CN117925626A - RNA nucleic acid aptamer for recognizing and binding mouse myoblasts and application thereof - Google Patents
RNA nucleic acid aptamer for recognizing and binding mouse myoblasts and application thereof Download PDFInfo
- Publication number
- CN117925626A CN117925626A CN202410067029.5A CN202410067029A CN117925626A CN 117925626 A CN117925626 A CN 117925626A CN 202410067029 A CN202410067029 A CN 202410067029A CN 117925626 A CN117925626 A CN 117925626A
- Authority
- CN
- China
- Prior art keywords
- aptamer
- nucleic acid
- seq
- rna molecule
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108091008104 nucleic acid aptamers Proteins 0.000 title claims abstract description 84
- 230000027455 binding Effects 0.000 title claims abstract description 26
- 210000003098 myoblast Anatomy 0.000 title claims abstract description 14
- 108091023037 Aptamer Proteins 0.000 claims abstract description 79
- 210000004027 cell Anatomy 0.000 claims abstract description 77
- 210000003205 muscle Anatomy 0.000 claims abstract description 10
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 10
- 238000011160 research Methods 0.000 claims abstract description 3
- 210000000663 muscle cell Anatomy 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 238000003384 imaging method Methods 0.000 claims description 5
- 239000002246 antineoplastic agent Substances 0.000 claims description 4
- 229940041181 antineoplastic drug Drugs 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 4
- 239000002086 nanomaterial Substances 0.000 claims description 4
- 230000002285 radioactive effect Effects 0.000 claims description 4
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000012216 screening Methods 0.000 abstract description 28
- 108091008103 RNA aptamers Proteins 0.000 abstract description 25
- 238000000034 method Methods 0.000 abstract description 21
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract 1
- 230000001575 pathological effect Effects 0.000 abstract 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 37
- 239000000047 product Substances 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 13
- 150000007523 nucleic acids Chemical class 0.000 description 11
- 238000011144 upstream manufacturing Methods 0.000 description 11
- 238000012163 sequencing technique Methods 0.000 description 9
- 239000011534 wash buffer Substances 0.000 description 9
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 8
- 102000053602 DNA Human genes 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 6
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 239000012148 binding buffer Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101150102573 PCR1 gene Proteins 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 101100519159 Arabidopsis thaliana PCR3 gene Proteins 0.000 description 3
- 108091008102 DNA aptamers Proteins 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 101100519158 Arabidopsis thaliana PCR2 gene Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000012203 high throughput assay Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 238000010187 selection method Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000023178 Musculoskeletal disease Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108091027568 Single-stranded nucleotide Proteins 0.000 description 1
- 208000010040 Sprains and Strains Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000000500 calorimetric titration Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 210000000626 ureter Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000036642 wellbeing Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- General Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Pathology (AREA)
- Public Health (AREA)
- Food Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
Abstract
The invention discloses an RNA aptamer for recognizing and combining mouse myoblasts and application thereof. The invention provides a complete set of RNA nucleic acid aptamer for recognizing and combining mouse myoblasts, which consists of all or part of 6 aptamers shown in SEQ ID No.1 to SEQ ID No. 6. These nucleic acid aptamers were obtained by a single round/oligoround screening method designed based on the traditional Cell-SELEX technology in combination with UMI and high throughput sequencing technology. The method can simultaneously obtain a plurality of nucleic acid aptamers with different targets, the nucleic acid aptamers have different binding modes on normal cells and tumor cells from different sources, and the molecular typing of the normal cells and the tumor cells from different sources can be realized according to the different binding modes. In addition, the invention has important significance for the research of the physiological and pathological conditions related to muscles and the diagnosis of treatment.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an RNA nucleic acid aptamer for recognizing and combining mouse myoblasts and application thereof.
Background
The aptamer is a single-stranded nucleotide (DNA or RNA molecule) capable of specifically binding to a target, has small molecular weight, is easy to design and synthesize, can be folded to form a specific conformation to specifically bind to the biological target, and is called as a chemical antibody. The aptamer has various advantages, such as simple synthesis, easy modification, stable chemical property, low immunogenicity, low cost and the like, can be used as a good novel efficient and stable molecular probe, and has great prospect in the fields of early diagnosis and accurate targeted therapy of cancers, drug therapy, biochemical sensing and the like.
The selection of target binding aptamer usually adopts a SELEX method, which can exponentially enrich the bound sequences from 10 13-1016 initial nucleic acid molecules, obtain candidate sequences of specific aptamer by sequencing technology, and then measure the binding between candidate aptamer and target by technical means such as surface plasmon resonance, isothermal calorimetric titration, etc. In the last decades, in order to obtain more excellent and richer aptamer, researchers have made a great deal of improvement on the basis of the SELEX method, and new SELEX methods including Cell-SELEX, capture-SELEX, GO-SELEX, GOLD-SELEX and the like are proposed, which objectively improve the screening efficiency, but repeated selection combined with the inherent properties of PCR amplification still faces the uncertainty associated with exponential enrichment caused by unavoidable artifacts. The causes of these artifacts are complex, including incomplete separation of non-nucleic acid aptamers, the effects of primer sequences, and sequence preference during PCR amplification, transcription, and reverse transcription.
In addition, researchers have attempted to circumvent the adverse effects of the SELEX process by shortening the time of selection, simplifying the procedure of aptamer selection, i.e., to separate aptamers by single round selection, which are mostly used for DNA aptamer acquisition. However, these methods have not found wider application than the SELEX method because of the variety, e.g., single round selection requires the discovery of aptamers from a large excess of non-aptamer nucleic acids as compared to SELEX, and existing single round selection methods have difficulty ensuring that the resulting aptamers have better binding properties. In addition, DNA itself is more stable than RNA aptamers and can be amplified directly by PCR, the process of each round of screening is not complex, and even a beginner can complete one round of aptamer selection within a day. And considering that accumulation of PCR errors during each repeated selection objectively increases sequence diversity, providing more opportunities for optimization of aptamer structure and acquisition of higher affinity aptamers, the SELEX method of DNA aptamers may be a more preferential selection in most cases than its single round of selection.
