CN109609505A - A kind of hammerhead ribozyme of the shearing RNA screened in vivo - Google Patents
A kind of hammerhead ribozyme of the shearing RNA screened in vivo Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
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Abstract
The invention belongs to technical field of molecular biology, and in particular to a kind of new method of the ribozyme of screening in vivo, and the ribozyme with catalysis RNA shear active screened by this method.The internal screening technique is using toxalbumin ibsC as reporter gene, and the expression of the shear action control toxic protein of the ribozyme obtained by vivo transcription mRNA corresponding on ibsC is to influence cell survival, to screen acquisition active ribozyme.This method utmostly reduces influence of the inactive RNA sequence to screening success rate without complicated in-vitro screening step, provides Screening Platform in simple, efficient Universal Cell for ribozyme evolution.The mode of action for the core enzyme-to-substrate that this method screening obtains is mainly intermolecular shear mode, shears suitable for catalysis extracellular, in prokaryotic cell, in eukaryocyte for the locus specificity of target substrates molecule, realizes that the expression to selected genes is lowered.
Description
Technical field
The present invention relates to a kind of RNA molecules with catalysis RNA shear active that screening in vivo obtains.
Background technique
Ribozyme is the single-chain nucleic acid macromolecular (Ribozyme) with catalysis.According to the size of its molecular weight, can divide
For two class of macromolecular ribozyme and small molecule ribozyme.Small ribozyme include hammerhead ribozyme (Hammerhead Ribozymes, HHRz),
Varkud satellite ribozyme, hepatitis delta virus ribozyme, hairpin ribozymes, winding-type ribozyme, gun type ribozyme, short axe type ribozyme
Deng, size between 30 to 150 nucleotide, oneself is catalyzed in specific position, intramolecular cleavage reaction occurs.Wherein, hammerhead shape core
The ribozyme of cleavage reaction can become catalysis point by transformation in the catalytic moleculars such as enzyme, hairpin ribozymes and hepatitis delta virus ribozyme
The ribozyme reacted between son.There is the characteristic of specific cleavage RNA using these ribozymes, ribozyme may be controlled in people's RNA virus disease
It treats and plays a significant role in preventing, in addition, the RNA of oncogene can also be used as the target of ribozyme.For the research of HIV-1
Show that a variety of HIV-1 diseased plants can be effectively suppressed in the Fairpin ribozyme that the RNA of HIV-1 is targeted in cell experiment and hammerhead ribozyme
Duplication, and be also that currently the only report enters clinical second phase experiment using the RNA of HIV-1 as the hammerhead ribozyme of target
Ribozyme.
But the application of ribozyme in vivo faces huge challenge.The ribozyme naturally found is all shearing in catalytic molecular
, the specific higher structure that the ability sheared in catalytic molecular depends on complete ribozyme molecule itself to be formed.But it is converted into
When the ribozyme sheared between catalytic molecular, the partial sequence of ribozyme can only be often kept, so that the catalytic efficiency of ribozyme declines.
For example the efficient shearing of hammerhead ribozyme (SEQ ID NO 5) in the cell relies on the interaction between prominent ring 1 and prominent ring 2
(Figure 1A), the catalytic structure that such interaction can stablize hammerhead ribozyme make hammerhead ribozyme in physiological conditions
Self cleavage can be efficiently catalyzed.But the interaction between prominent ring 1 and prominent ring 2 is in the hammerhead ribozyme of intermolecular shearing
Being impossible existing --- and it is found on target gene and meets Ribozyme cleavage sites and on the position of 6 nucleotide downstream
There are RNA possibility very little or the difficulty of specific composition sequence too big.Therefore it needs to screen not depending on this interaction completely
The hammerhead ribozyme that can be degraded by the mode efficient catalytic substrate RNA of intermolecular shearing, the core that adaptation intramolecular is sheared
Enzyme is evolved to adapt to the ribozyme of intermolecular shearing, could push the application of ribozyme in vivo as tool in this way.
In order to promote the application of HHRz, the catalysis that existing many researchs change ribozyme by way of in-vitro screening is special
Property.In-vitro screening can be from 1013-1015The order of magnitude in hangar, select the RNA with given activity points through excessive repeating query ring grizzly
Son optimizes the catalysis characteristics of HHRz with this.But in-vitro screening HHRz is mostly in high Mg2+It is carried out under concentration (10mM or higher)
, much higher than intracellular Mg2+Concentration (is less than 1mM), and in-vitro screening is difficult to simulate compound and the life of intracellular complexity
Object compartmentation.The ribozyme of in-vitro screening for shearing in vivo when may effect and bad, this also explains why most benefits
Wild type HHRz design is all based on the work of HHRz shearing cell target gene.But the sequence and knot of wild type HHRz
Structure is adapted with intramolecular shearing, this may be to cause the ribozyme tool designed based on wild type HHRz in diseases such as HIV-1
Effect and inapparent one of the major reasons in disease treatment.Therefore, it is necessary to obtain efficiently to urge by way of intracellular screening
Change the HHRz sheared between RNA molecule, ribozyme could be promoted in the development of related fields.
