베타-글로빈 전령 RNA 전구체는 분리하여, RNeasy Mini Kit(Qiagen)로 정제한 후 RNA 스플라이싱 연구에 사용하였다(도 1b, 좌측). Gene fragments of human beta-globin messenger RNA precursors including exon-2 / intron-2 / exon-3 were cloned using the plasmid pET32b (+) (Novagen) system and E. coli BL21 (DE3) (Real Biotech And induced transcription by 1 mM IPTG. Induced beta-globin messenger RNA precursors were isolated, purified with RNeasy Mini Kit (Qiagen) and used for RNA splicing studies (FIG. 1B, left).

전령 RNA 전구체 스플라이싱(엑손-2/인트론-2/엑손-3)은 전령 RNA 전구체 스플라이싱 키트(Proteinone, USA)의 HeLa 핵 추출물의 스플라이세오솜(spliceosome)에 의한 일련의 촉매 반응으로 수행하였다.Messenger RNA Precursor Splicing (Exon-2 / Intron-2 / Exon-3) is a series of catalytic reactions by spliceosome of HeLa nuclear extract of the Messenger RNA Precursor Splicing Kit (Proteinone, USA). Was performed.

실시예 2: 플라즈몬 및 SERS 프로브의 합성 Example 2: Synthesis of Plasmon and SERS Probes

플라즈몬 결합은 표면 증강 라만 산란(SERS), 산란 강도 및 국소 표면 플라즈몬 공명(LSPR)을 위한 집적 광학 구성을 이용한 RNA 스플라이싱을 모니터링 하여 탐색하였다. Plasmon binding was explored by monitoring RNA splicing using integrated optical construction for surface enhanced Raman scattering (SERS), scattering intensity and local surface plasmon resonance (LSPR).

플라즈몬 결합 프로브-1(P1)과 프로브-2(P2)는 엑손-2/인트론-2 및 인트론-2/엑손-3 사이의 경계 부위에 대응한다. Plasmon binding probe-1 (P1) and probe-2 (P2) correspond to the border site between exon-2 / intron-2 and intron-2 / exon-3.

5'-HS-(CH2)3CGTGGATCCTGAGAACTTCA-3'(P1): 서열번호 35'-HS- (CH 2 ) 3 CGTGGATCCTGAGAACTTCA-3 '(P1): SEQ ID NO: 3

5'-CCTGGGCAACGTGCTGGTCTG(CH2)3-HS-3'(P2): 서열번호 45'-CCTGGGCAACGTGCTGGTCTG (CH 2 ) 3 -HS-3 '(P2): SEQ ID NO: 4

티올-변형 프로브는 10㎕의 10N DTT로 10분간 상온에서 반응시킨 후, 초과 DTT를 50㎕의 에틸아세테이트로 3회 제거하여, 티올-변형 프로브로부터 티올-단편을 얻었다. 만들어진 프로브는 플라즈몬 프로브를 만들기 위해 50nm(0.05 mg/mL) 구연산-금나노 입자(AuNPs) 용액에서 즉시 반응시켰다(Kimling et al., J. Phys. Chem . B 110:15700, 2006). 1mL 구연산-금나노 입자 용액에 0.1M BSPP(bis(p-sulfonatophenyl)phenylphosphine dipotassium 염, 시그마 - 알드리치)를 첨가하여 리간드를 교환하였다. 과량의 BSPP는 원심분리로 제거하고, 입자는 10 mM Tris(pH 8.0), 40 mM NaCl 버퍼로 점진적으로 세척하여 두 부분으로 분리하였다. 한 부분은 P1 및 P2 프로브와 10시간 반응시킨 후, poly(ethylene glycol):금 나노입자들의 비율이 1:1,000이 되도록 poly(ethylene glycol) methyl ether thiol CH3O(CH2CH2O)nCH2CH2SH(시그마 알드리치, USA) 을 첨가하였다.The thiol-modified probe was reacted with 10 µl of 10N DTT at room temperature for 10 minutes, and then excess DTT was removed three times with 50 µl of ethyl acetate to obtain a thiol-fragment from the thiol-modified probe. The resulting probes were immediately reacted in 50 nm (0.05 mg / mL) citric acid-gold nanoparticles (AuNPs) solution to make plasmon probes (Kimling et al., J. Phys. Chem . B 110: 15700, 2006). Ligand exchange was performed by adding 0.1 M BSPP (bis (p-sulfonatophenyl) phenylphosphine dipotassium salt, Sigma-Aldrich) to 1 mL citric acid-gold nanoparticle solution. Excess BSPP was removed by centrifugation, and particles were separated into two portions by gradual washing with 10 mM Tris (pH 8.0), 40 mM NaCl buffer. One part was reacted with P1 and P2 probes for 10 hours, and then poly (ethylene glycol) methyl ether thiol CH 3 O (CH 2 CH 2 O) n was obtained so that the ratio of poly (ethylene glycol): gold nanoparticles was 1: 1,000. CH 2 CH 2 SH (Sigma Aldrich, USA) was added.

표면 증강 라만 산란(SERS) 스펙트럼의 라만 지문을 위해서는, 변형 금나노 입자가 1:50(AuNP/probe) 의 비율로 결합한 프로브-1(S1) 및 3' 말단에 Cy3가 결합한 프로브-2(S2)(서열번호 5, IDT DNA, USA)를 사용하였다. For Raman fingerprinting of the surface enhanced Raman scattering (SERS) spectrum, probe-1 (S1) with modified gold nanoparticles bound at a ratio of 1:50 (AuNP / probe) and probe-2 (S2 with Cy3 binding at the 3 'end) ) (SEQ ID NO: 5, IDT DNA, USA) was used.

5'-GTCTGGTCGTGCAACGGGTCC-Cy3-3'(S2): 서열번호 55'-GTCTGGTCGTGCAACGGGTCC-Cy3-3 '(S2): SEQ ID NO: 5

구체적으로, 엑손-2/인트론-2 및 인트론-2/엑손-3 사이의 경계 부위에 대응하는 플라즈몬 프로브는 50 nm 금나노 입자로 캡핑하여 결합비 1:50(AuNP:프로브)으로 하였으며, 표면 증강 라만 산란(SERS) 프로브는 3'-말단에 Cy3를 결합시켜 표면 증강 라만 산란(SERS)에 대한 라만 지문을 통한 RNA 스플라이싱용 프로브로 제작하였다. 이어서, 결합된 프로브는 티올-PEG, HS (CH2)11(OCH2CH2)3OH (OEG3) 및 1 μM BSA로 혼성화 버퍼(10 mM Tris. HCl, pH 8.0, 및 160 mM NaCl)와 RNA 스플라이싱 버퍼(RNA 스플라이 키트에 제공)에서 비활성화 시켰다. Specifically, the plasmon probe corresponding to the boundary region between exon-2 / intron-2 and intron-2 / exon-3 was capped with 50 nm gold nanoparticles to have a binding ratio of 1:50 (AuNP: probe), and the surface An enhanced Raman scattering (SERS) probe was constructed as a probe for RNA splicing through Raman fingerprinting for surface enhanced Raman scattering (SERS) by binding Cy3 to the 3'-end. The bound probe was then hybridized with thiol-PEG, HS (CH 2 ) 11 (OCH 2 CH 2 ) 3 OH (OEG3) and 1 μM BSA (10 mM Tris. HCl, pH 8.0, and 160 mM NaCl). It was inactivated in RNA splicing buffer (provided in the RNA splicing kit).