However, the selection of RNA aptamers differs significantly from the selection of DNA aptamers in that RNA instability and inability to directly amplify as templates for PCR means that reduction of the number of rounds of selection in RNA aptamer selection may be more significant. Meanwhile, based on the characteristic that the RNA aptamer can be prepared in large quantity through in vivo or in vitro transcription, the RNA aptamer has unique value based on in vivo regulation or in vitro large-scale production, and has important significance in improving the acquisition efficiency of the RNA aptamer. The current reports on single round screening methods for RNA aptamers are quite straightforward, including HT-seq studies using human genomic RNA libraries as initial libraries and single round selection methods for RNA aptamers based on RNase digestion and known sequence DNA vectors. However, these methods have respective limitations, so that we still lack RNA aptamer with good performance, and development of a single round screening method for RNA aptamer is still a significant task.
Muscle tissue is distributed throughout the vital tissues and organs of the human body and has a significant impact on the level of survival well-being of humans. Recent analysis of global disease burden data has shown that about 17.1 million people worldwide suffer from musculoskeletal diseases, including 150 or more disorders affecting the individual's motor system, ranging from short-lived problems that occur suddenly (such as fractures, sprains, and strains), to life-long disorders with continuously limited function and disability. Meanwhile, muscle tissue is a common administration route, and has important value besides the disease of the muscle tissue, and the screening of RNA aptamer targeted by muscle is also valuable.
Disclosure of Invention
The invention aims at providing an RNA aptamer for recognizing and binding mouse myoblasts and application thereof.
In a first aspect, the invention claims a set of nucleic acid aptamers.
The set of nucleic acid aptamers claimed in the invention is capable of recognizing and binding to mouse myoblasts, and in particular consists of all or part of the following six nucleic acid aptamers:
(A1) Aptamer 1: a single stranded RNA molecule shown in SEQ ID No. 1;
(A2) Aptamer 2: a single stranded RNA molecule shown in SEQ ID No. 2;
(A3) Aptamer 3: a single stranded RNA molecule shown in SEQ ID No. 3;
(A4) Aptamer 4: a single stranded RNA molecule shown in SEQ ID No. 4;
(A5) Aptamer 5: a single stranded RNA molecule shown in SEQ ID No. 5;
(A6) Aptamer 6: a single stranded RNA molecule shown in SEQ ID No. 6;
The moiety is 2 or more than 2 nucleic acid aptamers.
In a specific embodiment of the invention, C and U in each of the nucleic acid aptamer 1, the nucleic acid aptamer 2, the nucleic acid aptamer 3, the nucleic acid aptamer 4, the nucleic acid aptamer 5, and the nucleic acid aptamer 6 are modified by 2' -fluoro.
In a second aspect, the invention claims a set of aptamer derivatives.
The nucleic acid aptamer set derivative claimed in the invention is obtained by performing the following operations on each single nucleic acid aptamer in the nucleic acid aptamer set comprising the first aspect of the invention: and connecting fluorescein and/or an anti-tumor drug and/or a radioactive element and/or biological enzyme and/or biotin and/or a nanomaterial to one end or the middle (one position or a plurality of positions are connected) of the single aptamer to obtain the aptamer derivative with the same function as the single aptamer. In a specific embodiment of the present invention, fluorescein Isothiocyanate (FITC) is attached to the 5' -end of the single aptamer.
In a third aspect, the invention claims a nucleic acid aptamer.
The nucleic acid aptamer disclosed by the invention is used for identifying and combining mouse myoblasts, and can be specifically any one of the following:
(A1) Aptamer 1: a single stranded RNA molecule shown in SEQ ID No. 1;
(A2) Aptamer 2: a single stranded RNA molecule shown in SEQ ID No. 2;
(A3) Aptamer 3: a single stranded RNA molecule shown in SEQ ID No. 3;
(A4) Aptamer 4: a single stranded RNA molecule shown in SEQ ID No. 4;
(A5) Aptamer 5: a single stranded RNA molecule shown in SEQ ID No. 5;
(A6) Aptamer 6: a single stranded RNA molecule shown in SEQ ID No. 6.
In a specific embodiment of the invention, C and U in each of the nucleic acid aptamer 1, the nucleic acid aptamer 2, the nucleic acid aptamer 3, the nucleic acid aptamer 4, the nucleic acid aptamer 5, and the nucleic acid aptamer 6 are modified by 2' -fluoro.
In a fourth aspect, the invention claims aptamer derivatives.
The aptamer derivative claimed by the invention is obtained by connecting fluorescein and/or an anti-tumor drug and/or radioactive elements and/or biological enzymes and/or biotin and/or nano materials to one end or the middle of the aptamer in the third aspect, so as to obtain the aptamer derivative with the same function as the aptamer. In a specific embodiment of the present invention, fluorescein Isothiocyanate (FITC) is attached to the 5' -end of the aptamer.
In a fifth aspect, the invention claims a detection reagent for identifying and binding to muscle cells.
The detection reagent for identifying and binding to muscle cells claimed in the invention comprises the nucleic acid aptamer set described in the first aspect or the nucleic acid aptamer set described in the second aspect or the nucleic acid aptamer set described in the third aspect or the nucleic acid aptamer set described in the fourth aspect as an effective ingredient.
In a sixth aspect, the invention claims the use of a set of nucleic acid aptamers as described in the first aspect hereinbefore or a set of nucleic acid aptamer derivatives as described in the second aspect hereinbefore or a nucleic acid aptamer as described in the third aspect hereinbefore or a nucleic acid aptamer derivative as described in the fourth aspect hereinbefore for the manufacture of a product for or for the identification and binding of muscle cells.
