Ribonucleic acid purification
Disclosed herein are methods for purifying RNA comprising poly A. Also disclosed herein are compositions such as surfaces and oligonucleotides for purifying RNA comprising polyA. Other embodiments are also disclosed. Commercially-available resins having polythymidine oligonucleotide ligands typically contain less than 30 thymidine (2′deoxy) residues and some commercial resin suppliers utilize a distribution of dT chain lengths, not of a discreet length.
1. A method for purifying a ribonucleic acid (RNA) transcript comprising a polyA tail, the method comprising:
a) obtaining a first sample comprising the RNA transcript, wherein the first sample comprises RNA transcript and at least 5% impurities, and wherein the percentage of RNA transcript and the percentage of impurities are the inverse of each other;
b) contacting the first sample with a surface linked to twenty thymidines via a linker, wherein the surface linked to twenty thymidines comprises the structure:
or a salt thereof, under conditions such that the RNA transcript binds to the surface;
c) eluting the RNA transcript from the surface; and
d) collecting the RNA transcript in a second sample,
wherein the level of impurities is lower in the second sample than the level of impurities in the first sample.
2. The method of claim 1 , further comprising washing the surface with a solution after step b).
3. The method of claim 1 , further comprising preheating the first sample before step b).
4. The method of claim 1 , wherein one or more steps are performed using a batch process.
5. The method of claim 1 , wherein the sample comprises deoxyribonucleic acid (DNA) and the sample has not been subjected to DNase treatment.
6. The method of claim 1 , wherein the impurities comprise an RNA that does not comprise a polyA tail, DNA, a carbohydrate, a toxin, a polypeptide, and/or a nucleotide.
7. The method of claim 1 , wherein the contacting step is performed at a temperature of 65° C.
8. The method of claim 1 , wherein the contacting step is performed at a rate of 100 cm/h.
9. The method of claim 1 , wherein the RNA transcript and polyT/U bind one another via non-covalent bonding.
10. The method of claim 1 , wherein the first sample comprises a salt solution.
11. The method of claim 1 , wherein the first sample comprises a sodium chloride solution.
12. The method of claim 1 , wherein the elution step is performed with an elution buffer.
13. The method of claim 1 , wherein the elution step is performed at a temperature of 65° C.
14. The method of claim 1 , wherein the RNA transcript is the product of in vitro transcription using a non-amplified DNA template.
15. The method of claim 1 , wherein the RNA transcript is 100 to 10,000 nucleotides in length.
16. The method of claim 1 , wherein the method is repeated at least 2 times with the same surface.
17. The method of claim 1 , wherein the impurities comprise an endotoxin and the level of the endotoxin in the second sample is at least 50% lower than the level of endotoxin in the first sample.