다음으로, 스트랩타아비딘이 코팅된 유리(SMS, Arrayit)로 반응챔버를 만들고, 100μM 비오틴 포획 프로브(5'-GCTGCTGGTGGTCTACCCT-biotinylated-3': 서열번호 6)를 주입하여 2시간 동안 스트렙타비딘-비오틴 결합 반응을 시킨 후, 비특이적 반응을 최소화하기 위해 표면을 2mL 수퍼블록 버퍼(37516, Thermo Scientific)로 처리하였다. 그 다음, 10:1의 비율로 베타-글로빈 전령 RNA 전구체(200 μL, 1 ng/μL)를첨가하여, 10mM Tris-HCl(pH 7.5), 0.1mM EDTA의 37℃ 챔버에서 1시간 동안 변형 프로브와 배양하고 포획하였다. 포획 효율은 형광 프로브(5'-Cy3-GACCCGTTGCACGAC-3': 서열번호 7)로 모니터하였다.Next, the reaction chamber was made of straptavidin-coated glass (SMS, Arrayit), and 100 μM biotin capture probe (5'-GCTGCTGGTGGTCTACCCT-biotinylated-3 ': SEQ ID NO: 6) was injected for 2 hours to streptavidin- After the biotin binding reaction, the surface was treated with 2 mL superblock buffer (37516, Thermo Scientific) to minimize nonspecific reactions. Next, add the beta-globin messenger RNA precursor (200 μL, 1 ng / μL) at a ratio of 10: 1 to modify the probe for 1 hour in a 37 ° C. chamber of 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA. Were incubated and captured. Capture efficiency was monitored with a fluorescent probe (5'-Cy3-GACCCGTTGCACGAC-3 ': SEQ ID NO: 7).

실시예 3: 광학 구성 (Optical configuration)Example 3: Optical configuration

전령 RNA 전구체 단일분자의 RNA 스플라이싱은 산란 강도, 국소 표면 플라즈몬 공명 및 라만 산란의 집적 광학 구성으로 모니터링 하였다. 광학 구성은 암시야 콘덴서로 업라이트 이클립스 니켈 U 현미경 (니콘)에서 설정한 다음, 라만 산란용 Monora500i과 산란 강도용 Monora320i 쵤영 포트(Dongwoo Optron, 한국)를 통합하여, iXon EM+ CCD 카메라 (512 × 512 active pixel chip) (Andor, Oxford Instruments)와 연결하였다. 광원은 할로겐 또는 제논의 백색 광원을 사용하고, 레이저는 785nm 및 632.5nm의 소스를 사용하였다.RNA splicing of messenger RNA precursor monomolecules was monitored by scattering intensity, local surface plasmon resonance and integrated optical configuration of Raman scattering. The optical configuration was set on an upright Eclipse Nickel U microscope (Nikon) with a darkfield capacitor, then integrated Monora500i for Raman scattering and Monora320i shooting port (Dongwoo Optron, South Korea) for scattering intensity, iXon EM + CCD camera (512 × 512 active) pixel chip) (Andor, Oxford Instruments). The light source used a white light source of halogen or xenon, and the laser used sources of 785 nm and 632.5 nm.

실시예 4: RNA SplicingExample 4: RNA Splicing

반응챔버는 처음 15분 동안 1 mg/㎖ 비오틴 포획 프로브(Integrated DNA Technology)를 반응시키고, 300㎕의 T50 (50 mM NaCl/10반응챔버는 처음 15분 동안 1 mg/㎖ 비오틴 포획 프로브(Integrated DNA Technology)를 반응시키고, 300㎕의 T50 (50 mM NaCl/10 mM Tris, pH가 8.0)으로 세척하였다. 다음으로, 챔버를 45분간 SuperBlock (37516, Thermo Scientific)으로 반응시켜, 300㎕의 T50으로 다시 한번 세척한 후, 200㎕의 전령 RNA 전구체 분자 용액(5ng/㎕)으로 챔버를 반응시켰다. 그 결과, 전령 RNA 전구체 분자는 특정 포획 프로브가 코팅된 유리 표면에만 결합하고, 다른 mRNA 분자는 용액에 부유하였다. 변형된 유리 표면의 매우 높은 표면적으로 인해, 낮은 농도의 베타-글로빈 전령 RNA 전구체를 높은 비율로 농축 가능하다.The reaction chamber reacted with a 1 mg / ml biotin capture probe (Integrated DNA Technology) for the first 15 minutes, and 300 μl of T50 (50 mM NaCl / 10 reaction chamber had a 1 mg / ml biotin capture probe (Integrated DNA) for the first 15 minutes. Technology) and washed with 300 μl of T50 (50 mM NaCl / 10 mM Tris, pH 8.0) Next, the chamber was reacted with SuperBlock (37516, Thermo Scientific) for 45 minutes, followed by 300 μl of T50. After washing again, the chamber was allowed to react with 200 μl of messenger RNA precursor molecule solution (5 ng / μl), which resulted in messenger RNA precursor molecules bound only to a glass surface coated with a specific capture probe and other mRNA molecules in solution. Due to the very high surface area of the modified glass surface, it is possible to concentrate low concentrations of beta-globin messenger RNA precursors at high rates.