In a seventh aspect, the invention claims the use of a set of nucleic acid aptamers according to the first aspect hereinbefore or a set of nucleic acid aptamer derivatives according to the second aspect hereinbefore or a nucleic acid aptamer according to the third aspect hereinbefore or a nucleic acid aptamer derivative according to the fourth aspect hereinbefore or a detection reagent according to the fifth aspect hereinbefore for the preparation of a muscle-related physiopathological research product.
In an eighth aspect, the invention claims the use of a set of nucleic acid aptamers as described in the first aspect hereinbefore or a set of nucleic acid aptamer derivatives as described in the second aspect hereinbefore or a nucleic acid aptamer as described in the third aspect hereinbefore or a nucleic acid aptamer derivative as described in the fourth aspect hereinbefore or a detection reagent as described in the fifth aspect hereinbefore for the preparation of a product for imaging muscle cells or for imaging muscle cells.
In each of the above-described related aspects, the muscle cells are muscle fibroblasts. In a specific embodiment of the invention, the muscle cell is a mouse myoblast strain C2C12.
In a ninth aspect, the invention claims the use of a set of nucleic acid aptamers as described in the first aspect above or a set of nucleic acid aptamer derivatives as described in the second aspect above in any of the following:
(C1) Preparing a product for molecular typing of normal cells and tumor cells of different sources;
(C2) The normal cells and tumor cells of different origins are subjected to molecular typing.
In a specific embodiment of the present invention, the normal cells may be selected from the following: HEK-293T, SV-HUC-1, C2C12, DC2.4. The tumor cells of different origins may be selected from: HCT-8, MDA-MB-231, TOV21G, A549.
The invention is based on the traditional Cell-SELEX technology, combines UMI and high-throughput sequencing technology, and designs a method for rapidly acquiring RNA aptamer through single-round/low-round screening. According to the invention, a mouse myoblast strain C2C12 is taken as a target cell, a UMI sequencing library is constructed after single-round screening, high-throughput sequencing is carried out, and a plurality of nucleic acid aptamers capable of specifically recognizing the myoblast are selected. The aptamer obtained by screening has the characteristics of low immunogenicity, easiness in modification and marking, stable property and good reproducibility; compared with the antibody, the antibody has good stability and low synthesis cost. Subsequent characterization of the aptamer properties and identification of targets can be used to facilitate the discovery and discovery of tumor markers. Meanwhile, the aptamer can be used as a high-efficiency and high-penetrability molecular probe tool for early diagnosis of tumors, and can be combined with a drug to perform tumor targeted therapy.
Drawings
FIG. 1 is a basic flow chart of single-round/oligoround screening.
FIG. 2 shows the sequencing results and sequence characterization of the C2C12 cell RNA screening. A: single-round screening the homology of ten sequences before the result of product library construction and sequencing; b: selecting and primarily truncating the flow characterization results of the candidate aptamer and the target cell C2C 12; c: secondary structure of 5C 2C12 RNA aptamers fitted by mFOLD; d: flow-through assay of affinity of 5C 2C12 RNA aptamers; e: confocal characterization results of 5C 2C12 RNA aptamers with target cells C2C12 (Control is Control sequence).
FIG. 3 shows the flow-through binding of different C2C12 RNA aptamers to different cells. A: TOV21G cells; b: HEK-293T cells; c: a549 cells; d: SV-HUC-1 cells; e: MDA-MB-231 cells; f: C2C12 cells; g: DC2.4 cells.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The invention utilizes the Cell-SELEX technology, combines UMI and high-throughput sequencing technology, performs RNA screening by taking a randomly synthesized ssDNA sequence as an initial, takes a mouse myoblast strain C2C12 as a target Cell, aims at screening out a nucleic acid aptamer capable of specifically recognizing the C2C12 Cell strain, and establishes a single-round/oligoround RNA screening method. FIG. 1 is a basic flow chart of single-round/oligoround screening.
The cells required for the experiment were all derived from AMERICAN TYPE Culture Collection (ATCC), the specific culture conditions are shown in Table 1, 10% FBS and 100U/mL penicillin and streptomycin were added to the culture medium, and the culture medium was incubated in a constant temperature incubator at 37℃with carbon dioxide and a CO 2 concentration of 5%. The digestive juice used for cell passage is 0.25% Trypsin-EDTA, and the frozen stock solution used for frozen cell is commercial serum-free frozen stock solution.
TABLE 1 Each cell and culture conditions thereof
| Cell lines | Chinese name | Culture conditions |
| MDA-MB-231 | Human breast cancer cells | DMEM+10％FBS+1％P/S |
| HCT-8 | Human colorectal cancer cells | RPMI1640+10％FBS+1％P/S |
| TOV21G | Human ovarian cancer cells | 1640+10％FBS+1％P/S |
| HEK-293T | Human embryonic kidney cells | DMEM+10％FBS+1％P/S |
| SV-HUC-1 | Immortalized cell for human ureter epithelium | Ham's F-12K+10％ FBS+1％P/S |
| C2C12 | Mouse myoblasts | DMEM+10％FBS+1％P/S |
| DC2.4 | Mouse bone marrow derived dendritic cells | DMEM+10％FBS+1％P/S |
| A549 | Human non-small cell lung cancer cells | 1640+10％FBS+1％P/S |
Example 1, C2C12 RNA aptamer screening and preparation
1. Library and primer design
In the invention, when RNA aptamer screening is carried out, the design of a nucleic acid library and primers is as follows:
Random nucleic acid library (LibDD DNA): 5'-TAATACGACTCACTATAGGGAGGACGATGCG G-N40-CAGACGACTCGCCCGA-3' (SEQ ID No. 7); where N40 represents 40 consecutive N.