Summary of the invention
The technical solution for the internal screening ribozyme that the present invention establishes is (Fig. 1 C): using cell as screening vector, IbsC toxicity
Protein gene implements the signal reports screened in vivo, controls toxic protein by shear action of the ribozyme to toxic protein mRNA
Expression, and then cell survival is influenced, to judge whether that screening obtains active ribozyme.Specifically: building contains IbsC first
The plasmid library of toxic protein gene and ribozyme DNA library.Ribozyme is originated by different promoters from toxalbumin and is expressed.Ribozyme
Transcriptional expression is controlled by itself promoter and terminator, and ribozyme DNA library is building up to the anticodon loop sequence of tRNA gene
In to guarantee to transcribe the stability of resulting ribozyme.In order to realize the signal reports function of IbsC, the substrate-binding region of ribozyme
It is complementary with the resulting part mRNA of IbsC toxic protein genetic transcription, to form core enzyme-to-substrate (IbsC toxic protein gene) point
The substrate-binding region sequence of the model sheared between son, i.e., corresponding ribozyme DNA library should be with IbsC toxic protein gene order
Meet corresponding requirements.The expression of toxoprotein gene is by the promoter of itself, ribosome bind site, lactose operon sequence and end
Only son control；It is mentioned to slow down its expression speed in one additional protein gene of N-terminal amalgamation and expression of toxoprotein gene with this
The relative scale of high core enzyme-to-substrate is to enhance the shearing of ribozyme in vivo.Since ribozyme and reporter gene IbsC are expressed respectively,
The expression that ribozyme passes through intermolecular shearing property toxoprotein gene.Ribozyme HHRz with shear active can shear toxalbumin
MRNA causes its expression to be lowered, and bacterium can form monoclonal in screening flat board.But inactive ribozyme HHRzm cannot shear poison
Protein mRNA, toxoprotein gene overexpress so that bacterial death and cannot form clone (Fig. 1 C) in screening flat board, thus general
Nonactive sequence screening is rejected, and active ribozyme sequence is only filtered out.In this way, shear active only between catalytic molecular
Ribozyme just can be screened out, inactive ribozyme is directly removed from screening library, is a kind of positive sieve that can remove background
Select mode.
The structure of the ribozyme obtained by above-mentioned internal screening technique scheme is as shown in Figure 1B: by the first Binding Capacity area
Domain, big catalytic active center, loop-stem structure (stem 2 and prominent ring 2), small catalytic active center, the second substrate-binding region composition.Greatly
Catalytic active center, stem 2 and prominent ring 2, small catalytic active center constitute the catalytic core region of ribozyme.Big catalytic active center contains
Have 7 base CUGAUGA (SEQ ID NO 19), small catalytic active center contains 4 bases G AAA (SEQ ID NO 20), stem
2 and prominent ring 2 connect two catalytic active centers.Stem 2 forms stem structure by way of base pair complementarity, there is 3~8 bases
Pairing；The sequence length of prominent ring 2 is 3~8 bases；In a preferred approach, 2 sequence of stem 2 and prominent ring (calling " stem ring sequence " in the following text)
It can be SEQ ID NO21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID
NO 26, SEQ ID NO 27, SEQ ID NO 28, sequence described in SEQ ID NO 29.Two substrate-binding regions are respectively and in advance
First selected RNA substrate is combined by way of base pair complementarity, and substrate-binding region sequence can be according to different substrates
Sequence and occur to change accordingly；The length of substrate-binding region can change, only need to be with previously selected RNA substrate sequence knot
Conjunction forms stable duplex structure.
The shearing site of above-mentioned hammerhead ribozyme specific recognition is located at the first substrate-binding region and the second bottom of substrate
Between object bond area, sequence rules are " NUX ", and N indicates randomized bases (N=A or T or C or G), and X is indicated other than G
Other three bases (N=A or T or C), and specific cleavage occurs after X." NU " interacts with hammerhead ribozyme sequence to match
Exist to the form of base, " X " exists in the form of unpaired bases.
In order to generate the ribozyme of the higher shearing RNA of shear efficiency, a variety of substitutions can be used in stem 2 and prominent ring 2, lacked
Lose, insertion, duplication and other mutation method prepare the molecule for belonging to the open scope, as long as the molecule displays have gone out herein
The shear active of the locus specificity of description, then they just belong within the scope of this specification.
The characteristics of present invention screening gained ribozyme, is that cut mode is mainly intermolecular shearing, can also pass through building point
Shear mode in son plays a role.
In an embodiment, same ribozyme can shear the different substrate SEQ ID of sequence in prokaryotes
NO 10、SEQ ID NO 11。
In an embodiment, same ribozyme can shear the different substrate SEQ ID of sequence in eucaryote
NO 10、SEQ ID NO 12、SEQ ID NO 13。
Term definition: following term should be mentioned in text.
Ribozyme: ribozyme is a kind of single stranded RNA segment with catalysis, has efficient catalytic activity and structure recognition
Ability.Other vocabulary that can be used interchangeably herein with ribozyme are " catalytic activity RNA molecules ", " Ribozyme ", no matter these
Molecule is by being synthetically prepared or from organism or other sources, they are all construed as including its enzymatic activity
Part.
Base pair complementarity: refer to the single chain molecule of DNA or RNA or one section of nucleotide sequence and another few core in segment
The single-stranded sequence of thuja acid is substantially complementary to which by hydrogen bond, specifically heterozygosis is together.
Intramolecular shearing: ribozyme molecule catalysis substrate molecule is sheared, and ribozyme and substrate are on the same RNA.
Intermolecular shearing: ribozyme molecule catalysis substrate molecule is sheared, and ribozyme and substrate be not on the same RNA.
In-vitro screening: it in the reaction solution manually prepared, is filtered out from random oligonucleotide library and target molecule phase
The combinatorial chemistry technique of interaction (catalysis, in conjunction with etc.).
Into the cell/screening in vivo: it in protokaryon or eukaryocyte, is filtered out from random oligonucleotide library and target point
The technology of son interaction (catalysis, in conjunction with etc.).
Screening random dna library/screening random sequence library: being random with certain length by what is be chemically synthesized
DNA sequence dna (usually has 40 random sequences), the fixed sequence program that random sequence both ends have for PCR amplification.
Restriction enzyme: restriction enzyme is that one kind can identify specific deoxynucleotide sequence, and at every
The protease that phosphodiester bond in chain between two nucleotide of privileged site is cut.
Overexpression: overexpression refer to target gene under the starting of strong promoter, great expression in the cell.
Amalgamation and expression: amalgamation and expression refers to that the end 5' 3' by foreign protein genes and another gene is built into fusion
It is expressed, clone gene is made to be expressed as a part of fusion protein.