베타-글로빈 전령 RNA 전구체 포획 장치는 온도 제어기(TC-324B, Warner Instruments)를 이용하여 40℃의 최적 어닐링 온도로 조절하여, 혼성화 용액(10 mM Tris. HCl, pH 8.0 및 160 mM NaCl)에서 플라즈몬 프로브와 표면 증강 라만 산란(SERS) 프로브를 반응시켰다. 챔버는 RNA 스플라이싱 버퍼(20 mM HEPES-Na (pH 7.9), 20% Glycerol, 42 mM (NH4)2SO4, 0.5 mM DTT, 0.2 mM EDTA)로 평형화한 후, 인비트로 전령 RNA 전구체 스플라이싱을 위해 HeLa 세포 핵 추출물(P002-1, Protein One, 10 μL, 5 ng/μL)이 포함된 RNA 스플라이싱 버퍼 190㎕를 시간 0초 포인트에 챔버에 주입하였다. RNA 스플라이싱은 암시야 콘덴서로 업라이트 니콘 현미경 (이클립스의 Ni-U)을 사용하여 병렬로 모니터링 하였다. 데이터는 Shamrock 500i 분광기(Andor, Oxford Instruments)의 영상 포트에 Andor iXon EM+ CCD detector (512 × 512 pixel chip)가 장착된 100X Nikon 렌즈(N.A = 0.95)를 사용하여 기록하였다. RNA 스플라이싱은 플라즈몬 결합 모드에서 산란 강도, LSPR 및 라만 산란에 의해 검출하였다.The beta-globin messenger RNA precursor capture device was adjusted to an optimum annealing temperature of 40 ° C. using a temperature controller (TC-324B, Warner Instruments) to plasmon in the hybridization solution (10 mM Tris. HCl, pH 8.0 and 160 mM NaCl). The probe was reacted with a surface enhanced Raman scattering (SERS) probe. The chamber was equilibrated with RNA splicing buffer (20 mM HEPES-Na (pH 7.9), 20% Glycerol, 42 mM (NH 4 ) 2 SO 4 , 0.5 mM DTT, 0.2 mM EDTA), followed by in vitro messenger RNA precursor. For splicing, 190 μl of RNA splicing buffer containing HeLa cell nuclear extract (P002-1, Protein One, 10 μL, 5 ng / μL) was injected into the chamber at time point 0 seconds. RNA splicing was monitored in parallel using an upright Nikon microscope (Ni-U in Eclipse) as a darkfield condenser. Data was recorded using a 100X Nikon lens (NA = 0.95) equipped with an Andor iXon EM + CCD detector (512 x 512 pixel chip) in the imaging port of a Shamrock 500i spectrometer (Andor, Oxford Instruments). RNA splicing was detected by scattering intensity, LSPR and Raman scattering in plasmon binding mode.

4-1: 젤 전기영동에 의한 RNA 스플라이싱 확인4-1: Confirmation of RNA Splicing by Gel Electrophoresis

RNA 스플라이싱은 20 nM 전령 RNA 전구체, 30% 핵 추출물, 20 mM 염화칼륨, 2.5 mM 염화 마그네슘, 10 mM 크레아틴 포스페이트, 0.5 mM DTT, 0.4 units/l RNasin, 40 mM Tris-HCl (pH 8), 0.5 mM ATP 및 1% (v/v) DMSO를 40℃에서 10분간 반응시켜 수행하였다. mRNA 스플라이싱 산물, 즉, 전령 RNA 전구체와 DNA 프로브의 결합 입자는 젤 전기영동에 의해 분리하였다.RNA splicing includes 20 nM messenger RNA precursor, 30% nuclear extract, 20 mM potassium chloride, 2.5 mM magnesium chloride, 10 mM creatine phosphate, 0.5 mM DTT, 0.4 units / l RNasin, 40 mM Tris-HCl, pH 8, 0.5 mM ATP and 1% (v / v) DMSO were performed by reacting at 40 ° C for 10 minutes. mRNA splicing products, ie binding particles of messenger RNA precursor and DNA probe, were separated by gel electrophoresis.

그 결과, 도 2b 우측의 1% 아가로즈 젤은 금나노 입자 기능화 DNA 프로브(lane 1), 스플라이세오솜(spliceosome) 첨가 전 스플라이싱 반응(lane 2), 스플라이세오솜(spliceosome) 첨가 후 스플라이싱 반응(lane 3)을 나타낸다. 스플라이싱 혼합물은 인트론-2가 제거되어 이량체 형성을 나타내는 중간 밴드를 포함하며, 2개의 프로브가 플라즈몬 결합한 형태인 스플라이싱 mRNA는 850bp의 인트론-2가 제거되어 전령 RNA 전구체 보다 길이가 짧아졌다. 이는 금나노 입자 기능화 DNA 프로브보다 느려지고 전령 RNA 전구체와 혼성화 된 금나노 입자 기능화 DNA 프로브보다 빨라졌다. 이량체 밴드는 전기용출 시스템(Elutrap electroelution system, Whatman)으로 분리하여 투과형 전자현미경(TEM)으로 관찰하였다(도 2c)As a result, the 1% agarose gel on the right side of FIG. Post splicing reaction (lane 3). The splicing mixture contains an intermediate band showing dimer formation by removal of intron-2, and the splicing mRNA, in which two probes are plasmon-bound, is shorter than the messenger RNA precursor due to the removal of 850 bp of intron-2. lost. This is slower than gold nanoparticle functionalized DNA probes and faster than gold nanoparticle functionalized DNA probes hybridized with messenger RNA precursors. Dimer bands were separated by an electroelution system (Elutrap electroelution system, Whatman) and observed with a transmission electron microscope (TEM) (Fig. 2c).

