CN114752600B - Histidine-tagged aptamers - Google Patents

Histidine-tagged aptamers Download PDF

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CN114752600B
CN114752600B CN202210658986.6A CN202210658986A CN114752600B CN 114752600 B CN114752600 B CN 114752600B CN 202210658986 A CN202210658986 A CN 202210658986A CN 114752600 B CN114752600 B CN 114752600B
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aptamer
histidine
seq
tagged
affinity
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CN114752600A (en
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王赛
毛相朝
张玲
魏凯悦
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Ocean University of China
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Ocean University of China
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a histidine-tagged aptamer, belonging to the technical field of biological processing. The nucleotide sequence of the aptamer of the histidine tag is shown as SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO. 8. The histidine-tagged aptamer is obtained by taking 6H7-Original aptamer as an initial chain and performing affinity-enhanced transformation through the embedded cooperation of a core region, and has high affinity to a histidine tag. The histidine-tagged aptamer of the invention has great application potential.

Description

Histidine-tagged aptamers
Technical Field
The invention relates to a histidine-tagged aptamer, belonging to the technical field of biological processing.
Background
The histidine tag (his-tag) is a short peptide consisting of 6 histidine molecules, is a tool enzyme tag in a biological processing chain, and is generally connected to the N-terminal or C-terminal of a recombinant protein. In biotechnological processes, in particular in protein engineering and enzyme engineering, histidine tags are often attached to the protease termini in order to facilitate isolation and purification of proteases or polypeptides.
The aptamer (aptamer) is an oligonucleotide fragment obtained by in vitro screening through an exponential enrichment ligand evolution technology, has the biological characteristics of low cost, easy synthesis, modification, small batch difference, low immunogenicity and the like, can be quickly and closely combined with a corresponding target in a specificity manner, and can specifically recognize various targets. The aptamer serving as a chemical and biological sensing recognition molecule has been successfully applied to the fields of food safety analysis, biomedical research and the like.
The 6H7-Original aptamer is a nucleic acid aptamer aiming at histidine tag reported in the prior art, the dissociation equilibrium constant (Kd) between the aptamer and the histidine tag is 5.0 mu mol/L, the affinity for the histidine tag is limited, and the strengthening is necessary to obtain the nucleic acid aptamer with stronger affinity.
Disclosure of Invention
In view of the above prior art, the present invention provides several histidine-tagged aptamers. The invention takes 6H7-origin aptamer as an initial chain, and affinity enhancement modification is carried out through the embedded cooperation of a core region to obtain a plurality of nucleic acid aptamers with high affinity to histidine tags.
The invention is realized by the following technical scheme:
the nucleotide sequence of the aptamer of the histidine tag is shown as SEQ ID NO.4 or SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO. 8.
The application of the histidine-tagged aptamer in preparing a detection product for detecting histidine-tagged protein or polypeptide. The detection product is selected from a detection probe, a detection reagent, a detection kit or a detection sensor.
The histidine-tagged aptamer can be applied to detection, separation, enrichment or purification of histidine-tagged proteins or polypeptides.
The histidine-tagged aptamer is obtained by taking 6H7-Original aptamer as an initial chain and modifying by adopting a core region embedded synergistic strengthening method, and has the strengthening characteristics that: by predicting the secondary structure of the 6H7-Original aptamer, core region embedded cooperation is carried out according to the structural characteristics, so that the affinity of the aptamer and a target is improved. The affinity of the aptamer obtained by modifying the core region through the embedded synergistic strengthening method is obviously improved.
The histidine-tagged aptamer has high affinity to histidine tags, can be used for preparing detection products for detecting proteins or polypeptides with histidine tags, can be used for detecting, separating, enriching or purifying the proteins or polypeptides with histidine tags, and has important significance for detection, monitoring, separation, enrichment, purification and the like of protease in the actual biological processing process.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents and materials used in the following examples are conventional instruments, reagents and materials known in the art and are commercially available. Unless otherwise specified, the experimental methods and the detection methods in the following examples are conventional experimental methods and detection methods in the prior art.