LibDD upstream primer: 5'-TAA TAC GAC TCA CTA TAG GGA GGA CGA TGC GG-3' (SEQ ID No. 8).
LibDD downstream primer/reverse transcription primer: 5'-TCG GGC GAG TCG TCT G-3' (SEQ ID No. 9).
UMI library: 5'-TGGTGCGAATTCTGAGAGGT-N30-AGGT-TCGGGCGAGTCGTCTG-3' (SEQ ID No. 10); wherein N30 represents 30 consecutive N for distinguishing different sequences of the same UMI library, and four bases immediately after N30 are barcode sequences for distinguishing different UMI libraries.
UMI library upstream primer: 5'-TGGTGCGAATTCTGAGAGGT-3' (SEQ ID No. 11).
In the above sequences, N represents A, T, C, G random bases.
2. Solution preparation
1. Washing buffer solution: PBS buffer (pH=7.4), 1mM MgCl 2,1mM CaCl2, 4.5g/L D-glucose.
2. Binding buffer: consists of washing buffer with 1mg/mL of Bovine Serum Albumin (BSA) and 0.1mg/mL of tRNA (T8630, soy pal) and is prepared before each use.
3. Amplification of 5×mix: a premix solution containing a proper amount of primer, dNTPs, taq enzyme, 10 XBuffer and H 2 O is provided, wherein the primer is an upstream primer and a downstream primer of LibDD.
4. Library construction 5×mix: a premix containing a suitable amount of primers, dNTPs, taq enzyme, 10 XBuffer and H 2 O, wherein the primers are LibDD upstream primer and UMI library upstream primer.
In the invention, all used H 2 O is DEPC H 2 O, and all used consumables are subjected to high-temperature steam sterilization treatment.
3. RNA aptamer screening and identification thereof
1. Library concentration and RNA library preparation
(1) Library preparation: 200. Mu.L of DPBS was used to dissolve 2.4nmol LibDD library (SEQ ID No. 7), mixed well, denatured at 95℃for 5min, cooled on ice for 5min and renatured at room temperature for 10min.
(2) And (3) PCR extension: the synthesized ssDNA library (SEQ ID No. 7) was single-stranded by adding LibDD downstream primers (SEQ ID No. 9) under the following conditions: a) 95 ℃ for 5min; b) 59 ℃ for 5min; c) 72 ℃ for 5min; d) Short-term storage at-40deg.C.
(3) Transcription
Transcription system (20 μl) was formulated: the amount of dsDNA is calculated according to the length of the DNA sequence and the amount of the template added by PCR extension, and is obtained by dilution with DEPC H 2 O, and the system can be doubled and adjusted according to actual conditions. As shown in table 2. The prepared transcription system is then mixed and transcribed for 8-12h at 37 ℃.
TABLE 2 transcription System
(4) Degradation of the DNA template: to 20. Mu.L of the transcript was added 2.5. Mu.L of DNase I, 2.5. Mu.L of 10 Xreaction Buffer (EN 401-01, vazyme) and incubated at 37℃for 20min.
(5) RNA extraction and recovery
A) To 25. Mu.L of the product of the previous step was added 125. Mu.L of DEPC H 2 O, mixed well and transferred to a new 1.5mL EP tube.
B) To the EP tube was added 1 x volume of phenol-chloroform-isoamyl alcohol solution (volume ratio = 25:24:1, ph < 5.0) (EX 0125, G-CLONE) and vortexed.
C) Centrifuge at 15,000Xg for 10min at 4℃and transfer as much of the upper aqueous phase as possible to a new 1.5mL EP tube.
D) 1. Mu.L glycogen, 1/10 Xvolume of ammonium acetate (10 mol/L) and 2 Xvolume of ice-cold ethanol (100%) were added to the upper aqueous phase and allowed to settle for 2h at-80 ℃.
E) The product of the above step was removed and centrifuged at 15,000Xg for 15min at 4 ℃.
F) The supernatant was discarded, 300. Mu.L of ice-cold ethanol (75%) was added, and the pellet was vortexed to wash as thoroughly as possible, followed by centrifugation at 15,000Xg for 10min at 4 ℃.
G) And (5) vacuum spin drying. h) Add 50. Mu.L DPBS to dissolve the RNA product thoroughly, mix well and calibrate the RNA content using NanoDrop.
I) And (3) renaturation: denaturation at 95 ℃ for 5min, cooling on ice for 5min, renaturation at room temperature for 10min, and temporary storage at-80 ℃ for standby. The RNA obtained in this step is the initial RNA screening library.
2. Single round RNA aptamer screening
(1) Target cells (used after three generations of newly resuscitated C2C12 cells were cultured in a 10 cm. Times.2 cm dish) were prepared, and the cells were cultured until the confluency was 90%, and used in a good adherent state.
(2) The medium was aspirated off and washed twice with 2ml of wash buffer stored at 4 ℃.
(3) The prepared initial RNA screening library was added to 1300. Mu.L of binding buffer and resuspended and mixed well.
(4) The heavy suspension from the previous step was added to a C2C12 cell culture dish, incubated on ice for 1h, and mixed with shaking every five minutes.
(5) After the incubation, the supernatant was removed and gently washed 2 times with 2mL of wash buffer. Ensure that cells are not eluted in order to avoid loss of sequence.
(6) At 4℃2mL of the culture dish was addedReagent (15596026, invitrogen) digested for 1min, lysates were collected in 1.5mL EP tube and vortexed for 2-4min to allow more complete cell lysis. And (3) injection: the amount of TRIzol is optionally adjusted.
(7) To the EP tube, 1/5 Xvolume of chloroform was added, and the mixture was vortexed and then centrifuged at 15,000Xg for 10min at 4 ℃.