4-2: 색상 변화에 의한 RNA 스플라이싱 확인4-2: RNA Splicing Confirmation by Color Change

개별 금나노 입자는 비편광 백색광을 사용하여, 반응 챔버 내 용액에서 발생한 개별 입자의 광산란을 암시야 현미경으로 관찰하였다(도 2d). 금나노 입자는 전령 RNA 전구체에서 인트론이 제거되고 엑손이 연결되어 프로브가 근접하게 되면, 그린의 개별 금 나노 입자(도 2d, 좌측)에서 레드의 결합 금나노 입자(도 2d, 우측)로 색상이 변한다. DNA 프로브는 정확한 이중 가닥을 형성하기 위해 전령 RNA 전구체의 엑손/인트론 경계에 결합하며, 스플라이세오솜(spliceosome) 결합 전령 RNA 전구체의 접근에 영향을 받지 않는다. pET32a (+) 벡터의 T7 폴리머라제 대조군의 전령 RNA 전구체 분자의 색상은 변화가 관찰되지 않아, RNA 스플라이싱에 의해 색상이 변하는 것을 알 수 있었다. 플라즈몬 프로브는 RNA 스플라이싱으로 거리가 감소하고 전령 RNA 전구체 분자의 금나노 입자의 구성이 완전히 비가역적일 때, 일반적으로 플라즈본 결합을 생성하고 광학적 기록을 재생할 수 있다. 엑손-2(222bp) 및 엑손-3(126bp)가 인트론-2(851bp)로부터 분리되고, 엑손-2와 엑손-3이 연결되어, 2개의 프로브는 LSPR 및 SERS의 플라즈몬 부위의 증강으로 플라즈몬 결합 부위에 도달한다. HeLa 추출물 주입 포인트(point 0s to 360s)로부터 산란 강도가 서서히 증가하는 것은 점진적인 거리감소를 나타내며, 급격한 산란 강도의 증가는 스플라이싱 궤적 상 2개의 프로브가 매우 근접해 있을 때며 mRNA 형성을 위한 전령 RNA 전구체의 스플라이싱이 성공적인 것을 나타낸다(도 3)Individual gold nanoparticles were unpolarized white light, and the light scattering of the individual particles generated in the solution in the reaction chamber was observed with a dark field microscope (FIG. 2D). As the gold nanoparticles are removed from introns in the messenger RNA precursor and the exons are connected to the probe, the gold nanoparticles are colored from the individual gold nanoparticles of green (Fig. 2d, left) to the binding gold nanoparticles of red (Fig. 2d, right). Change. The DNA probe binds to the exon / intron boundary of the messenger RNA precursor to form the correct double strand and is not affected by the access of the spliceosome binding messenger RNA precursor. The color of the messenger RNA precursor molecule of the T7 polymerase control of the pET32a (+) vector was not observed, indicating that the color was changed by RNA splicing. Plasmon probes are generally capable of producing plasmon bonds and reproducing optical recordings when the distance is reduced due to RNA splicing and the composition of the gold nanoparticles of the messenger RNA precursor molecules is completely irreversible. Exon-2 (222 bp) and exon-3 (126 bp) are isolated from intron-2 (851 bp), exon-2 and exon-3 are linked, so that the two probes bind plasmons with enhancement of the plasmon sites of LSPR and SERS Reach the site. Slow increase in scattering intensity from the HeLa extract injection point (point 0s to 360s) indicates a gradual decrease in distance, with a sharp increase in scattering intensity when the two probes are very close together on the splicing trajectory and a messenger RNA precursor for mRNA formation Indicates that splicing of is successful (FIG. 3).

4-3: 국소 표면 플라즈몬 공명(LSRP) 및 SERS에 의한 RNA 스플라이싱 확인4-3: Confirmation of RNA Splicing by Local Surface Plasmon Resonance (LSRP) and SERS

RNA 스플라이싱의 결과는 국소 표면 플라즈몬 공명(LSRP)에서 플라즈몬 이동의 29nm 생성(도 3b) 및 SERS 스펙트럼(도 3c)을 서열번호 8/9 프라이머를 이용한 실시간 PCR로 확인하였다(도 3d).The results of RNA splicing were confirmed by real-time PCR with 29 nm generation (FIG. 3B) and SERS spectra (FIG. 3C) of plasmon shift in local surface plasmon resonance (LSRP) (FIG. 3D).

5'-cacagtctgcctagtacattac-3' (Forward) : 서열번호 85'-cacagtctgcctagtacattac-3 '(Forward): SEQ ID NO: 8

5'-ccctgatttggtcaatatgtgtac-3' (Reverse): 서열번호 95'-ccctgatttggtcaatatgtgtac-3 '(Reverse): SEQ ID NO: 9

금나노 입자는 단량체에서 이량체로 결합하여 산란 강도가 증가시키며, 전령 RNA 전구체의 스플라이싱과 연결된다. 결합 금나노 입자의 방향은 전령 RNA 전구체 스플라이싱의 움직임을 기반으로 반응 챔버 표면상에 표시되고, 인트론 단편이 용액내로 확산되면 실시간 PCR을 수행하였다. Gold nanoparticles bind dimers in monomers to increase scattering intensity and are linked to splicing of messenger RNA precursors. The orientation of bound gold nanoparticles was indicated on the reaction chamber surface based on the movement of messenger RNA precursor splicing, and real-time PCR was performed when the intron fragment diffused into solution.

금나노 입자의 비특이적 부위는 5% HS (CH2)11(OCH2CH2)3OH (OEG3) 및 1μM BSA로 차단하였다. 금 나노 입자는 SERS를 위한 플라즈몬 표면 및 플라즈몬 결합의 광학적 기록을 재생할 수 있다. 2개 이상의 결합체는 금나노 입자 표면에 비특이적 결합 차단에 의해 제거되며, 오직 하나의 프로브만 엑손/인트론 경계에 특이적으로 결합한다. 금나노 입자가 캡핑된 DNA 프로브의 밀도를 감소시키기 위해, 5% 이하의 금나노 입자로 캡핑될 때까지 반응 시간 및 분자 내 DNA 프로브의 농도를 조절하였다. RNA 스플라이싱 후, 2개의 금나노 입자의 결합은 안정화되어 연속적으로 시간 단위로 모니터링 된다. 용액 내 금나노 입자는 산란광의 편광으로 임의로 배향하고, 금나노 입자의 증가된 농도는 챔버내 RNA 스플라이싱 수율을 나타낸다. 상기 플랫폼은 스플라이세오솜(spliceosome)의 스플라이싱 부위 인식의 높은 민감도 및 특이성에 의한 것이다. RNA 스플라이싱은 대장균 추출물을 챔버로 주입하여 산란 강도를 관찰하였다.Nonspecific sites of gold nanoparticles were blocked with 5% HS (CH 2 ) 11 (OCH 2 CH 2 ) 3 OH (OEG3) and 1 μM BSA. Gold nanoparticles can reproduce optical recordings of plasmon surfaces and plasmon bonds for SERS. Two or more binders are removed by nonspecific binding blocking on the surface of the gold nanoparticles, and only one probe specifically binds to the exon / intron boundary. In order to reduce the density of the capped DNA probe, the reaction time and the concentration of the intramolecular DNA probe were adjusted until capped with 5% or less gold nanoparticles. After RNA splicing, the binding of the two gold nanoparticles is stabilized and continuously monitored in units of time. Gold nanoparticles in solution are optionally oriented with polarized light of scattered light, and the increased concentration of gold nanoparticles indicates RNA splicing yield in the chamber. The platform is due to the high sensitivity and specificity of splice site recognition of spliceosomes. RNA splicing was performed by injecting E. coli extract into the chamber to observe scattering intensity.

