RNA affinity purification
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.
1. A method of purifying a nucleic acid preparation, comprising:
contacting a nucleic acid preparation comprising messenger ribonucleic acid (mRNA) and double stranded RNA (dsRNA) with an RNase III enzyme that (i) is immobilized on a solid support, (ii) is catalytically inactive, and (iii) can specifically bind to the dsRNA; and
eluting a purified nucleic acid preparation comprising the mRNA from the solid support.
2. The method of claim 1 , wherein the mRNA is an in vitro-transcribed mRNA.
3. The method of claim 1 , wherein the RNase III enzyme is a thermostable RNase III enzyme.
4. The method of claim 3 , wherein the thermostable RNase III enzyme is a Thermotoga maritima RNase III enzyme.
5. The method of claim 1 , wherein the RNase III enzyme comprises an amino acid sequence having a modification at an amino acid position corresponding to E130 of the amino acid sequence of SEQ ID NO: 3.
6. The method of claim 5 , wherein the RNase III enzyme comprises the amino acid sequence of SEQ ID NO: 4.
7. The method of claim 1 , wherein the solid support comprises a carboxy-reactive resin or an amino-reactive resin.
8. The method of claim 1 , wherein the catalytically inactive RNase III enzyme has at least one amino acid mutation relative to a corresponding wild-type RNase III enzyme.
9. The method of claim 1 , wherein the purification is performed at a temperature of from 40° C. to 70° C.
10. The method of claim 1 , wherein the RNase III enzyme comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6.
11. An affinity purification method comprising:
passing a nucleic acid preparation over an affinity purification column to which an RNase III enzyme is immobilized, wherein the nucleic acid preparation comprises messenger ribonucleic acid (mRNA) and double stranded RNA (dsRNA), and wherein the RNase III enzyme is catalytically inactive and can specifically bind to the dsRNA; and
eluting a purified nucleic acid preparation comprising the mRNA from the solid support.
12. The method of claim 11 , wherein the RNase III enzyme is a thermostable RNase III enzyme.
13. The method of claim 12 , wherein the thermostable RNase III enzyme is a Thermotoga maritima RNase III enzyme.
14. The method of claim 13 , wherein the RNase III enzyme comprises an amino acid sequence having a modification at an amino acid position corresponding to E130 of the amino acid sequence of SEQ ID NO: 3.
15. The method of claim 11 , wherein the RNase III enzyme comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
16. The method of claim 11 , wherein the solid support comprises a carboxy-reactive resin or an amino-reactive resin.
17. The method of claim 11 , wherein the catalytically inactive RNase III enzyme has at least one amino acid mutation relative to a corresponding wild-type RNase III enzyme.
18. The method of claim 11 , wherein the purification is performed at a temperature of from 40° C. to 70° C.