The invention uses a biomembrane interference molecule interaction instrument to characterize and measure the affinity of the aptamer and the target. The principle of the instrument is a biological film mutual interference technology, which is an important position in the current research of the interaction between biological molecules. The specific determination method comprises the following steps: immobilizing the aptamer on the surface of the sensor through the binding of biotin and streptavidin; buffer solution required by reaction, aptamer with different concentrations and target with different concentrations marked by biotin are added into a 96-well plate, and the set program of the instrument is sensor balance 120 s, aptamer fixation 180 s, sensor balance 120 s, target combination 300 s, target dissociation 300 s, temperature 25 ℃ and frequency 2 Hz. The resulting binding-dissociation curve is fitted to obtain an affinity constant Kd value.
Example 16H 7 fortification of Original aptamers
6H7-origin aptamer, the nucleotide sequence of which is shown below:
5'-GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-3', as shown in SEQ ID NO. 1.
The experimental result of the biomembrane interference molecule interactor shows that the dissociation equilibrium constant (Kd) between the 6H7-Original aptamer and the histidine tag is 5.0 mu mol/L, and the lower the Kd value is, the higher the affinity between the two is.
In order to obtain the aptamer with better affinity with a target histidine tag, the core region embedded synergistic strengthening is carried out on the 6H7-Original aptamer: predicting the secondary structure of the 6H7-Original aptamer by using an online analysis tool, namely the UNAFold web server, and finding that the secondary structure has a stem-loop structure; combining autodock molecular docking simulation software to predict a core region fragment (represented by A, B, wherein A is 5 '-TGGTT-3' and is shown as SEQ ID NO.9, and B is 5'-GGGATTGGCCC-3' and is shown as SEQ ID NO. 10) of a target interacting with 6H7-origin aptamer, carrying out core region embedded synergistic modification on the 6H7-origin aptamer based on a secondary structure and a key position predicted by the molecular simulation software to obtain 7 modified nucleic acid aptamers, wherein the nucleic acid aptamers are respectively: 6H7-ABA obtained by embedding an A fragment on the 6H7-origin aptamer only, wherein the nucleotide sequence of the 6H7-ABA is shown as SEQ ID NO. 2; the nucleotide sequence of the 6H7-ABB obtained by embedding a B fragment on the 6H7-origin aptamer is shown as SEQ ID NO. 3; 6H7-ABAB obtained by embedding a cooperative A, B fragment on a 6H7-origin aptamer, wherein the nucleotide sequence of the ABAB is shown as SEQ ID NO. 4; 6H7-ABBA obtained by embedding a cooperative B, A fragment on a 6H7-origin aptamer, wherein the nucleotide sequence of the ABBA is shown as SEQ ID NO. 5; on the basis of 6H7-ABAB, 6H7-ABABA is obtained by embedding cooperative A segments, and the nucleotide sequence of the 6H7-ABA is shown in SEQ ID NO. 6; on the basis of 6H7-ABAB, 6H7-ABABB obtained by embedding cooperative B fragments is further embedded, and the nucleotide sequence of the 6H7-ABB is shown as SEQ ID NO. 7; the 6H7-ABAB is obtained by embedding a cooperative A, B fragment on the basis of 6H7-ABAB, and the nucleotide sequence of the 6H7-ABAB is shown in SEQ ID NO. 8.
The results of the 7 reinforced aptamer biofilm interference molecule interactors are shown in table 1, and show that: the two enhanced aptamers 6H7-ABA and 6H7-ABB have no good affinity with a target histidine tag, and the fact that the interaction between the aptamers and the target is influenced probably because the structure of 6H7 is greatly changed due to the inserted sequence is presumed. The affinity of the two strengthened aptamers 6H7-ABAB and 6H7-ABBA and the target histidine tag is improved, and the embedding coordination of the core region can improve the affinity of the aptamers and the target. The affinity of the three strengthened aptamers 6H7-ABABA, 6H7-ABABB and 6H7-ABABAB with a target histidine tag is remarkably improved, particularly the Kd value of the three strengthened aptamers 6H7-ABAB is as low as 0.0989 mu mol/L, compared with the aptamer 6H7-Original, the affinity is improved by 50.56 times, and the chimeric coordination of the fragments of the key bases is favorable for the interaction of the aptamers and the target.
TABLE 1
Figure 49023DEST_PATH_IMAGE002
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
<110> China oceanic university
<120> histidine-tagged aptamer
<141> 2022-06-09
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 1
gctatgggtg gtctggttgg gattggcccc gggagctggc 40
<210> 2
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 2
gctatgggtg gtctggttgg gattggccct ggttcgggag ctggc 45
<210> 3
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 3
gctatgggtg gtctggttgg gattggcccg ggattggccc cgggagctgg c 51
<210> 4
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 4
gctatgggtg gtctggttgg gattggccct ggttgggatt ggccccggga gctggc 56
<210> 5
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 5
gctatgggtg gtctggttgg gattggcccg ggattggccc tggttcggga gctggc 56
<210> 6
<211> 61
<212> DNA
<213> Artificial Sequence
<400> 6
gctatgggtg gtctggttgg gattggccct ggttgggatt ggccctggtt cgggagctgg 60
c 61
<210> 7
<211> 67
<212> DNA
<213> Artificial Sequence
<400> 7
gctatgggtg gtctggttgg gattggccct ggttgggatt ggcccgggat tggccccggg 60
agctggc 67
<210> 8
<211> 72
<212> DNA
<213> Artificial Sequence
<400> 8
gctatgggtg gtctggttgg gattggccct ggttgggatt ggccctggtt gggattggcc 60
ccgggagctg gc 72
<210> 9
<211> 5
<212> DNA
<213> Artificial Sequence
<400> 9
tggtt 5
<210> 10
<211> 11
<212> DNA
<213> Artificial Sequence
<400> 10
gggattggcc c 11