4-4: RNA 스플라이싱의 동력학4-4: Dynamics of RNA Splicing

전령 RNA 전구체 스플라이싱의 동력학은 10분 동안 측정하였다. 챔버 내로 프리-RNA 스플라이싱 키트 주입하면 2.5~7분 사이에 인트론이 제거되고 10분 후 완전히 제거되었다. 종래의 방법에서는 3'-엑손 다음의 인트론이 15분에 제거된다(Zeng et al., Mol . Cell Biol . 20:8290, 2000; Aoufouchi et al., Cell. 85:415, 1996). 이 결과는 RNA 스플라이싱 시간이 스플라이싱 개시부터 인트론의 스플라이싱까지의 스플라이세오솜(spliceosome) 조립을 포함한 전체 스플라이싱의 총 시간임을 나타낸다. 전령 RNA 전구체 스플라이싱에서 2.5분 지연은 RNA 스플라이싱의 오류를 수정하는 시간이다. 또한, 다양한 스플라이싱의 시간은 5'의 스플라이싱 부위나 인트론의 길이에 의존하며, 또는 인핸서 및 사일런스의 친화력에 의존한다(Wang et al., RNA 14:802, 2008). 인트론 제거의 다양한 속도는 초기 스플라이싱 부위의 인식, 스플라이싱 부위의 페어링, 엑손의 적절한 이음 등의 진행 전에, 삽입 속도의 조절과 같은 스플라이세오솜(spliceosome) 조립의 단계에서의 속도 차이 때문이다. 다른 이유는, 스플라이싱 진행을 억제하기 위한 인트론 내의 세린/아르기닌(SR) 단백질의 결합 때문이다(Wang et al., RNA 14:802, 2008). 표면 증강 라만 산란(SERS)-기반 분석에서, 전령 RNA 전구체 스플라이싱의 실시간 동력학은 RNA 스플라이싱 진행 동안 용액 내 표면 증강 라만 산란(SERS) 신호 측정에 의해 조사되며, 후속 시간에 따른 전령 RNA 전구체 스플라이싱 라만 스펙트럼 변화는 30 및 10초 간격 및 노출 시간에서 기록하였다. 표면 증강 라만 산란(SERS) 태그 분자(Cy3)의 라만 피크는 1589, 1383 및 1120 cm-1 기본 특징 피크에서 모니터링 되었다(도 4a). 전령 RNA 전구체 스플라이싱 과정 동안 시간별 스펙트럼 측정은 도 4b에 나타냈다. Kinetics of messenger RNA precursor splicing was measured for 10 minutes. Injection of the pre-RNA splicing kit into the chamber removed the introns between 2.5-7 minutes and completely after 10 minutes. In conventional methods, the intron following the 3'-exon is removed at 15 minutes (Zeng et al., Mol . Cell Biol . 20: 8290, 2000; Aoufouchi et al., Cell . 85: 415, 1996). This result indicates that RNA splicing time is the total time of total splicing, including spliceosome assembly, from splicing initiation to splicing of introns. The 2.5 minute delay in messenger RNA precursor splicing is the time to correct errors in RNA splicing. In addition, the time of various splicing depends on the length of the 5 'splicing site or intron, or on the affinity of the enhancer and the silence (Wang et al., RNA 14: 802, 2008). The varying rates of intron removal can be attributed to differences in rates at the stage of spliceosome assembly, such as control of insertion rate, prior to progression of initial splicing sites, pairing of splicing sites, proper splice of exons, etc. Because. Another reason is the binding of serine / arginine (SR) proteins in introns to inhibit splicing progression (Wang et al., RNA 14: 802, 2008). In surface enhanced Raman scattering (SERS) -based assays, the real-time kinetics of messenger RNA precursor splicing is investigated by surface enhanced Raman scattering (SERS) signal measurements in solution during RNA splicing progression, followed by messenger RNA over time. Precursor splicing Raman spectral changes were recorded at 30 and 10 second intervals and exposure time. Raman peaks of the surface enhanced Raman scattering (SERS) tag molecule (Cy3) were monitored at 1589, 1383 and 1120 cm −1 basic feature peaks (FIG. 4A). Hourly spectral measurements during the messenger RNA precursor splicing process are shown in FIG. 4B.

SERS 강도는 금나노 입자 표면에 도달하는 P1프로브 및 P2-Cy3 프로브의 라만 다이(dye)의 수에 비례하므로, 스플라이세오솜(spliceosome)의 상대적 활성을 반영하고 전령 RNA 전구체 스플라이싱의 효율을 나타낸다. 정규화된 표면 증강 라만 산란(SERS) 강도 변화는 전령 RNA 전구체 스플라이싱 효율의 직접적인 지표이다(도 4b). 도 4에 나타난 바와 같이, 스플리세오솜 복합체를 함유하는 HeLa 추출물 주입 전 3분 동안은 모든 피크의 강도가 정상곡선을 나타냈다. RNA 스플라이싱을 위한 플라즈몬 결합 및 표면 증강 라만 산란(SERS) 기반 분석의 감도는 0.1 μg/μl에서 5 μg/μl 농도 범위의 전령 RNA 전구체 샘플의 플라즈몬 이동 및 SERS 강도 변화에 의해 측정된다. 그러나, 스플리세오솜의 활성시 반응챔버에서 전령 RNA 전구체의 스플라이싱을 나타내는 초기 4~5분에 각 라만 피크의 상당한 증가가 관찰된다. 스플리세오솜 0.1μg/μl 농도에서 라만 신호증가는 10분 종점에서 정체기에 도달한다. 플라즈몬 이동 및 정규화 표면 증강 라만 산란(SERS) 강도의 변화는 도 4c에 나타냈다. 예상한 바와 같이, 라만 신호의 증가 속도는 전령 RNA 전구체의 농도와 비례하는데, 이는 전령 RNA 전구체의 농도가 높아지면 전령 RNA 전구체의 스플라이싱 속도가 증가하기 때문이다. 검출은 위한 범위는 0.1 μg/μl에서 5 μg/μl로 설정하였으며, 0.1 μg/μl 보다 낮은 전령 RNA 전구체 스플라이싱은 주어진 검출 시간 내에 변화가 없었고, 음성 대조군인 대장균 추출물의 10분간 기록 역시 SERS 강도의 변화가 관찰되지 않았다. 이 결과는, RNA 형성의 자기조립 또는 반응 용액의 금나노 입자와 라만 다이(dye)의 자기 결합보다는 스플리세오솜 활성 기반의 전령 RNA 전구체 스플라이싱 분석에서 라만 신호가 증가하는 것을 입증하였다.SERS intensity is proportional to the number of Raman dies of the P1 probe and P2-Cy3 probe reaching the surface of the gold nanoparticles, thus reflecting the relative activity of the spliceosome and the efficiency of messenger RNA precursor splicing Indicates. Normalized surface enhanced Raman scattering (SERS) intensity change is a direct indicator of messenger RNA precursor splicing efficiency (FIG. 4B). As shown in FIG. 4, the intensity of all peaks showed a normal curve for 3 minutes before the injection of HeLa extract containing the spliceosome complex. Plasmon binding and surface enhancement for RNA splicing The sensitivity of Raman scattering (SERS) based assays is measured by plasmon shift and SERS intensity change of messenger RNA precursor samples ranging from 0.1 μg / μl to 5 μg / μl concentration. However, a significant increase in each Raman peak is observed in the initial 4-5 minutes, indicating splicing of messenger RNA precursors in the reaction chamber upon activation of the spliceosome. Raman signaling increases at a concentration of 0.1 μg / μl of spliteosome to reach plateau at the 10 minute end point. Plasmon shift and normalized surface enhanced Raman scattering (SERS) intensity changes are shown in FIG. 4C. As expected, the rate of increase of the Raman signal is proportional to the concentration of the messenger RNA precursor because the higher the concentration of the messenger RNA precursor increases the splicing rate of the messenger RNA precursor. The detection range was set from 0.1 μg / μl to 5 μg / μl, and messenger RNA precursor splicing lower than 0.1 μg / μl did not change within a given detection time, and 10 minutes recording of the negative control E. coli extract was also performed. No change in intensity was observed. This result demonstrated an increase in Raman signals in messenger RNA precursor splicing assays based on spliceosome activity rather than self-assembly of RNA formation or magnetic binding of Raman die with gold nanoparticles in the reaction solution.