(8) The upper aqueous phase was transferred to a fresh 1.5mL EP tube, and an appropriate amount of DNase I was added and incubated at 37℃for 30-60min.
(9) RNA was extracted as described above, followed by 7.5. Mu.L of DEPC H 2 O for reconstitution.
(10) The RNA obtained in the above step was reverse transcribed into cDNA according to the reagent instructions using the first strand cDNA synthesis kit (E6560S, NEB).
(11) PCR amplification
PCR1 system: 20. Mu.L of cDNA+40. Mu.L of the above step was amplified to 5 Xmix+140. Mu.L of DEPC H 2 O. Amplification procedure: a) 95 ℃ for 3min; b) Amplifying for 10 cycles at 95 ℃,30s+58 ℃,30s+72 ℃,30 s; c) 72 ℃ for 5min; d) 4 ℃, and preserving for a short period.
Cycle number optimization PCR2: mu.L of PCR1 product+10. Mu.L amplified 5 Xmix+39. Mu.L DEPC H 2 O was split into 5 tubes of 10. Mu.L each. The temperature and time conditions of each PCR procedure were the same as those of PCR1, with the number of cycles ranging from 7 to 15, 2 cycles apart. After completion of PCR2, nucleic acid denaturing PAGE gel (8%) electrophoresis was performed: to each sample was added 1 volume of 2 Xurea loading buffer, denatured at 95℃for 10min, cooled to 4℃and loaded and electrophoresed at 300V constant pressure for 10-15min, and the bands of the PAGE gel were visualized by a gel imager (ImageQuant 800, cytiva (GE)), and the optimal number of cycles was confirmed according to the target band yield and the presence or absence of bands.
PCR3: PCR3 (PCR 3 system: 10. Mu.L of PCR1 product+100. Mu.L of amplified 5 Xmix+390. Mu.L of DEPC H 2 O) was performed under optimized conditions (optimal cycle number), and after completion the product was pooled into a new 1.5mL EP tube for gel recovery.
(12) Agarose gel recovery
A) A3% agarose gel (pre-added Gelred dye) was prepared.
B) To the PCR3 product, 100. Mu.L of 6 Xloading buffer was added, and the mixture was mixed and loaded.
C) At a constant voltage of 300V, running the gel for about 40min until the strips separate significantly.
D) The target band at a position of about 88bp in length was observed and cut under a UV lamp, and weighed.
E) The agarose gel recovery kit (DC 301-01, vazyme) was used for gel recovery according to the reagent instructions. And (3) injection: DEPC H 2 O was used for elution during the last step of the recovery process.
F) The dsDNA content was calibrated using NanoDrop (NANODROP ONE, thermo Scientific).
G) Spin-dry under vacuum, add DEPC H 2 O and re-dissolve to 0.1mg/mL.
(13) Transcription and RNA recovery are carried out according to the related steps, and the obtained RNA is a single-round screening product library.
3. UMI library construction and high throughput assays
(1) UMI library construction
Based on the RNA characteristics, we add an additional library construction step before DNA sequencing. In this step, we incubated the single round of RNA screening product library with target cells (C2C 12 cells), then sorted the living cells, and reverse transcribed using UMI library as primers, and the resulting cDNA was PCR-submitted to the company for sequencing. Wherein the sequences of the UMI library are: 5'-TGGTGCGAATTCTGAGAGGT-N30-AGGT-TCGGGCGAGTCGTCTG-3' (SEQ ID No. 10); where N30 represents 30 consecutive N (N represents A, T, C, G random bases), each sequence in the library contains only one copy. By adding UMI sequences by reverse transcription, we strive to reduce the effect of PCR preferential amplification and the like on sequencing, reflecting the binding difference of RNA and target cells as truly as possible. The operation of this section includes:
a) The library was screened using the first round of RNA and after the end of incubation according to the relevant step in step (1), the cells were resuspended using 200. Mu.L of wash buffer.
B) Live cell sorting was performed using a flow cytometer (MoFlo Astrios EQS, beckman Coulter).
C) RNA was extracted as described in the previous relevant steps.
D) cDNA was obtained by reverse transcription using a first strand cDNA synthesis kit (E6560S, NEB) using a specific UMI library as primers according to the reagent instructions.
E) PCR amplification and detection of the band of interest was performed using an 8% nucleic acid denaturing PAGE gel.
In this procedure, the primers used for PCR amplification were LibDD upstream primers and UMI library upstream primers, i.e., library 5 Xmix was used for the experiment.
(2) High throughput assay
After the PCR amplification reaction, a PCR band without a non-specific band, with a clear target band and a bright lane was selected for amplification. After amplification, the amplified product was sequenced by Hangzhou Repu Gene technologies Co.
After high throughput sequencing is complete, the data is initially processed using Python. An effective sequence should contain the complete LibDD ssDNA library sequence structure (upstream and downstream fixed segments, middle random sequence segments), the four base sequence of the barcode sequence (AGGT in the present invention) for distinguishing between different UMI libraries, the UMI library random region (N30) for distinguishing between different sequences in the same UMI library, and the upstream fixed sequence complement. In the analysis, the fixed sequences at both ends of the sequence, i.e., the upstream fixed segment of LibDD ssDNA library sequences and the complementary sequence of the upstream fixed sequences of the UMI library, are first removed. And then cutting according to the first ten base sequences of the downstream fixed section of the LibDD ssDNA library sequence, splitting data according to whether the second half sequence contains the barcode, counting the occurrence times of the sequences corresponding to the first half in the data containing the barcode, and outputting a result. It is noted that in order to reflect as much as possible the reality of RNA binding to cells, the count value of a single effective sequence in sequencing is considered to be once when counted after resolution. The distribution of the first ten sequences in the nucleic acid sequence after completion of statistics is shown in Table 3, and most of the random sequences are concentrated at about 40 bases in length, which corresponds to the designed library sequences.