Claims (2)

1. A histidine-tagged aptamer characterized by: the nucleotide sequence is shown in SEQ ID NO. 6.
2. A histidine-tagged aptamer, characterized in that: the nucleotide sequence is shown in SEQ ID NO. 8.
CN202210658986.6A 2022-06-13 2022-06-13 Histidine-tagged aptamers Active CN114752600B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102719430A (en) * 2012-06-13 2012-10-10 湖南大学 Nucleic acid aptamer molecular beacon probe for detecting histidine-tag recombinant proteins and detection method thereof
CN103149110A (en) * 2013-02-26 2013-06-12 浙江大学 Method for detecting bitter substance dina based on receptor sensor
CN109055382A (en) * 2018-08-27 2018-12-21 温州医科大学附属第医院 Aptamer and its application of 6X histidine tag can be specifically bound

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005024042A2 (en) * 2003-09-04 2005-03-17 The Regents Of The University Of California Aptamers and methods for their in vitro selection and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102719430A (en) * 2012-06-13 2012-10-10 湖南大学 Nucleic acid aptamer molecular beacon probe for detecting histidine-tag recombinant proteins and detection method thereof
CN103149110A (en) * 2013-02-26 2013-06-12 浙江大学 Method for detecting bitter substance dina based on receptor sensor
CN109055382A (en) * 2018-08-27 2018-12-21 温州医科大学附属第医院 Aptamer and its application of 6X histidine tag can be specifically bound

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads;Öznur Kökpinar等;《Biotechnol Bioeng》;20110512;第108卷(第10期);第2372页右栏第2段 *
Development of a chimeric aptamer and an AuNPs aptasensor for highly sensitive and specific identification of Aflatoxin B1;YanYang 等;《Sensors and Actuators B: Chemical》;20200905;第319卷;摘要，第2页第2.3节，第3.1节，第4页第3.3节 *
核酸适配体筛选的改良与优化策略;李世雨 等;《生物技术通报》;20170712;第33卷(第11期);67-75页 *
适配体芯片技术及研究进展;王旭 等;《分析试验室》;20210203;第40卷(第4期);479-488页 *

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