4-5: RNA 스플라이싱의 억제4-5: Inhibition of RNA Splicing

소분자는 스플리세오솜 조립의 동력학, 전령 RNA 전구체의 변화 속도를 억제한다. 따라서, 인비보 및 인비트로에서 스플리세오솜의 일반적인 mRNA 스플라이싱 억제제(33)인, 이소깅제틴(isoginkgetin, 5458-19-6, 칼 바이오 켐)으로 전령 RNA 전구체 스플라이싱에 대한 억제를 추가로 연구하였다. 33μM 이소깅제틴의 RNA 스플라이싱 억제는 37℃에서 0~10분 동안 플라즈몬 결합 및 SERS 신호의 산란 강도를 이용하여 측정하였다. 단일 분자 수준에서 높은 시간 해상도의 전령 RNA 전구체 스플라이싱의 접근은 프로브 및 이소깅제틴을 통한 RNA 스플라이싱의 속도를 정량하여 나타냈다. 이러한 RNA 스플라이싱 결과는 산란 강도를 측정하는 것이 합리적이다. 두가지 대표적인 산란 현상인 산란 강도 및 라만 산란은 도 5a 및 도 5b에 나타냈다. 매우 근접한 두 개의 플라즈몬 프로브로 RNA 스플라이싱의 변화를 반영할 수 있는 라만 산란 및 산란 강도는 산란 궤적 강도 변화를 포함한다. 산란 강도(도 5a) 및 라만 산란(도 5b) 억제제의 존재와 부재의 RNA 스플라이싱을 비교하여 플라즈몬 결합의 평균 강도의 증가를 나타냈다. 따라서, RNA 스플라이싱 활성에 대한 산란 강도는, 스플라이싱 시간의 작용에서 억제제의 친화력(도 5c) 및 스플리오세솜 복합체에 대한 억제제 활성으로 설명된다. 억제 진행 곡선(레드 곡선, 도 5c)으로부터 반응 시간의 작용에 대한 억제(%)는 억제제의 존재 유무(도 5d)에 따른 모든 시간의 mRNA 산물의 축적을 계속 측정하여 결정하였다. 이는, 이소깅제틴에 의한 스플리세오솜의 자기 조립의 불안정화를 나타낸다. 따라서, t1/2의 스플라이싱에서 관찰된 성장 억제제 농도의 증가는 엑손 - 인트론 경계 부위에 스플리세오솜의 낮은 결합력에 의해 설명된다.Small molecules inhibit the kinetics of spliceosome assembly, the rate of change of messenger RNA precursors. Thus, inhibition against messenger RNA precursor splicing with isoginkgetin (5458-19-6, Cal Biochem), a common mRNA splicing inhibitor 33 of spliceosome in vivo and in vitro. Was further studied. RNA splicing inhibition of 33 μM isogingzetin was measured using plasmon binding and scattering intensity of the SERS signal at 37 ° C. for 0-10 minutes. The approach of high time resolution messenger RNA precursor splicing at the single molecule level has been shown by quantifying the rate of RNA splicing through probes and isogingzetin. These RNA splicing results are reasonable to measure scattering intensity. Two representative scattering phenomena, scattering intensity and Raman scattering, are shown in FIGS. 5A and 5B. Raman scattering and scattering intensity, which can reflect changes in RNA splicing with two very close plasmon probes, include scattering trajectory intensity changes. Comparison of scattering intensity (FIG. 5A) and Raman scattering (FIG. 5B) inhibitors with and without RNA splicing showed an increase in the average intensity of plasmon binding. Thus, scattering intensity for RNA splicing activity is explained by the affinity of the inhibitor in the action of splicing time (FIG. 5C) and the inhibitory activity against the spliocesome complex. The inhibition (%) on the action of reaction time from the inhibition progress curve (red curve, FIG. 5C) was determined by continually measuring the accumulation of mRNA product at all times with or without inhibitor (FIG. 5D). This indicates instability of the self-assembly of the spliceosome by isogingzetin. Thus, the increase in growth inhibitor concentration observed in t1 / 2 splicing is explained by the low binding force of spliceosome to the exon-intron boundary region.

실시예 5: 데이터 분석Example 5: Data Analysis

산란 강도, 플라즈몬 결합 및 라만 산란 기반 RNA 스플라이싱 모니터링을 위해, 소프트웨어 Solis T를 이용하여 Andor CCD 검출기로 데이터를 기록하였다. 모든 결합 쌍은 플라즈몬 이동 및 라만 이동을 나타내는 완만 또는 급진적으로 강도가 증가하는 것으로 나타낸다. 스플라이세오솜(spliceosome) 복합체 주입 후 모든 기록 과정은 0초로 세팅한 강도의 점진적인 하락을 포함한다. 단지 결합 쌍은 RNA 스플라이싱 후 이량체의 플라즈몬 이동과 플라즈몬 강도의 결합을 나타내는 분석을 포함한다. 레일리 산란과 라만 산란의 산란 및 스펙트럼 데이터는 OriginPro 프로그램(version 8.6)으로 그래프 하였다. 이러한 과정은 스플라이세오솜(spliceosome) 복합체의 첨가와 관찰 강도 증가 사이의 시간차에 의해 정의되는 스플라이싱 시간 결정을 OriginPro 프로그램으로 추가 분석하였다For scattering intensity, plasmon binding and Raman scattering based RNA splicing monitoring, data was recorded with the Andor CCD detector using software Solis T. All binding pairs are shown to increase slowly or radically, indicating plasmon shift and Raman shift. All recordings following the spliceosome complex injection included a gradual drop in intensity set to zero seconds. Only binding pairs include assays that show the binding of dimer's plasmon migration and plasmon intensity after RNA splicing. Scattering and spectral data of Rayleigh scattering and Raman scattering are plotted with the OriginPro program (version 8.6). This process was further analyzed by the OriginPro program for splicing time determinations defined by the time difference between the addition of spliceosome complexes and the increase in observed intensity.

