Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Enzyme complex and method for regulating nuclease sequence selectivity

A nuclease and compound technology, applied in the field of bioengineering, can solve the problems of lack of versatility and heavy workload

Active Publication Date: 2017-12-19
PEKING UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have a large workload and are not universal

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Enzyme complex and method for regulating nuclease sequence selectivity
  • Enzyme complex and method for regulating nuclease sequence selectivity
  • Enzyme complex and method for regulating nuclease sequence selectivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1

[0031] In this embodiment, the target DNA strand sequence is:

[0032] 5'-TAT CTG CAC TAGATG CAC CTT-3';

[0033] The unrelated DNA strand sequence is:

[0034] 5'-CGGAAGACAGATGCUAAGTGCTTGACCTTCCG-3';

[0035] The sequence of the all-thio DNA template strand (S-DNA) of the synthetic target DNA sequence is:

[0036] 5'-AAAAAAAAAAAGGTGCATCTAGTGCAGATA-3';

[0037] For the principle of combining the above S-DNA with DNase I to form DNase I@S-DNA and sequence selectivity determination, please refer to figure 1 .

[0038] In the present invention, in order to effectively indicate the cleavage of DNase I / RNaseA to the target DNA / RNA sequence and other irrelevant sequences to be cleaved, the applicant has used two methods:

[0039] 1) Fluorescent probe indication method. Such as figure 1 As shown in , both the intended cleavage target sequence and the unrelated sequence are labeled with a fluorescent group and a quencher group, and high-efficiency FRET ca...

Embodiment 2

[0061] Embodiment 2

[0062] In this example,

[0063] The target RNA strand sequence is: 5'-UAUCUGCACUAGAUGCACCUU-3';

[0064] The unrelated RNA strand sequence is: 5'-UUGUACUACACAAAAGUACUG-3';

[0065] The synthetic all-thio-RNA sequence is:

[0066] 5'-AUACAGCUAAGGUGCAUCUAGUGCAGAUA-3'.

[0067] The specific implementation steps are as follows:

[0068] 1) Mix 1 μL of 2 mg / mL RNaseA and 1.60 nmol of all-thio-RNA strands in 50 μL of buffer, the composition of the buffer is: 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.2. Incubate the solution at 37 °C 1 hour.

[0069] 2) The solution obtained in 1) was ultrafiltered using an ultrafiltration tube with a cut-off molecular weight of 30 kD and a treatment volume of 500 μL. The buffer used for ultrafiltration is the same as that used in 1). The rotating speed used for the first ultrafiltration is 5000g, and the ultrafiltration time is 15min. The second ultrafiltration rotating speed is 3000g, and the time is 15min...

Embodiment 3

[0089] The applicant has used the SPDP method to carry out covalent coupling reaction after DNase I is combined with S-DNA, and the reaction steps are as follows:

[0090]1. Use 0.05M sodium phosphate buffer solution (containing 0.15M sodium chloride, pH 7.2) to prepare a DNaseI solution with a concentration of 10mg / mL. To 1 mL of the DNase I solution, 25 μL of 6.2 mg / mL SPDP solution dissolved in DMSO was added, and left overnight at room temperature.

[0091] 2. Ultrafiltration to remove excess SPDP, the buffer solution used for ultrafiltration consists of: 0.05M sodium phosphate, 0.15M sodium chloride, 0.01M EDTA, pH 7.2.

[0092] 3. Mix the S-DNA labeled with -SH at the end and the DNase I solution obtained in step 2 at a molar ratio of 1:1, and leave it overnight at room temperature.

[0093] 6. Ultrafiltration to remove free DNase I and S-DNA.

[0094] The DNase I@S-DNA covalent complex prepared by the above steps also exhibited specific cleavage of the S-DNA complemen...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

An enzyme composite and a method for regulating nuclease sequence selectivity are disclosed. A novel chemical regulation means is developed, nucleases (DNase and RNase) which don't have sequence specificity originally are allowed to obtain sequence selectivity, and the sequence selectivity of the enzymes can be regulated flexibly according to requirements of a needed cleavage sequence. Accordingly, a new tool for molecular biology study, medicine development, development of genetic engineering techniques, and the like is provided.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, relates to the field of artificially regulating the properties of enzymes, and in particular relates to an enzyme complex and a method for regulating nuclease sequence selectivity. Background technique [0002] Artificially regulating the substrate selectivity and catalytic activity of enzymes is of great significance in life science research and bioengineering applications. On the one hand, as natural biocatalysts, enzymes have strong catalytic performance, especially in catalyzing macromolecular reactions. At present, there are very few types of artificially synthesized catalysts, and enzymes have irreplaceable and extensive applications. On the other hand, the enzyme preparation cycle is long, the yield is small, and the types of existing enzymes are limited, which are far from meeting the needs of practical applications. Therefore, the transformation of existing enzymes to obtain new ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96C12N9/22
CPCC12N9/22C12N9/96C12Y301/13005
Inventor 赵美萍肖先金吴曈勃苏昕
Owner PEKING UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com