TABLE 3 Single round of screening sequencing results for C2C12 cell RNA aptamer screening
| Name of the name | Sequence (both ends fixed sequence region is omitted here) | Length of | Copy number | Ordering of |
| CCRNA-1 | 5′-UGGACUGGGGAUGUCAAUGGAAAACACUUUGUGCGCGCCC-3′ | 40 | 69 | 1 |
| CCRNA-2 | 5′-CCAACUCAUUCGGUCGUUUCGGAGAAAUGACCGGUCGGCC-3′ | 40 | 53 | 2 |
| CCRNA-3 | 5′-CGGGAACCUAUGUCAAUCGUAAAGGUUUAGGGUUCUGCGC-3′ | 40 | 44 | 3 |
| CCRNA-4 | 5′-CAGGUAACGUAAACAGUUGCACUUCUUGCGUUCCCUGCGG-3′ | 40 | 34 | 4 |
| CCRNA-5 | 5′-CACUAUCGGUGGCUGGCGUUUUACCAGCAGAUUCGUGCCC-3′ | 40 | 32 | 5 |
| CCRNA-6 | 5′-CACACAGAUGAAUCGCAUCGUUCGGUCGUUUCCACGUGUG-3′ | 40 | 31 | 6 |
| CCRNA-7 | 5′-GCAGAAUCAAUGCGGUUCCUUCGUGGAACCGGUUUGGCCC-3′ | 40 | 31 | 7 |
| CCRNA-8 | 5′-UCCGUGCGGUAUCAUGACGUGUUGACGUCGAGUGGUUGGC-3′ | 40 | 27 | 8 |
| CCRNA-9 | 5′-GGUCGUGAACCUAUUCCACGUGUAGUGGGUUCUGUCCCGC-3′ | 40 | 24 | 9 |
| CCRNA-10 | 5′-CAGAUACAGCCUAUGUCGUUCUUGGUGCAUAAUGUGUGCC-3′ | 40 | 23 | 10 |
Example 2 characterization of C2C12 aptamer candidates
1. Binding of aptamer candidates to target cells
Based on the sequencing conditions of high throughput sequencing, we selected 6 potential aptamer sequences based on sequence enrichment and homology analysis, as shown in Table 3 and FIG. 2A. Based on its possible secondary structure, we performed a preliminary truncation of the sequence after truncation, see table 4, which fits the structure shown in fig. 2C. Aptamers were labeled with Fluorescein Isothiocyanate (FITC) at the 5' end and their binding to target cells (C2C 12 cells) was determined by flow cytometry. Wherein the flow cytometry experiment is specifically performed as follows:
(1) Nucleic acid sequence preparation: aptamer RNA (lyophilized powder, synthesized by Bei Xin Biotechnology Inc. of Suzhou) or control sequence (DNA, lyophilized powder, synthesized by Shanghai Biotechnology Inc.) was centrifuged at 8,000Xg for 1min at room temperature, dissolved using DPBS and quantified to 5. Mu. Mol.L -1 with NanoDrop. And carrying out variofying on the quantified candidate aptamer according to the following steps: a) 95 ℃ for 5min; b) On ice for 5min; c) Room temperature for 10min. According to the number of nucleic acid sequences, 1.5ml clean EP tube is used, corresponding marks (one for each sequence) are made, 4 mu l of nucleic acid sequence is respectively taken out of the corresponding EP tube, the temporary storage is carried out at 4 ℃ in a dark place, the rest nucleic acid sequence is split into 50 mu l/tube and then is placed at-80 ℃ for storage, and the nucleic acid sequence can be directly used in the subsequent flow cytometry and other experiments.
Note that: the control Sequence (Random Sequence) involved in this step is: cg gcg act tcc act gag tgg ctg ggt agg ggt agg cgg gtt agg gtg tgt cgt cg. All reference sequences referred to in this example are those unless otherwise specified.
(2) Cell preparation: target cells (C2C 12 cells, cultured to 90% confluence and good adherent state) were digested with an enzyme-free digestion solution, washed twice with a washing buffer (see example 1, the same as below), resuspended with a binding buffer (see example 1, the same as below), and counted with a living cell counter to obtain a cell suspension. Based on the counting results, the binding buffer was supplemented to a cell concentration of 3X 10 5/96. Mu.l.
(3) Incubation: mu.l of the cell suspension was taken separately in each of the EP tubes described in step (1), mixed with gentle shaking and incubated on ice for 30min, and the shaking mixed once every 5min.
(4) Washing: after the incubation was completed, the cells were resuspended by centrifugation, removal of the supernatant, followed by washing 2 times with 200. Mu.l of wash buffer, and after completion 200. Mu.l of wash buffer.
(5) And (3) detecting: detection was performed using Attune NXT flow cytometer (Thermo Scientific). After the experiment was completed, the data was processed with flowjo_v10 software.
As shown in FIG. 2B, the six sequences all bound to different degrees on the target cell C2C12, indicating that the six sequences obtained by the screening were all nucleic acid aptamers to the C2C12 cell. And (3) injection: the control Sequence (Random Sequence) involved in this step is: cg gcg act tcc act gag tgg ctg ggt agg ggt agg cgg gtt agg gtg tgt cgt cg. All reference sequences referred to in this example are those unless otherwise specified.
Table 4, CCRNA aptamer sequences and affinities
Note that: the numbers in the sequence names are the abundance ranks of the sequenced nucleic acid aptamers; the C base and U base in the aptamer sequence are modified by 2' -Fluoro.