본 발명에 따른 산란 강도, 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 기반으로 광학 현미경을 통해 단일 입자에서의 RNA 스플라이싱을 검출 및 모니터링하는 방법은 높은 감도, 시간 단축 및 저비용으로 인해, RNA 스플라이싱, 스플라이싱 억제제의 스크리닝 및 RNA 나노기술과 같은 스플라이싱을 심도있게 연구하는 플랫폼으로 유용하다.Based on scattering intensity, localized surface plasmon resonance and surface enhanced Raman scattering according to the present invention, a method for detecting and monitoring RNA splicing in a single particle via optical microscopy has been found to provide high sensitivity, time savings and low cost. It is useful as a platform for in-depth study of splicing such as splicing, screening of splicing inhibitors and RNA nanotechnology.

이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. 본 발명의 단순한 변형 내지 변경은 이 분야의 통상의 지식을 가진 자에 의하여 용이하게 이용될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

전자파일 첨부하였음.Electronic file attached.

Claims (24)

다음 단계를 포함하는 RNA 스플라이싱의 검출 및 모니터링 방법:Methods for detecting and monitoring RNA splicing comprising the following steps: (a) 표적 유전자를 발현 벡터에 클로닝하고 세포, 조직 또는 인간을 제외한 생체 내로 벡터를 형질전환시켜, 재조합 표적 전령 RNA 전구체의 전사체를 생성하는 단계;(a) cloning the target gene into the expression vector and transforming the vector in vivo except cells, tissues or humans to generate transcripts of the recombinant target messenger RNA precursor; (b) 플라즈몬 프로브 또는 표면 증강 라만 산란(SERS)-태그 프로브를 상기 재조합 표적 전령 RNA 전구체의 전사체와 반응시키거나, 직접 세포, 조직 또는 인간을 제외한 생체 내로 도입시켜, 상기 플라즈몬 프로브 또는 표면 증강 라만 산란(SERS)-태그 프로브를 전령 RNA 전구체의 엑손/인트론 및 인트론/엑손의 경계에 부착시키는 단계; 및(b) a plasmon probe or surface enhanced Raman scattering (SERS) -tag probe is reacted with a transcript of said recombinant target messenger RNA precursor or introduced directly into vivo, excluding cells, tissues or humans to enhance said plasmon probe or surface Attaching a Raman scattering (SERS) -tag probe to the boundaries of exons / introns and introns / exons of the messenger RNA precursor; And (c) 광학 현미경을 통해 산란 강도, 레일리 산란의 국소 표면 플라즈몬 공명 및 표면 증강 라만 산란을 측정하여, RNA 스플라이싱을 분석하여 검출하는 단계.(c) analyzing and detecting RNA splicing by measuring scattering intensity, local surface plasmon resonance of Rayleigh scattering and surface enhanced Raman scattering through an optical microscope. 제1항에 있어서, 상기 (b) 단계는 인-비트로(in vitro) 또는 인-비보(in vivo)에서 수행하는 것을 특징으로 하는 방법.The method of claim 1, wherein step (b) is performed in-vitro or in vivo. 제1항에 있어서, 상기 RNA 스플라이싱 분석은 나노입자 결합 패턴을 포함하는 레일리 산란의 국소 표면 플라즈몬 공명 방법, 적어도 하나 이상의 나노입자와의 일차원 결합 패턴을 포함하는 산란 강도의 방법 및 전자기 표면 증강 패턴을 형성하는 표면 증강 라만 산란(SERS)-태그 프로브와 나노입자 결합의 표면 증강 라만 산란(SERS) 방법으로 구성된 군에서 선택되는 하나로 수행하는 것을 특징으로 하는 방법.The method of claim 1, wherein the RNA splicing assay comprises a method of local surface plasmon resonance of Rayleigh scattering comprising a nanoparticle binding pattern, a method of scattering intensity comprising a one-dimensional binding pattern with at least one nanoparticle and an electromagnetic surface enhancement And a surface enhanced Raman scattering (SERS) -tag probe forming a pattern and a surface enhanced Raman scattering (SERS) method of nanoparticle binding. 제3항에 있어서, 상기 레일리 산란의 국소 표면 플라즈몬 공명, 산란 강도 및 표면 증강 라만 산란(SERS)은 광학 현미경 챔버의 고체상 또는 용액상에서 발생하는 것을 특징으로 하는 방법.The method of claim 3, wherein the local surface plasmon resonance, scattering intensity and surface enhanced Raman scattering (SERS) of Rayleigh scattering occur in the solid or solution phase of an optical microscope chamber. 제3항에 있어서, 상기 결합 패턴은 전령 RNA 전구체 분자의 인트론 수에 따른 나노입자를 포함하는 프로브의 결합 패턴인 것을 특징으로 하는 방법.The method of claim 3, wherein the binding pattern is a binding pattern of a probe including nanoparticles according to the number of introns of a messenger RNA precursor molecule. 제1항에 있어서, 상기 표면 증강 라만 산란(SERS)-태그 프로브는 라만 다이(Raman dye)가 태그된 것을 특징으로 하는 방법.The method of claim 1, wherein the surface enhanced Raman scattering (SERS) -tagged probe is tagged with a Raman dye. 제6항에 있어서, 상기 라만 다이(Raman dye)는 시아닌-3(Cyanine-3), 시아닌-5(Cyanine-5), 시아닌-5.5(Cyanine-5.5), 시아닌 7(Cyanine 7), 4-아미노티오페놀(4-aminothiophenol), 4-메틸벤젠티올(4-methylbenzenethiol), 2-나프탈렌티올(2-naphthalenethiol), 로다민-5-(and-6)-이소티오시아네이트(rhodamine-5-(and-6)-isothiocyanate), 테트라메틸로다민-5-이소티오시아네이트(tetramethylrhodamine-5-isothiocyanate), 로다민 B 로다민 6G 닐 블루(rhodamine B Rhodamine 6G nile blue), FAM 및 TAMRA으로 구성된 군에서 선택되는 것을 특징으로 하는 방법.The method according to claim 6, wherein the Raman dye is Cyanine-3 (Cyanine-3), Cyanine-5 (Cyanine-5), Cyanine-5.5 (Cyanine-5.5), Cyanine 7 (Cyanine 7), 4- 4-aminothiophenol, 4-methylbenzenethiol, 2-naphthalenethiol, rhodamine-5- (and-6) -isothiocyanate (rhodamine-5- (and-6) -isothiocyanate, tetramethylrhodamine-5-isothiocyanate, rhodamine B rhodamine 6G nile blue, FAM and TAMRA Characterized in that it is selected from the group. 