Detailed description of the invention
Fig. 1 is the internal screening plan for the ribozyme sheared between catalytic molecular of the specific embodiment 1 based on toxalbumin reporter gene
Summary and the selection result.A. the general structure for the ribozyme sheared in catalytic molecular；B. the structure for the ribozyme sheared between catalytic molecular is logical
Formula；C. the internal screening strategy for the ribozyme sheared between catalytic molecular: the artificial synthesized library containing random dna sequence passes through limit
Property restriction endonuclease processed is building up on carrier, passes through the expression of RNA shearing property toxalbumin between catalytic molecular；D. screening random dna text
Library design: there is the random sequence of 7 bases and 6 bases respectively on big catalytic active center and prominent ring；E. the TX-2 screened
(SEQ ID NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9) are in pH7.4, magnesium ion 0.5mM, 37 DEG C of items
The shear ability of substrate is significantly increased compared with wild type ribozyme (SEQ ID NO 6) under part；What the screening F. predicted obtained
The secondary structure of the hammerhead ribozyme of intermolecular interaction.
Fig. 2 is the table that the ribozyme that the screening of specific embodiment 2 obtains lowers red fluorescent protein and Spinach in Escherichia coli
It reaches.A. double fluorescence shear reporter gene schematic diagram in Escherichia coli.B. the ribozyme of acquisition is screened to red in Escherichia coli
The downward ability of fluorescin (SEQ ID NO 10) expression.C. ribozyme is under RNA molecule Spinach (SEQ ID NO 11)
Tune ability.
Fig. 3 is that the ribozyme that the screening of specific embodiment 3 obtains lowers red fluorescent protein in the table of human carcinoma cell line (Hela)
It reaches.
Fig. 4 is that the ribozyme that the screening of specific embodiment 4 obtains strikes drop to zebra fish gene expression.A. the ribozyme of acquisition is screened
Red fluorescent protein is lowered to express in zebra fish.B. the ribozyme for screening acquisition lowers zebra fish nacre (SEQ ID NO 12)
The expression of (control pigmentation) gene.C. the ribozyme for screening acquisition lowers (the control tail portion zebra fish ntl (SEQ ID NO 13)
Development) gene expression.
Fig. 5 is the structure and active vitro characterization for the higher ribozyme of activity that the screening of specific embodiment 5 obtains.A.TX-2 is again
Screening is with hangar (SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33).B. the TX- screened
2m11 (SEQ ID NO 14), TX-2m12 (SEQ ID NO 15) and TX-2m13 (SEQ ID NO 16) are in pH7.4, magnesium ion
0.5mM, the shear ability of substrate is significantly increased compared with TX-2 under the conditions of 37 DEG C；The secondary structure of corresponding ribozyme prediction.
C.TX-9 is screened again with hangar (SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37).D. it sieves
The TX-9m5 (SEQ ID NO 17) and TX-9m11 (SEQ ID NO 18) of choosing are under the conditions of pH7.4, magnesium ion 0.5mM, 37 DEG C
The shear ability of substrate is significantly increased compared with TX-9；The secondary structure of corresponding ribozyme prediction.
Specific embodiment
Explain the present invention in detail with reference to embodiments.Embodiment is for the ease of better understanding the present invention, but simultaneously
Non- limitation of the present invention.
The internal screening for the ribozyme that embodiment 1. is sheared between the catalytic molecular based on toxalbumin reporter gene
Naturally occurring hammerhead ribozyme (Figure 1A) is the ribozyme sheared in catalytic molecular, highly conserved is urged there are two
Change activated centre (big catalytic active center, small catalytic active center), by 3 loop-stem structures (stem 1, prominent ring 1, stem 2, prominent ring 2,
Stem 3, prominent ring 3) it is connected, GUC is one of ribozyme cleavage recognition site, clipped position (place shown in Figure 1A scissors) after GUC.The core
The efficient shearing of enzyme in the cell relies on the interaction between prominent ring 1 and prominent ring 2.After riving at prominent ring 3, it can be formed point
The ribozyme sheared between son, but still need to retain the interaction between prominent ring 1 and prominent ring 2 --- the shear efficiency pole of ribozyme after deletion
It reduces greatly and is unfavorable for intermolecular shearing.And when being used between intracellular molecules shear by such ribozyme, to meet prominent ring 1 and dashing forward
The such interaction difficulty of ring 2 is too big.It would therefore be desirable to screen the interaction that do not dashed forward between ring 1 and prominent ring 2
And the hammerhead ribozyme of the energy intermolecular shearing of efficient catalytic.
Currently, the screening of ribozyme mostly uses the mode of in-vitro screening to carry out, but in-vitro screening HHRz is mostly in high Mg2+Concentration
It is carried out under (10mM or higher), much higher than intracellular Mg2+Concentration (is less than 1mM), and in-vitro screening is difficult to simulate cell
The compound of interior complexity and biological compartmentation, so that often activity reduces when the ribozyme of in-vitro screening for shearing in vivo
Or it loses.Therefore, the present invention establishes the screening that the technology screened in a set of new ribozyme body is used for hammerhead ribozyme, mainly
The design of construction and screening strategy including DNA library.
DNA library is constructed based on naturally occurring hammerhead ribozyme structure.It is expected that the bottom of the ribozyme screened
Object bond area and RNA substrate by base pair complementarity in conjunction with, structure similar to intramolecular shear in ribozyme molecule formed
Stem 1 and stem 3.The substrate-binding region of ribozyme can occur to change (Figure 1B) accordingly to be used for according to the sequence of different substrates
The shearing of different genes sequence --- no longer need to the base composition for retaining original stem 1 and stem 3.For reach screening catalytic molecular between
Shear the purpose of ribozyme, we based on this structure in the big catalytic active center of hammerhead ribozyme (SEQ ID NO 19) and
Prominent ring 2 (CAAAUG is shown in SEQ ID NO 21) introduces 7 bases respectively and the random sequence of 6 bases constructs corresponding DNA
Library --- the intracellular screening (Fig. 1 D) with hangar 1 and with hangar 2, for ribozyme.