2. Aptamer affinity assay
Five sequences which bind strongly to the target cells (C2C 12 cells) were selected based on the above results and their affinities were characterized. The aptamer with different concentrations is respectively incubated with 3×10 5 C2C12 cells on ice for 30min, and the cells are detected by a flow cytometer after centrifugation, washing and resuspension. Flow cell experimental data were processed with flowjo_v10 software and plotted with GRAPHPAD PRISM software, with the X-axis being RNA concentration (nM) and the Y-axis being the average fluorescence intensity after deduction of the cell autofluorescence values. The equilibrium dissociation constant KD of the aptamer was calculated using the formula y=b max X/(kd+x), where B max is the average fluorescence intensity at maximum specific binding, fitted to the KD value at the same time.
As shown in FIG. 2D and Table 4, their equilibrium dissociation constants (KD values) are 90.75+ -12.08 nM, 168.57 + -23.86 nM, 4.58+ -0.81 nM, 82.94+ -18.31 nM and 103.12+ -27.43 nM, respectively, indicating their strong affinity with the target cells (C2C 12 cells).
3. Binding of the co-Jiao Biaozheng aptamer
After digestion with pancreatin and preparation of cell suspension, counting with a counter, 10-20 ten thousand C2C12 cells were taken in the copoly Jiao Min, and after addition of 2ml of suitable medium, were cultivated for 24-48 hours at 37℃in a 5% CO 2 environment until the cells were completely adherent and grown to 70% -80%. The confocal dish was removed, washed three times with DPBS, and subsequently stained at 37℃for 15-20 minutes with 1X hochest diluted with DPBS. DPBS wash dye was added followed by 200nM of FITC-labeled RNA aptamer at the 5' -end or control sequence diluted in binding buffer and incubated at 4℃for 30min. The washing buffer was used for three times and fixed with 4% paraformaldehyde at room temperature.
Microscopic imaging was performed using a nikon single photon confocal microscope, fluorescence IMAGEs of FITC and hochest channels were acquired, and post IMAGE processing was performed using NIS-ELEMENT VIEWER and IMAGE J. As a result, as shown in FIG. 2E, fluorescence binding signals were observed on confocal images of the remaining five sequences compared to the control sequence, indicating that all five sequences were nucleic acid aptamers to C2C12 cells.
4. Nucleic acid aptamer for molecular typing of cells
Further, we performed flow cytometry characterization on more cell lines using five nucleic acid aptamers, wherein flow cytometry experiments were specifically performed as described above. As shown in FIG. 3, binding of the six sequences on DC2.4 cells was not apparent. CCRNA-2a, CCRNA-3a and CCRNA-6a have relatively strong binding on C2C12 and various cancer cells, and are not obviously bound in various normal cell lines, so that the binding targets thereof can be highly expressed on the surfaces of the cancer cells. CCRNA-1a was more clearly associated with each cell line, suggesting that its target may have relatively high stable expression in different cells. CCRNA-4a and CCRNA-5a bind more clearly only on the surface of C2C12 cells, suggesting that their targets may be specific for expression on muscle fibroblasts. In summary, our results further demonstrate that by the oligo-round/single-round RNA aptamer cell screening method, we can obtain multiple different target nucleic acid aptamers simultaneously, which have different binding patterns on normal cells and tumor cells of different sources, and can realize molecular typing of normal cells and tumor cells of different sources according to the different binding patterns.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. A set of nucleic acid aptamers consisting of all or part of the following six nucleic acid aptamers:
(A1) Aptamer 1: a single stranded RNA molecule shown in SEQ ID No. 1;
(A2) Aptamer 2: a single stranded RNA molecule shown in SEQ ID No. 2;
(A3) Aptamer 3: a single stranded RNA molecule shown in SEQ ID No. 3;
(A4) Aptamer 4: a single stranded RNA molecule shown in SEQ ID No. 4;
(A5) Aptamer 5: a single stranded RNA molecule shown in SEQ ID No. 5;
(A6) Aptamer 6: a single stranded RNA molecule shown in SEQ ID No. 6;
The moiety is 2 or more than 2 nucleic acid aptamers.
2. The nucleic acid aptamer is any one of the following six nucleic acid aptamers:
(A1) Aptamer 1: a single stranded RNA molecule shown in SEQ ID No. 1;
(A2) Aptamer 2: a single stranded RNA molecule shown in SEQ ID No. 2;
(A3) Aptamer 3: a single stranded RNA molecule shown in SEQ ID No. 3;
(A4) Aptamer 4: a single stranded RNA molecule shown in SEQ ID No. 4;
(A5) Aptamer 5: a single stranded RNA molecule shown in SEQ ID No. 5;
(A6) Aptamer 6: a single stranded RNA molecule shown in SEQ ID No. 6.
3. The set of nucleic acid aptamers of claim 1 or the nucleic acid aptamer of claim 2, wherein: c and U in the aptamer 1 are modified by 2' -fluoro; and/or
C and U in the aptamer 2 are modified by 2' -fluoro; and/or
C and U in the aptamer 3 are modified by 2' -fluoro; and/or
C and U in the aptamer 4 are modified by 2' -fluoro; and/or
C and U in the aptamer 5 are modified by 2' -fluoro; and/or
C and U in the aptamer 6 are modified by 2' -fluoro.
4. A set of aptamer derivatives, characterized in that: the nucleic acid aptamer derivative is obtained by carrying out the following operations on each single nucleic acid aptamer in the nucleic acid aptamer set according to claim 1 or 3: and connecting fluorescein and/or antitumor drugs and/or radioactive elements and/or biological enzymes and/or biotin and/or nanomaterials to one end or the middle of the single aptamer to obtain the aptamer derivative with the same function as the single aptamer.
