Removal of DNA fragments in mRNA production process
The present invention describes methods of removing DNA from an RNA transcript during the mRNA production process. The method embodies procedures for obtaining an in vitro transcription product, and removing any DNA from the product. The DNA can be removed by adding either free DNase or a resin containing immobilized DNase to the product, and recovering the RNA transcript. Alternatively, the DNA template used in the in vitro transcription reaction is labeled. After transcription, the product is applied to a resin that is configured to bind the label, and the RNA transcript is recovered. To detect whether any residual impurities are left in the RNA transcript product, the product is subjected to nuclease digestion and subsequently to liquid chromatography-tandem mass spectrometry analysis to quantitate any residual DNA. The present invention demonstrates efficient and effective methods of isolating an RNA transcript from an in vitro transcription product.
1. A method for removing a DNA template from a sample comprising said DNA template and an RNA transcript, comprising:
(a) providing a sample comprising
(b) digesting the DNA template by contacting the sample with a DNase enzyme to produce a sample comprising digested DNA template and the RNA transcript;
(c) contacting a portion of the sample of step (b) with a second nuclease under conditions that promote nucleotide digestion to obtain a digested sample;
(d) analyzing the digested sample by liquid chromatography-tandem mass spectrometry; and
(e) detecting the presence or absence of residual deoxynucleotides in the digested product based on the liquid chromatography-tandem mass spectrometry analysis.
2. The method of claim 1 , wherein the RNA transcript is the product of in vitro transcription using a non-amplified DNA template.
3. The method of claim 1 , wherein the RNA transcript is a full length RNA transcript.
4. The method of claim 1 , wherein the RNA transcript comprises chemically modified mRNA molecules.
5. The method of claim 1 , wherein the RNA transcript is between 100 and 10,000 nucleotides in length.
6. The method of claim 1 , wherein the method further comprises separating the digested DNA template from the RNA transcript by a process that comprises a centrifugation, an ultrafiltration, a precipitation, a liquid chromatography, a gel electrophoresis, or a vacuum column step.
7. The method of claim 1 , wherein the DNase is DNase I.
8. The method of claim 7 , wherein 2 units of DNase I are added for each μg of DNA template in the sample.
9. The method of claim 7 , wherein the DNase I is incubated with the sample at 37° Celsius for one hour.
10. The method of claim 1 , wherein the DNase is attached to a solid support.
11. The method of claim 10 , wherein the solid support is selected from the group consisting of a solid phase resin, controlled pore glass, polystyrene, polystyrene divinyl benzene, cellulose, polyacrylamide, silica, polymethacrylate, and dextran based polymers.