Enzymatic methods to generate high yields of sequence specific RNA oligonucleotides with extreme precision

Patent metadata

Field	Value
Publication number	US-12325873-B2
Application number	US-202318152367-A
Country	US
Kind code	B2
Filing date	Jan 10, 2023
Priority date	Apr 24, 2019
Publication date	Jun 10, 2025
Grant date	Jun 10, 2025

Abstract

Described herein are synthetic methods for producing sequence-specific RNA oligonucleotides that eliminate impurities produced in prior art methods. In one aspect, an end-protected capture DNA complementary to a portion of the product RNA is employed. In another aspect, the template DNA is covalently or noncovalently linked to the RNA polymerase, either directly or through the use of a nontemplate DNA. In a third aspect, a flow chamber is employed. All of the methods can be used in combination.

First claim

The invention claimed is:

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.

2. The method of claim 1 , wherein the reaction chamber is in operable communication with a downstream chamber comprising an immobilized reagent that specifically binds full-length product RNA, wherein the immobilized reagent does not bind less than full-length RNA and double-stranded RNA impurities.

3. The method of claim 1 , wherein the support is a microparticle or a bead.

4. The method of claim 1 , wherein RNA synthesis is conducted under low salt conditions of 0 to 50 mM.

5. The method of claim 1 , wherein RNA synthesis is conducted under high salt conditions of 50 to 1000 mM.

6. The method of claim 1 , wherein the reaction mixture further comprises a capture DNA that is 3′ end protected, and is complementary to 8 to 20 nucleotides at the 3′ end of the product RNA.

7. The method of claim 6 , wherein the 3′ end of the capture DNA comprises 3′ amino, 3′-deoxy (H), 3′-phosphorylation, 3′-O-methyl, 3′-fluorophore, 3′-biotin, 3′-azide, 3′-fluoro, 3′-PEG, or a 3′-mismatched base.

8. The method of claim 1 , further comprising purifying the product RNA.

9. The method of claim 8 , further comprising removing the capture DNA after product RNA synthesis.

10. 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 covalently 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 a haloalkane dehalogenase tag, and wherein the nontemplate DNA is covalently linked to the haloalkane dehalogenase tag of the RNA polymerase via a 3 ′ or 5 ′halogen-modification.

11. The method of claim 10 , wherein the reaction chamber is in operable communication with a downstream chamber comprising an immobilized reagent that specifically binds full-length product RNA, wherein the immobilized reagent does not bind less than full-length RNA and double-stranded RNA impurities.

12. The method of claim 10 , wherein the support is a microparticle or a bead.

13. The method of claim 10 , wherein RNA synthesis is conducted under low salt conditions of 0 to 50 mM.

14. The method of claim 10 , wherein RNA synthesis is conducted under high salt conditions of 50 to 1000 mM.

15. The method of claim 10 , wherein the reaction mixture further comprises a capture DNA that is 3′ end protected, and is complementary to 8 to 20 nucleotides at the 3′ end of the product RNA.

16. The method of claim 15 , wherein the 3 ′ end of the capture DNA comprises 3′ amino, 3′-deoxy (H), 3′-phosphorylation, 3′-O-methyl, 3′-fluorophore, 3′-biotin, 3′-azide, 3′-fluoro, 3′-PEG, or a 3′-mismatched base.

17. The method of claim 10 , further comprising purifying the product RNA.

18. The method of claim 17 , further comprising removing the capture DNA after product RNA synthesis.

Assignees

• Univ Massachusetts

Inventors

• Martin Craig
• Cavac Elvan
• Malagodapathiranage Kithmie Harshana
• Sui shuo
• Perry Sarah L
• Gholamalipour Yasaman

Classifications

• C12Q1/6865

  Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS] · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N9/1247 Primary

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q2563/131

  the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin · CPC title