US 12,325,873 B2
Enzymatic methods to generate high yields of sequence specific RNA oligonucleotides with extreme precision
Craig Martin, Amherst, MA (US); Elvan Cavac, Northampton, MA (US); Kithmie Harshana Malagodapathiranage, Amherst, MA (US); Shuo Sui, Sunderland, MA (US); Sarah L. Perry, Belchertown, MA (US); and Yasaman Gholamalipour, Worcester, MA (US)
Assigned to University of Massachusetts, Boston, MA (US)
Filed by University Of Massachusetts, Boston, MA (US)
Filed on Jan. 10, 2023, as Appl. No. 18/152,367.
Application 18/152,367 is a continuation of application No. 16/857,563, filed on Apr. 24, 2020, granted, now 11,578,348.
Claims priority of provisional application 62/933,119, filed on Nov. 8, 2019.
Claims priority of provisional application 62/837,906, filed on Apr. 24, 2019.
Prior Publication US 2023/0265477 A1, Aug. 24, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12N 9/12 (2006.01); C12N 15/10 (2006.01); C12Q 1/6865 (2018.01)
CPC C12P 19/34 (2013.01) [C12N 9/1247 (2013.01); C12N 15/10 (2013.01); C12Q 1/6865 (2013.01)] 18 Claims
 
1. A method of synthesizing a product RNA, the method comprising
preparing a reaction mixture comprising a functional template DNA for the product RNA, and an RNA polymerase under conditions for RNA synthesis, wherein the RNA polymerase is noncovalently linked to a nontemplate DNA that is 10 nucleotides or longer in length, and wherein the nontemplate DNA is complementary to a minimum of 10 nucleotides of the template DNA upstream of the transcription start site, or wherein the nontemplate DNA is complementary to all or part of the entire template strand DNA in the coding region, wherein the nontemplate DNA and the RNA polymerase are immobilized to a solid support, and wherein the reaction mixture and the solid support are contained in a reaction chamber of a fluidic chip, the fluidic chip comprising fluidic paths for reagent delivery and product RNA removal, and
synthesizing the product RNA in the reaction mixture and continuously removing the product RNA from the reaction chamber while flowing fresh RNA synthesis reagents into the reaction chamber,
wherein the RNA polymerase comprises an avidin-binding peptide, the nontemplate DNA comprises a 3′ or 5′ biotin label respectively, and the RNA polymerase is noncovalently linked to the nontemplate DNA via a bead support that binds both the avidin-binding peptide and the biotin.