In order to guarantee that the new hammerhead ribozyme that screening obtains can adapt to intracellular environment as far as possible, the present invention is using thin
Screening technique intracellular.The size in screening library is that screening is successfully crucial.Therefore, substantial amounts and transfection are needed when intracellular screening
Reactor of the high-efficient cell as ribozyme.Here, we select Escherichia coli are this to be widely used in culture and conversion
Cell of the cell as screening ribozyme, contained ribozyme is consistent in each bacterium, can be distinguished with other sequences and
Facilitate the screening of active ribozyme.Again since the cleavage reaction of ribozyme in the cell is sightless, it would be desirable to a report base
Reflect that the shear active of ribozyme is screened with convenience and high-efficiency because providing screening signal.IbsC gene (SEQ ID NO 1) coding one
Kind cytotoxin albumen (SEQ ID NO 2), overexpression can lead to cell death.The mutation of IbsC tolerable height is without shadow
Its toxicity is rung, and one section of albumen of its N-terminal amalgamation and expression nor affects on its activity, therefore IbsC is an ideal ribozyme screening
Reporter gene.
It is as shown in Figure 1 C with the intracellular screening strategy that IbsC toxic protein gene implements screening signal reports in vivo, pass through
Expression of the ribozyme to the shear action control toxic protein of toxic protein mRNA, and then cell survival is influenced, to judge whether
Screening obtains active ribozyme.Specifically: plasmid library of the building containing IbsC toxic protein gene and ribozyme DNA library first.
Due to being intermolecular shearing, ribozyme needs to be originated by different promoters from toxalbumin expresses (Fig. 1 C).The transcriptional expression of ribozyme
It is controlled by itself promoter (T7P) and terminator (T7T), since ribozyme only needs to transcribe, ribozyme does not have ribosomes collection
The translational controls element such as coincidence point (RBS) and initiation codon.Since Escherichia coli are prokaryotes, mRNA translation and degradation process
Be it is synchronous, to extend ribozyme half-life period in the cell, ribozyme DNA library is building up to the anticodon of tRNA gene by we
Guarantee the stability of the resulting ribozyme of transcription in ring sequence.In order to realize the signal reports function of IbsC, the substrate knot of ribozyme
Conjunction region needs can be complementary with the resulting part mRNA of IbsC toxic protein genetic transcription, to form core enzyme-to-substrate (IbsC poison
Property protein gene) intermolecular shearing model, i.e., the substrate-binding region sequence of corresponding ribozyme DNA library should be with IbsC toxicity
Protein gene sequence meets corresponding requirements.The expression of toxoprotein gene is by the promoter (T7P) of itself, ribosome bind site
(RBS), lactose operon sequence (lacO) and terminator (T7T) control；Toxalbumin only has 19 amino acid, to slow down its expression
Speed, in one green fluorescence protein gene (EGFP) (Fig. 1 C) of N-terminal amalgamation and expression of toxoprotein gene, so as to improve ribozyme
Relative scale with substrate is to enhance the shearing of ribozyme in vivo.Since ribozyme and reporter gene IbsC are expressed respectively, ribozyme is logical
Cross the expression of intermolecular shearing property toxoprotein gene.Ribozyme HHRz with shear active, which can shear toxalbumin mRNA, to be caused
It, which is expressed, lowers, and bacterium can form monoclonal in screening flat board.But inactive ribozyme HHRzm cannot shear toxalbumin
MRNA, toxoprotein gene overexpress so that bacterial death and clone (Fig. 1 C) cannot be formed in screening flat board, thus by non-live
Property sequence screening reject, only filter out active ribozyme sequence.
It is as follows with hangar sequence to meet the ribozyme DNA that IbsC toxic protein mRNA is substrate:
With hangar 1 (SEQ ID NO 3)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
NNNNNNNGTCCCAAATAGGACGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGC
GCCAGGATCC
With hangar 2 (SEQ ID NO 4)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCA
GCTGATGAGTCCNNNNNNGGACGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGG
GCGCCAGGATCCN indicates randomized bases (N=A or T or C or G).There is XbaI and HindIII restriction enzyme at sequence both ends
Site is for being inserted into carrier.
Due to longer with hangar sequence, two segments synthesis are generally divided into, the last period is sense strand sequence, and latter section is anti-
Adopted chain-ordering, the two is in the complementation of 3 '-end parts, and being mutually primer by polymerase chain reaction amplification is full-length gene, and PCR is anti-
Answer system and PCR condition as follows:
50 μ lPCR reaction systems:
PCR condition is: 95 DEG C of preheating 1min；95 DEG C of denaturation 30s, 51 DEG C of renaturation 30s, 72 DEG C of extension 40s, 18 recycle.
Ribozyme after 37 DEG C of digestion 3h of XbaI and HindIII restriction enzyme, leads to the full length sequence PCR product of hangar
T4 ligase is crossed to be connected on the carrier pET32 that identical endonuclease digestion is crossed.Carrier containing ribozyme fragments is transformed into competence
In cell (Bl21 (DE3)), after 37 DEG C are shaken bacterium 1h, 4000rpm is centrifuged 5 minutes, is collected all bacterium solutions and is applied to containing ammonia benzyl
In the screening flat board of penicillin and IPTG, it is placed in 37 DEG C of constant incubators and is incubated overnight.Active ribozyme (HHRz) can be with
It shears RNA and toxalbumin is prevented to express, form clone in screening flat board.Picking monoclonal simultaneously send sequencing to screen ribozyme to determine
Sequence, obtain preferred ribozyme TX-2 (SEQ ID NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9).
