Granted Patent B2

US 12,618,102 · App. 17/528,967 · Granted May 5, 2026

Inventors: Niall Armes (Cambridge, GB); Hannah Williams (Cambridge, GB); Matthew Forrest (Cambridge, GB); Mathew Parker (Cambridge, GB); Sidong Liu (Cambridge, GB); Lauren Parker (Cambridge, GB)

Assignee: Biocrucible Limited

C12Q1/6848C07K1/14C12Q1/6853C07K2319/00

Abstract

Claims (16)

1 . A fusion protein comprising a first polypeptide fused to a second polypeptide, wherein the first polypeptide comprises of a Recombinase Polymerase Amplification (RPA) component selected from a recombinase agent, a single-strand stabilizing agent, a polymerase, and a recombinase loading protein, further wherein the second polypeptide comprises of at least one functional intrinsically-disordered region (IDR) which comprises the amino acid sequence of any one of SEQ ID NOs: 1-43.

2 . The fusion protein of claim 1 , wherein the second polypeptide consists of at least one functional intrinsically-disordered region (IDR).

3 . The fusion protein of claim 1 , wherein the fusion protein is capable of causing liquid-liquid demixing and the formation of a plurality of phase-separated aqueous compartments.

4 . The fusion protein of claim 1 , wherein the first polypeptide consists of an RPA component selected from a recombinase agent, a single-strand stabilizing agent, a polymerase, and a recombinase loading protein.

5 . The fusion protein of claim 1 , wherein the RPA component is:

(a) a recombinase agent selected from the group consisting of: UvsX, T4 UvsX, T6 UvsX, RB18 UvsX, E. coli phage wV7 UvsX, Shigella phage CB8 UvsX, Shigella phage Shfl2 UvsX, E. coli phage AR1 UvsX, phage vB_EcoM_G4507 UvsX, Shigella phage SHFML-11 UvsX, Escherichia phage vB_EcoM_DalCa UvsX, E. coli RecA, E. coli RadA, E. coli RadB, E. coli Rad 51 or any functional analog, homolog or derivative thereof, and any combination thereof;

(b) a recombinase loading protein selected from the group consisting of UvsY, E. coli RecO, E. coli RecR or any functional analog, homolog or derivative thereof, and any combination thereof;

(c) a polymerase selected from the group consisting of eukaryotic pol-«, eukaryotic pol-β, eukaryotic pol-8, eukaryotic pol-¿, Bacillus stearothermophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, S. aureus DNA polymerase I (Sau polymerase), E. coli DNA polymerase I Klenow fragment, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V, bacteriophage T4 gp43 DNA polymerase, bacteriophage T7 DNA polymerase, and bacteriophage Phi-29 DNA polymerase, or any functional analog, homolog or derivative thereof, and any combination thereof; or

(d) a single strand stabilizing agent is selected from the group consisting of Gp32, E. coli SSB protein, phage T4 Gp32 protein, phage Rb69 Gp32, phage vB_EcoM NBG1 Gp32, or any functional analog, homolog or derivative thereof, and any combination thereof.

6 . The fusion protein of claim 1 , wherein said RPA component is a single strand stabilizing agent that is a Gp32 protein, and wherein the fusion protein has: (i) an amino acid sequence having at least 80% identity to SEQ ID NO:120 or any one of SEQ ID NOs: 65 to 88; or (ii) the amino acid sequence of any one of SEQ ID NO: 120 or any one of SEQ ID NOs: 65 to 88.

7 . The fusion protein of claim 1 , wherein the second polypeptide is fused to the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, or any amino acid position along a length of the first polypeptide.

8 . An isolated nucleic acid molecule encoding a fusion protein as defined in any one of claim 1-3, 4 or 5-7 .

9 . A kit comprising a fusion protein as defined in any one of claim 1-3, 4 or 5-7 .

10 . The kit of claim 9 , wherein the kit further comprises a recombinase agent, a recombinase loading protein, a polymerase, first and second nucleic acid primers, an exonuclease, a buffer, and/or a source of multivalent metal ions.

11 . The kit of claim 9 , wherein the first polypeptide of the fusion protein consists of an RPA component selected from the group consisting of: a recombinase agent, a single-strand stabilizing agent, a polymerase, and a recombinase loading protein, and wherein the kit further comprises an recombinase agent, an recombinase loading protein, a polymerase, first and second nucleic acid primers, an exonuclease, a buffer, and/or a source of multivalent metal ions.

12 . The kit of claim 10 , wherein all components are provided in lyophilized form.

Assignments (1)

Continuity (2)

References Cited (122)