Method for synthesizing selectively labeled RNA
The invention relates to a method for synthesizing a selectively labeled RNA, and an apparatus for performing the method. Specific segments or discrete residues within the RNA may be selectively labeled, and different segments may include different labels.
1. A method for synthesizing a RNA, comprising performing an initiation stage, an elongation stage, and a termination stage, wherein:
(a) the initiation stage comprises:
(i) providing a solid phase comprising a DNA template, wherein the DNA is attached to a solid substrate;
(ii) providing a first liquid phase comprising a RNA polymerase and ribonucleoside triphosphates (rNTPs);
(iii) mixing the solid phase and first liquid phase;
(iv) incubating the solid phase and first liquid phase at 4-37° C. for at least 10 minutes to initiate synthesis of the RNA;
(v) pausing the RNA synthesis by incubating the solid phase and first liquid phase at 0-5° C. for at least 10 minutes, whereupon the solid phase comprises the RNA polymerase and the RNA being synthesized; and
(vi) separating the solid phase from the first liquid phase;
(b) the elongation stage comprises:
(i) providing a second liquid phase comprising rNTPs;
(ii) mixing the solid phase and second liquid phase;
(iii) incubating the solid phase and second liquid phase at 4-37° C. for at least 10 minutes to elongate the RNA;
(iv) pausing the RNA synthesis by incubating the solid phase and second liquid phase at 0-5° C. for at least 10 minutes;
(v) separating the solid phase from the second liquid phase; and
(vi) repeating steps (i)-(v) of part (b) n times, wherein n is equal to 1-100, and wherein the rNTPs in the second liquid phase are the same or different in each repeat;
(c) the termination stage comprises:
(i) providing a third liquid phase comprising rNTPs;
(ii) mixing the solid phase with the third liquid phase;
(iii) incubating the solid phase and third liquid phase at 4-37° C. for at least 10 minutes; and
(iv) pausing the RNA synthesis by incubating the solid phase and third liquid phase at 0° C. for at least 10 minutes;
wherein steps (a)-(c) are repeated m times, wherein m is equal to 1-100, and wherein rNTPs of at least one of the first liquid phase, the second liquid phase, or third liquid phase comprise a label.
2. The method of claim 1 , wherein the solid substrate is a bead comprising a gel, glass, or a synthetic polymer.
3. The method of claim 2 , wherein the bead has a diameter of 5-100 μm.
4. The method of claim 1 , wherein the RNA polymerase is T7 RNA polymerase.
5. The method of claim 1 , wherein the label is selected from the group consisting of 13 C/ 15 N, 2 H, Cy3, Cy5, a fluorophore, a heavy atom, and a chemical modification.