Use 5'-32P label substrate and 100pM the RNA (active ribozyme HHRz) with catalytic activity pH7.4, magnesium from
It is reacted 4 hours for 37 DEG C under conditions of sub- 0.5mM, cleaved products is separated by 10% PAGE glue, calculate the percentage (figure of cutting
2E).Under conditions of close to physiologic ionic concentration and pH, compared with wild type ribozyme (SEQ ID NO 6), the excellent of acquisition is screened
Select ribozyme TX-2 (SEQ ID NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9) that there is substrate better
Shear effect (Fig. 2 F).The original series of the prominent ring 2 of wild type ribozyme and stem 2 are GUCCCAAAUAGGAC (SEQ ID NO 21),
Screen obtain TX-2, TX-5 and TX-9 stem ring sequence be respectively GUCCGGUAGCGGAC (SEQ ID NO 22),
GUCCAGUGCUGGAC (SEQ ID NO 26) and GUCCGAGGACGGAC (SEQ ID NO 27) (Fig. 2 F).
The ribozyme that the screening of embodiment 2. obtains lowers the expression of red fluorescent protein and Spinach in Escherichia coli.
Obtained ribozyme tool is screened in vivo there are two substrate-binding region (the first substrate-binding region and the second Binding Capacity
Region), positioned at the two sides in catalytic core region, two substrate-binding regions are mutual with the sequence of previously selected RNA substrate respectively
It mends, core catalysis region then remains unchanged.The present embodiment selects red fluorescent protein RNA (SEQ IDNO 10) and Spinach's
One Duan Xulie (SEQ ID NO 11) is used as target RNA, investigates work energy of the preferred ribozyme of screening acquisition in prokaryotic cell
Power.Corresponding plasmid construction is as shown in Figure 2 A, and other conditions are referring to embodiment 1.
Red fluorescent protein mCherry is building up to the downstream green fluorescent protein EGFP, and ribozyme can pass through intermolecular shearing
The expression of red fluorescence is lowered in reaction, but the egfp expression of upstream is unaffected as internal reference (Fig. 2A).Screening obtains
Ribozyme TX-2 (SEQ ID NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9) compared with wild type ribozyme,
There is better shear efficiency (Fig. 2 B) to red fluorescent protein RNA.
Spinach is a kind of RNA molecule, it can issue under laser excitation green with small molecule compound formation compound
Color fluorescence then destroys its structural intergrity without green fluorescence after the RNA of ribozyme cleavage Spinach.Screen the ribozyme obtained
TX-2 (SEQ ID NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9) are right compared with wild type ribozyme
The RNA of Spinach has better shear efficiency (Fig. 2 C).
The ribozyme that the screening of embodiment 3. obtains lowers red fluorescent protein in the expression of human carcinoma cell line (Hela).
It screens the preferred ribozyme obtained and preferably shearing effect is shown to several genes in the Escherichia coli of prokaryotic cell
Rate.The present embodiment tests downward of the ribozyme to the red fluorescent protein (SEQ ID NO 10) expressed in the human cell line of eukaryon
Ability.The ribozyme that screening obtains is building up on carrier for expression of eukaryon pmCherry by restriction enzyme SalI and HindIII,
The carrier containing ribozyme of recombination is gone the extracting of endotoxin plasmid extraction kit to obtain plasmid.5 microgram recombinant vectors use2000 (liposomes 2000) transfection mentions the Hela cell for the previous day being laid on 6 orifice plates.After transfection 24 hours, pancreas
Enzymic digestion obtains single cell suspension, red fluorescence intensity (Moflo XDP, the excitation wavelength of flow cytometry analysis cell
561nm, launch wavelength 620nm).Compared with original ribozyme, the preferred ribozyme TX-2 (SEQ ID NO 7) of intracellular screening,
TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO9), which has the expression of red fluorescent protein, preferably lowers ability (figure
3)。
The ribozyme that the screening of embodiment 4. obtains strikes drop to zebra fish gene expression
1nl contains the plasmid microinjection zebra of screening ribozyme (targeting red fluorescent protein gene (SEQ ID NO 10))
Fish fertilized egg fluorescence microscope and records the red fluorescence expression of fertilized egg cell after 48h, the ribozyme TX-2 (SEQ of screening
ID NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9) can effectively reduce red fluorescent gene zebra fish by
Expression (Fig. 4 A) in smart ovum.
1nl contains the matter of screening ribozyme (targeting nacre gene --- control cytochromes are calm (SEQ ID NO 12))
Grain microinjection zebra fish fertilized egg fluorescence microscope and records pigmentation situation after 50h.Real-time fluorescence is fixed after for 24 hours
Measure the mRNA level in-site of PCR detection nacre gene.Ribozyme TX-2 (SEQ ID NO 7), the TX-5 (SEQ ID NO 8) of screening and
TX-9 (SEQ ID NO 9) can effectively reduce expression (Fig. 4 B) of the nacre gene in zebra fish.
The plasmid that 1nl contains screening ribozyme (targeting ntl gene --- control tail portion development (SEQ ID NO 13)) is micro-
Zebra fish fertilized egg is injected, fluorescence microscope and records pigmentation situation after 28h.Ribozyme TX-2 (the SEQ ID of screening
NO 7), TX-5 (SEQ ID NO 8) and TX-9 (SEQ ID NO 9) can effectively reduce expression of the ntl gene in zebra fish
(Fig. 4 C).
Embodiment 5. screens acquired active ribozyme again to obtain the higher ribozyme of activity.
In order to generate the ribozyme of the higher shearing RNA of shear efficiency, the effect for improving ribozyme can be continued by screening again
Rate.We in the position of prominent ring 2 according to 30% frequency of mutation introduce mutation, and (original base frequency of occurrences is 70%, other three
The frequency that a base occurs all is 10%) (Fig. 5 A and C), in the stem 2 for the preferred ribozyme TX-2 and TX-9 that first round screening obtains
Introduce completely random mutation (tri- bases of A, T, G, C occur possibility frequency be all be 25%) (Fig. 5 A and C), re-start core
Screening obtains the higher ribozyme mutant of multiple activity (specific method is referring to embodiment 1) in second wheel cells of enzyme, preferably
For TX-2m11 (SEQ ID NO 14, stem ring sequence are SEQ ID NO 23), TX-2m12 (SEQ ID NO 15, stem ring sequence
For SEQ ID NO 24), TX-2m13 (SEQ ID NO 16, stem ring sequence are SEQ ID NO 25), TX-9m5 (SEQ ID NO
17, stem ring sequence is SEQ ID NO 28) and TX-9m11 (SEQ ID NO 18, stem ring sequence are SEQ ID NO 29).
Meeting IbsC toxic protein mRNA is the as follows with hangar sequence based on the screening DNA again of ribozyme TX-2 of substrate:
With hangar 3 (SEQ ID NO 30)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
CTGATGAGTCCNNNNNNGGACGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGC
GCCAGGATCC
With hangar 4 (SEQ ID NO 31)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
CTGATGANNNNGGTAGCNNNNGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGGGC
GCCAGGATCC
With hangar 5 (SEQ ID NO 32)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
CTGATGANNNNNGGTAGCNNNNNGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGG
GCGCCAGGATCC
With hangar 6 (SEQ ID NO 33)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
CTGATGANNNNNNGGTAGCNNNNNNGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTC
GGGCGCCAGGATCC
Meeting IbsC toxic protein mRNA is the as follows with hangar sequence based on the screening DNA again of ribozyme TX-9 of substrate:
With hangar 7 (SEQ ID NO 34)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCA
GCTGATGAGTCCNNNNNNGGACGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGG
GCGCCAGGATCC is with hangar 8 (SEQ ID NO 35)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCA
GCTGATGANNNNGAGGACNNNNGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGG
GCGCCAGGATCC is with hangar 9 (SEQ ID NO 36)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
CTGATGANNNNNGAGGACNNNNNGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTCGG
GCGCCAGGATCC
With hangar 10 (SEQ ID NO 37)
TCTAGAGCCCGGATAGCTCAGTCGGTAGAGCAGCGGCCGTACTTCCACCAACGAATTCCATGAATCCAG
CTGATGANNNNNNGAGGACNNNNNNGAAACGCACCGAATCTAATACGGCCGCGGGTCCAGGGTTCAAGTCCCTGTTC
GGGCGCCAGGATCC
Use 5'-32Condition of the RNA with catalytic activity of substrate and 100pM that P is marked in pH7.4, magnesium ion 0.5mM
Lower 37 DEG C are reacted 2 hours, are separated cleaved products by 10% PAGE glue, are calculated the percentage of cutting.The preferred ribozyme of screening
TX-2m11, TX-2m12, TX-2m13, TX-9m5 and TX-9m11 are under conditions of close to physiologic ionic concentration and pH, to substrate
With better shear effect (Fig. 5).
The screening of the comprehensive analysis first round and the second wheel screen the sequence and structural information of resulting ribozyme, and the present invention newly screens
Ribozyme be located at the base of stem 2 and prominent ring 2 and delete, replacement and extend, may all significantly improve the shear active of ribozyme, stem 2
Sequence has 3~8 base pairings, and the sequence length for ring 2 of dashing forward is 3~8 bases；Compared to naturally occurring hammerhead ribozyme
The base of (Figure 1A), stem 2 and prominent ring 2 is deleted, replaces and is extended, and the shear active of ribozyme may be all significantly improved.
Sequence table
<110>Chengdu Inst. of Biology, Chinese Academy of Sciences
<120>a kind of hammerhead ribozyme of the shearing RNA screened in vivo
<130> 2018
<141> 2019-01-14
<160> 37
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213> escherichia coli
<400> 1
atgatgcgac ttgtcatcat actgattgta ctgttactca taagtttcag cgcttattaa 60
<210> 2
<211> 19
<212> PRT
<213> escherichia coli
<400> 2
Met Met Arg Leu Val Ile Ile Leu Ile Val Leu Leu Leu Ile Ser Phe
1 5 10 15
Ser Ala Tyr
<210> 3
<211> 156
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (70)..(76)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (70)..(70)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (71)..(71)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (72)..(72)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (73)..(73)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (74)..(74)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (75)..(75)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (76)..(76)
<223> n is a, c, g, t or u
<400> 3
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagn nnnnnngtcc caaataggac gaaacgcacc gaatctaata cggccgcggg 120
tccagggttc aagtccctgt tcgggcgcca ggatcc 156
<210> 4
<211> 156
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (81)..(86)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (82)..(82)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (83)..(83)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (84)..(84)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (85)..(85)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (86)..(86)
<223> n is a, c, g, t or u
<400> 4
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgagtcc nnnnnnggac gaaacgcacc gaatctaata cggccgcggg 120
tccagggttc aagtccctgt tcgggcgcca ggatcc 156
<210> 5
<211> 69
<212> RNA
<213> Artificial Sequence
<400> 5
gcagguacau ccagcugaug agucccaaau aggacgaaac gcgcuucggu gcguccugga 60
uuccacugc 69
<210> 6
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (35)..(45)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
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<223> n is a, c, g, t or u
<220>
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<223> n is a, c, g, t or u
<220>
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<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
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<223> n is a, c, g, t or u
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<223> n is a, c, g, t or u
<220>
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<223> n is a, c, g, t or u
<220>
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<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
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<223> n is a, c, g, t or u
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<223> n is a, c, g, t or u
<220>
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<220>
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<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, t or u
<400> 6
nnnnnnnnnc ugaugagucc caaauaggac gaaannnnnn nnnnn 45
<210> 7
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
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<222> (45)..(45)
<223> n is a, c, g, t or u
<400> 7
nnnnnnnnnc ugaugagucc gguagcggac gaaannnnnn nnnnn 45
<210> 8
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (35)..(45)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (35)..(35)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (38)..(38)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, t or u
<400> 8
nnnnnnnnnc ugaugagucc agugcuggac gaaannnnnn nnnnn 45
<210> 9
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (35)..(45)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (35)..(35)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (38)..(38)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, t or u
<400> 9
nnnnnnnnnc ugaugagucc gaggacggac gaaannnnnn nnnnn 45
<210> 10
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 10
ccccgacuac uugaagcugu c 21
<210> 11
<211> 20
<212> RNA
<213> Artificial Sequence
<400> 11
uuacucauaa guuucagcgc 20
<210> 12
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 12
gagaugcucg aguacaguca c 21
<210> 13
<211> 21
<212> RNA
<213> Artificial Sequence
<400> 13
ucggaaauau gucugccuca a 21
<210> 14
<211> 44
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (34)..(44)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (35)..(35)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (38)..(38)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<400> 14
nnnnnnnnnc ugaugagucc gguagggacg aaannnnnnn nnnn 44
<210> 15
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (35)..(45)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (35)..(35)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (38)..(38)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, t or u
<400> 15
nnnnnnnnnc ugaugagucc gguaggggac gaaannnnnn nnnnn 45
<210> 16
<211> 49
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (39)..(49)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (46)..(46)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (47)..(47)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (48)..(48)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (49)..(49)
<223> n is a, c, g, t or u
<400> 16
nnnnnnnnnc ugaugagucc aggguagccu ggacgaaann nnnnnnnnn 49