제1항에 있어서, 상기 플라즈몬 프로브는 RNA 분자에 병렬로 결합하는 것을 특징으로 하는 방법.The method of claim 1, wherein the plasmon probe binds in parallel to the RNA molecule. 제8항에 있어서, 상기 프로브는 표적 유전자 전령 RNA 전구체 서열과 50~100%의 상동성을 갖는 것을 특징으로 하는 방법.The method of claim 8, wherein the probe has 50-100% homology with a target gene messenger RNA precursor sequence. 제8항에 있어서, 상기 프로브는 전령 RNA 전구체 분자의 수에 따라 2~100개 이상의 나노입자를 포함하는 것을 특징으로 하는 방법.The method of claim 8, wherein the probe comprises from 2 to 100 or more nanoparticles, depending on the number of messenger RNA precursor molecules. 제10항에 있어서, 상기 나노입자는 금, 은, 백금 및 구리로 구성된 군에서 선택되는 어느 하나인 것을 특징으로 하는 방법.The method of claim 10, wherein the nanoparticles are any one selected from the group consisting of gold, silver, platinum and copper. 제8항에 있어서, 상기 프로브는 RNA 스플라이싱 후, 1~80nm의 공간 분리를 갖는 것을 특징으로 하는 방법.The method of claim 8, wherein the probe has a spatial separation of 1 to 80 nm after RNA splicing. 제1항에 있어서, 상기 전령 RNA 전구체는 전령 RNA 전구체, 마이크로 RNA 전구체, hnRNA, rRNA, tRNA 및 viral RNA로 구성된 군에서 선택되는 것을 특징으로 하는 방법.The method of claim 1, wherein the messenger RNA precursor is selected from the group consisting of messenger RNA precursor, micro RNA precursor, hnRNA, rRNA, tRNA and viral RNA. 제1항에 있어서, 상기 RNA 스플라이싱은 엑손 옆에 적어도 하나 이상의 인트론을 함유하고 있는 전령 RNA 전구체 분자 또는 프리-마이크로 RNA에서 인트론이 제거되는 것을 특징으로 하는 방법.The method of claim 1, wherein said RNA splicing removes introns from pre-micro RNA or messenger RNA precursor molecules that contain at least one intron next to an exon. 제14항에 있어서, 상기 인트론의 제거는 스플라이세오솜(spliceosom)의 절단 메커니즘에 의해 수행되는 것을 특징으로 하는 방법.The method of claim 14, wherein the removal of the intron is performed by a cleavage mechanism of spliceosom. 제1항에 있어서, 상기 RNA 스플라이싱은 나노입자를 이용한 전령 RNA 전구체의 동력학 모니터링에 의해 수행되는 것을 특징으로 하는 방법.The method of claim 1, wherein said RNA splicing is performed by kinetic monitoring of messenger RNA precursors using nanoparticles. 제16항에 있어서, 상기 나노입자는 금, 은, 백금 및 구리로 구성된 군에서 선택되는 어느 하나인 것을 특징으로 하는 방법.The method of claim 16, wherein the nanoparticles are any one selected from the group consisting of gold, silver, platinum and copper. 제17항에 있어서, 상기 나노입자는 안정제를 추가로 함유하는 것을 특징으로 하는 방법.18. The method of claim 17, wherein the nanoparticles further contain a stabilizer. 제18항에 있어서, 상기 나노입자와 안정제는 티올기가 직접 결합하는 것을 특징으로 하는 방법.The method of claim 18, wherein the nanoparticles and the stabilizer is characterized in that the thiol group directly bonds. 제18항에 있어서, 상기 안정제는 티올폴리에틸렌글리콜(PEG), 폴리에틸렌글리콜(PEG) 유도체, 티올올리고에틸렌글리콜, 세틸트리메틸암모늄브로마이드 및 폴리스티렌술폰산으로 구성된 군에서 선택되는 것을 특징으로 하는 방법.19. The method of claim 18, wherein the stabilizer is selected from the group consisting of thiol polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, thiol oligoethylene glycol, cetyltrimethylammonium bromide and polystyrenesulfonic acid. 제1항에 있어서, 상기 세포는 원핵세포, 진핵세포, 정상세포 및 암세포로 구성된 군에서 선택되는 것을 특징으로 하는 방법.The method of claim 1, wherein the cell is selected from the group consisting of prokaryotic cells, eukaryotic cells, normal cells and cancer cells. 제1항에 있어서, 상기 광학 현미경은 암시야 콘덴서; 스펙트럼 그래프용 분광기; 신호처리용 CCD 카메라; 레일리 산란용 할로겐 또는 제논의 백색 광원; 표면 증강 라만 산란(SERS) 신호 검출용 레이저 소스; 및 RNA 스플라이싱 분석용 반응 챔버를 포함하는 것을 특징으로 하는 방법.The optical microscope of claim 1, further comprising: a dark field condenser; Spectrograph for spectral graph; CCD camera for signal processing; White light sources of halogen or xenon for Rayleigh scattering; Laser sources for surface enhanced Raman scattering (SERS) signal detection; And a reaction chamber for RNA splicing analysis. 제22항에 있어서, 상기 챔버는 전령 RNA 전구체 포획을 위한 비오틴-DNA 프로브가 수직 방향으로 고정된 스트렙타비딘 분자가 코팅되어 있는 것을 특징으로 하는 방법.23. The method of claim 22, wherein the chamber is coated with streptavidin molecules in which a biotin-DNA probe for messenger RNA precursor capture is immobilized in a vertical direction. 제22항에 있어서, 상기 챔버는 표면 증강 라만 산란(SERS) 신호 검출용 플라즈몬 나노입자-프로브 혼합물을 함유하는 것을 특징으로 하는 방법.23. The method of claim 22, wherein said chamber contains plasmon nanoparticle-probe mixture for surface enhanced Raman scattering (SERS) signal detection.
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