Methods for in vitro transcription
AssigneeArcturus Therapeutics, Inc.
InventorsBrett Dallas HILL, Kyoung-Joo Jenny CHOI
Life of this patent
Estimating the term…
Abstract
Disclosed herein are methods of producing transcribed RNA product with increased yield and reduced RNA degradation.
Claims31
1-107. (canceled)
A method for producing a single-stranded RNA product, the method comprising:
- a) reacting a transcription reaction mixture comprising a buffer solution comprising Mg2+, a DNA template, ribonucleoside tri-phosphates (rNTPs), and RNA polymerase;
- wherein the rNTPs comprise adenine (A), cytosine (C), guanine (G), and uracil (U) nucleosides, or analogs thereof, at a molar ratio that is within 5-10% of the ratio of A:C:G:T in the DNA template;
- and
- b) controlling the rate of the transcription reaction of step (a) to be about 10 mM rNTP/hour to about 25 mM rNTP/hour, thereby producing the single-stranded RNA product.
The method of claim, 108 further comprising:
- c) stopping the transcription reaction by digesting the DNA template with deoxyribonuclease (DNase) or quenching the RNA polymerase with a chelator;
- thereby producing the single-stranded RNA product.
A method for producing a single-stranded RNA product, the method comprising:
- a) reacting a transcription reaction mixture comprising a buffer solution comprising Mg2+, a DNA template, ribonucleoside tri-phosphates (rNTPs), and RNA polymerase;
- wherein the rNTPs comprise adenine (A), cytosine (C), guanine (G), and uracil (U) nucleosides, or analogs thereof, at a molar ratio that is within 5-10% of the ratio of A:C:G:T in the DNA template, and wherein the transcription reaction results in the depletion of rNTPS, and
- b) stopping the transcription reaction prior to a point in which the rNTPs have been depleted to between about 0% to 35% of the original amount of rNTPs in the reaction mixture;
- thereby producing the single-stranded RNA product.
A method for reducing the degradation of an RNA transcript having a size larger than 4000 nucleotides, comprising reacting a transcription reaction mixture comprising a buffer solution comprising Mg2+, a DNA template, ribonucleoside tri-phosphates (rNTPs), and RNA polymerase;
- wherein the rNTPs comprise adenine (A), cytosine (C), guanine (G), and uracil (U) nucleosides, or analogs thereof;
- with a chelator.
A composition for in vitro transcription of a nucleic acid, the composition comprising:
- a) ribonucleotide triphosphates (rNTPs), wherein the rNTPs consist of adenine (A), cytosine (C), guanine (G), and uracil (U) at a molar ratio of about 10:13:13:6 (A:C:G:U);
- b) Mg2+;
- c) a DNA template;
- and
- d) RNA polymerase.
Dependencies are inferred from the claim text.