<210> 17
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (35)..(45)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (35)..(35)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (38)..(38)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (45)..(45)
<223> n is a, c, g, t or u
<400> 17
nnnnnnnnnc ugaugagucc gaggaaggac gaaannnnnn nnnnn 45
<210> 18
<211> 44
<212> RNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (1)..(9)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (34)..(44)
<223> n is a, c, g, or u
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (35)..(35)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (36)..(36)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (38)..(38)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (39)..(39)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (40)..(40)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (43)..(43)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (44)..(44)
<223> n is a, c, g, t or u
<400> 18
nnnnnnnnnc ugaugagucc gaggaggacg aaannnnnnn nnnn 44
<210> 19
<211> 7
<212> RNA
<213> Artificial Sequence
<400> 19
cugauga 7
<210> 20
<211> 4
<212> RNA
<213> Artificial Sequence
<400> 20
gaaa 4
<210> 21
<211> 14
<212> RNA
<213> Artificial Sequence
<400> 21
gucccaaaua ggac 14
<210> 22
<211> 14
<212> RNA
<213> Artificial Sequence
<400> 22
guccgguagc ggac 14
<210> 23
<211> 13
<212> RNA
<213> Artificial Sequence
<400> 23
guccgguagg gac 13
<210> 24
<211> 14
<212> RNA
<213> Artificial Sequence
<400> 24
guccgguagg ggac 14
<210> 25
<211> 18
<212> RNA
<213> Artificial Sequence
<400> 25
guccagggua gccuggac 18
<210> 26
<211> 14
<212> RNA
<213> Artificial Sequence
<400> 26
guccagugcu ggac 14
<210> 27
<211> 14
<212> RNA
<213> Artificial Sequence
<400> 27
guccgaggac ggac 14
<210> 28
<211> 14
<212> RNA
<213> Artificial Sequence
<400> 28
guccgaggaa ggac 14
<210> 29
<211> 13
<212> RNA
<213> Artificial Sequence
<400> 29
guccgaggag gac 13
<210> 30
<211> 156
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (81)..(86)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (82)..(82)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (83)..(83)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (84)..(84)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (85)..(85)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (86)..(86)
<223> n is a, c, g, t or u
<400> 30
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgagtcc nnnnnnggac gaaacgcacc gaatctaata cggccgcggg 120
tccagggttc aagtccctgt tcgggcgcca ggatcc 156
<210> 31
<211> 155
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (77)..(80)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (87)..(90)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (77)..(77)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (78)..(78)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (79)..(79)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (80)..(80)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (87)..(87)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (88)..(88)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (89)..(89)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (90)..(90)
<223> n is a, c, g, t or u
<400> 31
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgannnn ggtagcnnnn gaaacgcacc gaatctaata cggccgcggg 120
tccagggttc aagtccctgt tcgggcgcca ggatc 155
<210> 32
<211> 158
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (77)..(81)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (88)..(92)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (77)..(77)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (78)..(78)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (79)..(79)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (80)..(80)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (88)..(88)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (89)..(89)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (90)..(90)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (91)..(91)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (92)..(92)
<223> n is a, c, g, t or u
<400> 32
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgannnn nggtagcnnn nngaaacgca ccgaatctaa tacggccgcg 120
ggtccagggt tcaagtccct gttcgggcgc caggatcc 158
<210> 33
<211> 160
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (77)..(82)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (89)..(94)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (77)..(77)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (78)..(78)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (79)..(79)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (80)..(80)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (82)..(82)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (89)..(89)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (90)..(90)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (91)..(91)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (92)..(92)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (93)..(93)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (94)..(94)
<223> n is a, c, g, t or u
<400> 33
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgannnn nnggtagcnn nnnngaaacg caccgaatct aatacggccg 120
cgggtccagg gttcaagtcc ctgttcgggc gccaggatcc 160
<210> 34
<211> 156
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (81)..(86)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (82)..(82)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (83)..(83)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (84)..(84)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (85)..(85)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (86)..(86)
<223> n is a, c, g, t or u
<400> 34
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgagtcc nnnnnnggac gaaacgcacc gaatctaata cggccgcggg 120
tccagggttc aagtccctgt tcgggcgcca ggatcc 156
<210> 35
<211> 156
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (77)..(80)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (87)..(90)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (77)..(77)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (78)..(78)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (79)..(79)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (80)..(80)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (87)..(87)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (88)..(88)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (89)..(89)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (90)..(90)
<223> n is a, c, g, t or u
<400> 35
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgannnn gaggacnnnn gaaacgcacc gaatctaata cggccgcggg 120
tccagggttc aagtccctgt tcgggcgcca ggatcc 156
<210> 36
<211> 158
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (77)..(81)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (88)..(92)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (77)..(77)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (78)..(78)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (79)..(79)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (80)..(80)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (88)..(88)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (89)..(89)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (90)..(90)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (91)..(91)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (92)..(92)
<223> n is a, c, g, t or u
<400> 36
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgannnn ngaggacnnn nngaaacgca ccgaatctaa tacggccgcg 120
ggtccagggt tcaagtccct gttcgggcgc caggatcc 158
<210> 37
<211> 160
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (77)..(82)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (89)..(94)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (77)..(77)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (78)..(78)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (79)..(79)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (80)..(80)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (81)..(81)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (82)..(82)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (89)..(89)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (90)..(90)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (91)..(91)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (92)..(92)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (93)..(93)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (94)..(94)
<223> n is a, c, g, t or u
<400> 37
tctagagccc ggatagctca gtcggtagag cagcggccgt acttccacca acgaattcca 60
tgaatccagc tgatgannnn nngaggacnn nnnngaaacg caccgaatct aatacggccg 120
cgggtccagg gttcaagtcc ctgttcgggc gccaggatcc 160
Claims (9)
1. shearing the strategy of ribozyme between a kind of intracellular screening catalytic molecular, which is characterized in that using cell as screening vector, malicious egg
White IbsC is reporter gene, constructs the plasmid library containing IbsC toxic protein gene and ribozyme DNA library, and plasmid library is turned
After entering cell, the expression of toxic protein is controlled by ribozyme the shear action of toxic protein mRNA, and then influence cell survival,
To screen acquisition active ribozyme.
2. shearing the strategy of ribozyme between intracellular screening catalytic molecular according to claim 1, which is characterized in that have and cut
Cutting active ribozyme HHRz and can shearing toxalbumin mRNA causes its expression to be lowered, and bacterium can form Dan Ke in screening flat board
It is grand；But inactive ribozyme HHRzm cannot shear toxalbumin mRNA, toxoprotein gene overexpress so that bacterial death and cannot be
Clone is formed in screening flat board, so that nonactive sequence is therefrom screened rejecting, only filters out active ribozyme sequence.
3. plasmid library according to claim 1, which is characterized in that ribozyme is from toxalbumin by different promoter initiate tables
Up to and independently express；The transcriptional expression of ribozyme is controlled by itself promoter and terminator, and ribozyme DNA library is building up to
Guarantee the stability of the resulting ribozyme of transcription in the anticodon loop sequence of tRNA gene；The substrate-binding region of ribozyme with
The resulting part mRNA of IbsC toxic protein genetic transcription is complementary, is sheared with being formed between ribozyme and IbsC toxic protein gene molecule
Model；The expression of toxoprotein gene is by the promoter of itself, ribosome bind site, lactose operon sequence and terminator control
System, in one additional protein gene of N-terminal amalgamation and expression of toxoprotein gene, so as to improve core enzyme-to-substrate relative scale with
Enhance the shearing of ribozyme in vivo.
4. a kind of ribozyme with locus specificity endonuclease activity, which is characterized in that by the first substrate-binding region, greatly
Catalytic active center, loop-stem structure (stem 2 and prominent ring 2), small catalytic active center, the second substrate-binding region composition；Big catalysis
7 base CUGAUGA (SEQ ID NO 19) is contained in activated centre, and small catalytic active center contains 4 bases G AAA (SEQ ID
NO 20), stem 2 and prominent ring 2 connect two catalytic active centers.
5. ribozyme according to claim 4, which is characterized in that the shearing site of specific recognition is located at the first bottom of substrate
Between object bond area and the second substrate-binding region, sequence rules be " NUX ", N indicate randomized bases (N=A or T or C or
G), X indicates other three bases (N=A or T or C) other than G, and specific cleavage occurs after X；" NU " and hammerhead shape core
Enzyme sequence interaction exists in the form of matching base, and " X " exists in the form of unpaired bases.
6. ribozyme according to claim 4, which is characterized in that the ribozyme has the first substrate-binding region and the second substrate
The bond area substrate-binding region Liang Ge, positioned at the two sides in catalytic core region；It is selected respectively with preparatory two substrate-binding regions
Fixed RNA substrate is combined by way of base pair complementarity, and substrate-binding region sequence can be according to the sequence of different substrates
And corresponding variation occurs；The length of substrate-binding region can change, only need in conjunction with previously selected RNA substrate sequence shape
At stable duplex structure.
7. ribozyme according to claim 4, which is characterized in that stem 2 forms stem structure by way of base pair complementarity,
There are 3~8 base pairings；The sequence length of prominent ring 2 is 3~8 bases；In a preferred approach, stem 2 and 2 sequence of prominent ring can be with
For SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO24, SEQ ID NO 25, SEQ ID NO
26, any sequence described in SEQ ID NO 27, SEQ ID NO 28 or SEQ ID NO 29.
8. ribozyme according to claim 4, which is characterized in that cut mode is mainly intermolecular shearing.
9. ribozyme according to claim 4, which is characterized in that suitable for being directed in extracellular, prokaryotic cell, in eukaryocyte
The catalysis of the locus specificity of target substrates molecule is sheared, and realizes that the expression to selected genes is lowered.
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Cited By (2)
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| CN116286926A (en) * | 2023-02-28 | 2023-06-23 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Construction of double-fluorescence reporter plasmid and application of double-fluorescence reporter plasmid in detection of intracellular c-di-GMP level of escherichia coli |
| CN116286926B (en) * | 2023-02-28 | 2023-12-29 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Construction of double-fluorescence reporter plasmid and application of double-fluorescence reporter plasmid in detection of intracellular c-di-GMP level of escherichia coli |
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Application publication date: 20190412 |