5. A nucleic acid aptamer derivative characterized by: the aptamer derivative is obtained by connecting fluorescein and/or an anti-tumor drug and/or radioactive elements and/or biological enzymes and/or biotin and/or nano materials to one end or the middle of the aptamer in the claim 2 or 3, and has the same function as the aptamer.
6. A detection reagent for identifying and binding to a muscle cell, comprising as an active ingredient the nucleic acid aptamer set of claim 1 or3 or the nucleic acid aptamer set derivative of claim 4 or the nucleic acid aptamer set of claim 2 or3 or the nucleic acid aptamer derivative of claim 5.
7. Any of the following applications:
(B1) Use of a set of nucleic acid aptamers according to claim 1 or 3 or a set of nucleic acid aptamer derivatives according to claim 4 or a nucleic acid aptamer according to claim 2 or 3 or a nucleic acid aptamer derivative according to claim 5 for the preparation of a product for or for the identification and binding of muscle cells;
(B2) Use of a set of nucleic acid aptamers according to claim 1 or 3 or a set of nucleic acid aptamer derivatives according to claim 4 or a nucleic acid aptamer according to claim 2 or 3 or a nucleic acid aptamer derivative according to claim 5 or a detection agent according to claim 6 for the preparation of a muscle-related physiopathological research product;
(B3) Use of a set of nucleic acid aptamers according to claim 1 or 3 or a set of nucleic acid aptamer derivatives according to claim 4 or a nucleic acid aptamer according to claim 2 or 3 or a nucleic acid aptamer derivative according to claim 5 for the preparation of a product for imaging muscle cells or for imaging muscle cells.
8. The detection reagent according to claim 6 or the use according to claim 7, characterized in that: the muscle cells are muscle fibroblasts;
further, the muscle cell is a mouse myoblast strain C2C12.
9. Use of the set of nucleic acid aptamers of claim 1 or 3 or the set of nucleic acid aptamer derivatives of claim 4 in any of the following:
(C1) Preparing a product for molecular typing of normal cells and tumor cells of different sources;
(C2) The normal cells and tumor cells of different origins are subjected to molecular typing.
10. The use according to claim 9, characterized in that: the normal cells are selected from the following: HEK-293T, SV-HUC-1, C2C12, DC2.4; and/or
The tumor cells of different origins are selected from: HCT-8, MDA-MB-231, TOV21G, A549.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410067029.5A CN117925626A (en) | 2024-01-17 | 2024-01-17 | RNA nucleic acid aptamer for recognizing and binding mouse myoblasts and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410067029.5A CN117925626A (en) | 2024-01-17 | 2024-01-17 | RNA nucleic acid aptamer for recognizing and binding mouse myoblasts and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117925626A true CN117925626A (en) | 2024-04-26 |
Family
ID=90753158
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410067029.5A Pending CN117925626A (en) | 2024-01-17 | 2024-01-17 | RNA nucleic acid aptamer for recognizing and binding mouse myoblasts and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117925626A (en) |
-
2024
- 2024-01-17 CN CN202410067029.5A patent/CN117925626A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7303244B2 (en) | Methods for generating aptamers with improved off-rates | |
| CN108034658B (en) | Nucleic acid aptamer for detecting human uveal melanoma cells | |
| WO2003002758A1 (en) | Nucleic acid ligands to complex targets | |
| CN110004147B (en) | Aptamer of epithelial cell adhesion molecule EpCAM screened from human plasma and preparation method and application thereof | |
| CN101538613A (en) | Molecular probe related to disease and method for producing the same | |
| CN117925627A (en) | RNA nucleic acid aptamer for recognizing and binding to human colorectal cancer cells and application thereof | |
| CN113215167A (en) | Aptamer and application thereof in detection of cells infected by iridovirus of micropterus salmoides | |
| CN109402127B (en) | Group of high-affinity nucleic acid aptamers capable of being specifically bound with connective tissue growth factor and application of high-affinity nucleic acid aptamers | |
| CN106032534B (en) | A kind of rna aptamer and its screening technique in conjunction with Non-small cell lung carcinoma cell-specific | |
| CN117925626A (en) | RNA nucleic acid aptamer for recognizing and binding mouse myoblasts and application thereof | |
| CN117925600A (en) | Single-round/low-round screening method for RNA aptamer | |
| CN109337908B (en) | Nucleic acid aptamer group specifically binding to gliotoxin and application thereof | |
| CN112501174A (en) | RNA aptamer specifically bound with CD 44-hyaluronic acid binding domain protein and screening method and application thereof | |
| CN112029771A (en) | Aptamer specifically binding to meperidine and application thereof | |
| CN114317544B (en) | Aptamer specifically binding to CD133, screening method and application thereof | |
| CN107532216A (en) | The synchronous detection of oligonucleotides and its kit and purposes of correlation | |
| CN109554369B (en) | Use of aptamers for recognition and binding of alkaline phosphatase heterodimers | |
| CN114736951B (en) | Construction method of high-throughput sequencing library of small molecular RNA | |
| Feng et al. | Chemo-versus Immuno-Precipitation of G-Quadruplex-DNA (G4-DNA): A Direct Comparison of the Efficiency of the Antibody BG4 versus the Small-Molecule Ligands TASQs for G4 Affinity Capture | |
| CN112553209B (en) | Human non-small cell lung cancer aptamer truncation and application thereof | |
| CN110951735B (en) | Long-chain non-coding RNA lnc-PMIF, small interfering RNA thereof and application thereof | |
| WO2023202030A1 (en) | Method for constructing high-throughput sequencing library of small rna | |
| CN113817740B (en) | Application research of nucleic acid aptamer recognition and PTPRF (positive temperature coefficient PRF) and related functions thereof | |
| CN117187250A (en) | Aptamer and application thereof related to recognition and binding of thyrotropin receptor protein | |
| US20240052412A1 (en) | Method for detecting rna structure at whole transcriptome level and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |