METHOD FOR SYNTHESIZING SELECTIVELY LABELED RNA

摘要

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.

主权项

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 5-30 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., 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 5-20 minutes to elongate the RNA;(iv) pausing the RNA synthesis by incubating the solid phase and second liquid phase at 0-5° C. for 5-30 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 5-30 minutes; and(iv) pausing the RNA synthesis by incubating the solid phase and third liquid phase at 0° C. for 5-30 minutes;wherein steps (a)-(c) are repeated multiple times, and wherein rNTPs of at least one of the first liquid phase, the second liquid phase, or third liquid phase comprise a label.