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Mini-ILL RNASES, methods for changing specificity of RNA sequence cleavage by Mini-ILL RNASES, and uses thereof

A specificity and sequence technology, applied in Mini-III RNase, changing the specificity of Mini-III RNase cleaving RNA sequence and its application field

Inactive Publication Date: 2018-11-23
BIOTECH INNOVATIONS SP ZOO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

from Bacillus subtilis ( Bacillus subtilis )'s RNase Mini-III has a domain of this type, but it lacks the dsRNA-binding domain typical of other known members of the RNase III superfamily

Method used

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  • Mini-ILL RNASES, methods for changing specificity of RNA sequence cleavage by Mini-ILL RNASES, and uses thereof
  • Mini-ILL RNASES, methods for changing specificity of RNA sequence cleavage by Mini-ILL RNASES, and uses thereof
  • Mini-ILL RNASES, methods for changing specificity of RNA sequence cleavage by Mini-ILL RNASES, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Cloning of sequences encoding specific Mini-III RNases

[0148] Microorganisms were purchased in lyophilized form from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) (Table 1). After suspending the lyophilized product in 500 µl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0), the suspension was extracted with phenol (saturated with 100 mM Tris-HCl, pH 8.5). The aqueous phase was re-extracted with a phenol:chloroform (1 / 1 v / v) mixture, followed by nucleic acid precipitation by adding 50 μl of 3M sodium acetate pH 5.2 and 1 mL of ethanol. The precipitated nucleic acid was centrifuged (12000g, 10 min, 4°C) and the liquid was removed. The precipitate was washed with 1 mL of 70% ethanol and dried. The obtained DNA was suspended in 20 µl of TE buffer.

[0149] Table 1. Origin of cloned sequences encoding Mini-III RNases

[0150]

[0151] Table 2. Primer sequences used to amplify genomic DNA encoding specific Mini-III RNases...

Embodiment 2

[0157] Protein was expressed and purified from a recombinant plasmid encoding wild-type Mini-III RNase.

[0158] Transform Escherichia coli strain BL21 (DE3) (F–ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1sam7 ) with the recombinant plasmid carrying the Mini-III nuclease gene obtained in Example 1 nin5]). Transformation was carried out according to Example 1. Transformants were selected on LB solid medium supplemented with 50 μg / mL kanamycin and 1% glucose. Inoculate selected transformant colonies in LB liquid medium and incubate with shaking at 37°C for 5 hours. Then, inoculate in 500 mL liquid ZY (Studier 2005) supplemented with kanamycin to a concentration of 100 μg / mL 25 mL of culture grown in LB medium and incubated with shaking at 37 °C for 24 h. The culture was centrifuged at 5000 g for 10 min at 4 °C and suspended in STE buffer (0.1 M NaCl, 10 mM Tris-HCl pH 8.0 , 1mM EDTA pH 8.0), and centrifuged again. The pellet was suspended in 20mL lysis so...

Embodiment 3

[0162] Determining optimal reaction conditions for in vitro cleavage of dsRNA substrates with purified enzymes

[0163] To determine the optimal conditions for the reaction buffer, limited cleavage of the Φ6 phage genome was performed in the buffers listed in Table 3.

[0164] table 3

[0165]

[0166] In this experiment, 1.5 μg of dsRNA was used, as well as 3.3 μg of BsMiniIII obtained in Example 2, 80 ng of CkMiniIII, 23.5 μg of CrMiniIII, 5 μg of CtMiniIII, 0.8 μg of FnMiniIII, 1.1 μg of FpMiniIII, 2.1 μg of SeMiniIII, 0.185 μg of TmMiniIII, 11.5 ng of TtMiniIII. Aliquots corresponding to 0.5 μg dsRNA were collected after 5, 10 and 15 minutes of reaction, except for TtMiniIII and SeMiniIII, which had reaction times of 2, 4 and 6 minutes, and 20, 40 and 60 minutes, respectively.

[0167] The cleavage products were separated by electrophoresis on a 1.5% agarose gel supplemented with ethidium bromide at a final concentration of 0.5 μg / mL. The buffer that produced the mos...

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Abstract

The object of the invention is a Mini-Ill RNase with amino acid sequence comprising an acceptor part, and a transplantable a4 helix, and a transplantable a5b-a6 loop, which form structures of a4 helixand a5b-a6 loop, respectively, in the Mini-Ill RNase structure, wherein the fragments which form structures of a4 helix and a5b-a6 loop, respectively, correspond structurally to respective structuresof a4 helix and a5b-a6 loop formed by amino acid sequence fragments 46-52 and 85-98, respectively, of Mini-Ill RNase from Bacillus subtilis shown in SEQ ID NO: 1, wherein the said Mini- Ill RNase exhibits sequence specificity in dsRNA cleavage being dependent only on a ribonucleotide sequence of the substrate, and independent from an occurrence of secondary structures in the substrate's structure, and independent from a presence of other assisting proteins, and wherein the Mini-Ill RNase is not the Mini-Ill protein from Bacillus subtilis of SEQ ID NO: 1, nor SEQ ID NO: 1 with D94R mutation. The invention also relates to a method of obtaining a chimeric Mini-Ill RNase, a Mini-Ill RNase encoding construct, a cell with a Mini-Ill RNase encoding gene, use of Mini-Ill RNase for dsRNA cleavage,as well as a method of dsRNA cleavage depending only on a ribonucleotide sequence.

Description

[0001] describe. technical field [0002] The subject of the present invention are novel native and chimeric Mini-III RNases whose amino acid sequence comprises a receptor part and a graftable α4 helix forming the structures of α4 helix and α5b-α6 loop respectively in the Mini-III RNase structure and parts of the graftable α5b-α6 loop, where the Mini-III RNase exhibits sequence specificity in dsRNA cleavage that is dependent only on the ribonucleotide sequence of the substrate and independent of the substrate structure. The appearance of the order structure, and does not depend on the existence of other auxiliary proteins, and wherein said Mini-III RNase is not the Mini-III protein from Bacillus subtilis of SEQ ID NO: 1, nor the SEQ ID NO with D94R mutation :1. The invention also relates to methods for obtaining chimeric Mini-III RNases, Mini-III RNase-encoding constructs, cells with Mini-III RNase-encoding genes, the use of Mini-III RNases for dsRNA cleavage and Methods fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22
CPCC12N9/22C12N15/52C12Y301/26003
Inventor J.布尼克基K.斯克沃内克D.格洛M.库尔克斯卡
Owner BIOTECH INNOVATIONS SP ZOO
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