US2022290125A1PendingUtilityA1

Rna affinity purification

69
Assignee: MODERNATX INCPriority: Dec 13, 2016Filed: Jun 2, 2022Published: Sep 15, 2022
Est. expiryDec 13, 2036(~10.4 yrs left)· nominal 20-yr term from priority
Inventors:Edward J. Miracco
C12Q 1/44C12Y 301/27004C12N 11/14C12N 15/1006C12N 15/10C12N 9/22C12Q 1/6806C12Y 301/26003
69
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Claims

Abstract

Provided herein, in some embodiments, are methods of purifying a nucleic acid preparation. The methods may comprise contacting a nucleic acid preparation comprising messenger ribonucleic acid with an RNase III enzyme that is immobilized on a solid support and binds to double-stranded RNA contaminants.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method of purifying a nucleic acid preparation, comprising
 contacting a nucleic acid preparation comprising messenger ribonucleic acid (mRNA) with an RNase III enzyme that is immobilized on a solid support, under conditions that result in binding of the RNase III enzyme to double-stranded RNA.   
     
     
         2 . The method of  claim 1 , wherein the mRNA is an in vitro-transcribed mRNA. 
     
     
         3 . The method of  claim 1  or  2 , wherein the preparation further comprises double-stranded RNA (dsRNA). 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the RNase III enzyme is catalytically inactive. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the RNase III enzyme is a thermostable RNase III enzyme. 
     
     
         6 . The method of  claim 5 , wherein the thermostable RNase III enzyme is a  Thermotoga maritima  RNase III enzyme. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the RNase III enzyme comprises an amino acid sequence identified by SEQ ID NO: 3. 
     
     
         8 . The method of any one of  claims 1 - 6 , wherein the RNase III enzyme comprises an amino acid sequence having a modification at an amino acid position corresponding to E130 of the sequence identified by SEQ ID NO: 3. 
     
     
         9 . The method of  claim 8 , wherein the RNase III enzyme comprises an amino acid sequence identified by SEQ ID NO: 4. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the solid support comprises a carboxy-reactive resin or an amino-reactive resin. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the mRNA comprises at least one chemical modification. 
     
     
         12 . The method of  claim 11 , wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio15 pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the mRNA comprises a 5′ terminal cap. 
     
     
         14 . The method of  claim 14 , wherein the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp. 
     
     
         15 . A method, comprising:
 performing an in vitro transcription reaction in the presence of a template nucleic acid to produce an in vitro transcription product; and   contacting the in vitro transcription product with an RNase III enzyme that is immobilized on a solid support.   
     
     
         16 . The method of  claim 15 , wherein the in vitro transcription product comprises messenger RNA (mRNA). 
     
     
         17 . The method of  claim 15  or  16 , wherein the in vitro transcription product further comprises double-stranded RNA (dsRNA). 
     
     
         18 . The method of any one of  claims 15 - 17 , wherein the RNase III enzyme is catalytically inactive. 
     
     
         19 . The method of any one of  claims 15 - 18 , wherein the RNase III enzyme is a thermostable RNase III enzyme. 
     
     
         20 . The method of  claim 19 , wherein the thermostable RNase III enzyme is a  Thermotoga maritima  RNase III enzyme. 
     
     
         21 . The method of any one of  claims 15 - 20 , wherein the RNase III enzyme comprises an amino acid sequence identified by SEQ ID NO: 3. 
     
     
         22 . The method of any one of  claims 15 - 20 , wherein the RNase III enzyme comprises an amino acid sequence having a modification at an amino acid position corresponding to E130 of the sequence identified by SEQ ID NO: 3. 
     
     
         23 . The method of  claim 22 , wherein the RNase III enzyme comprises an amino acid sequence as identified by SEQ ID NO: 4. 
     
     
         24 . The method of any one of  claims 15 - 23 , wherein the solid support comprises a carboxy-reactive resin or an amino-reactive resin. 
     
     
         25 . The method of any one of  claims 16 - 24 , wherein the mRNA comprises at least one chemical modification. 
     
     
         26 . The method of  claim 25 , wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio15 pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methylpseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. 
     
     
         27 . The method of any one of  claims 16 - 26 , wherein the mRNA comprises a 5′ terminal cap. 
     
     
         28 . The method of  claim 27 , wherein the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp. 
     
     
         29 . A composition comprising RNA purified according to a method that comprises contacting a preparation comprising messenger ribonucleic acid (mRNA) with an RNase III enzyme that is immobilized on a solid support. 
     
     
         30 . The composition of  claim 29 , wherein the composition is substantially free of double-stranded RNA. 
     
     
         31 . A composition comprising RNA prepared according to a method that comprises
 performing an in vitro transcription reaction in the presence of a template nucleic acid to produce an in vitro transcription product; and   contacting the in vitro transcription product with an RNase III enzyme that is immobilized on a solid support.   
     
     
         32 . The composition of  claim 31 , wherein the composition is substantially free of double-stranded RNA. 
     
     
         33 . A method of purifying a product of an in vitro transcription reaction, comprising
 contacting a product of an in vitro transcription reaction comprising messenger ribonucleic acid (mRNA) with a catalytically inactive thermostable RNase III enzyme that is immobilized on a solid support, under conditions that result in binding of the RNase III enzyme to double-stranded RNA.   
     
     
         34 . The method of  claim 33 , wherein the catalytically inactive thermostable RNase III enzyme has an amino acid sequence identified by SEQ ID NO: 4. 
     
     
         35 . A catalytically inactive thermostable RNase III enzyme having an amino acid sequence identified by SEQ ID NO: 4.

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