US-12618102-B2 - Biochemical reaction methods and reagents comprising intrinsically disordered regions

Abstract

The invention relates to processes for performing biochemical reactions, such as in an aqueous in vitro reaction system. The processes involve macromolecules, particularly polypeptides, comprising one or more functional intrinsically disordered regions (IDRs). The invention also relates to IDR-macromolecules, including IDR-polypeptides, including macromolecules or polypeptides comprising a tagged amino acid sequence which comprises or consists of one or more functional IDRs. Such functional IDRs are capable of increasing the efficiency of the biochemical reaction. The invention relates to kits comprising any such macromolecules and polypeptides. The invention further relates to processes for stimulating or enhancing liquid-liquid demixing in a solution using any such macromolecules and polypeptides, including in combination with multivalent metal ions, thereby providing reagents capable of increasing the efficiency of a biochemical reaction.

Inventors

• Niall ARMES
• HANNAH WILLIAMS
• Matthew Forrest
• Mathew Parker
• Sidong Liu
• Lauren Parker

Assignees

• Biocrucible Limited

Dates

Publication Date

20260505

Application Date

20211117

Claims (12)

1. 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. 2 . The fusion protein of claim 1 , wherein the second polypeptide consists of at least one functional intrinsically-disordered region (IDR).
3. 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. 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. 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. 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. 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. 8 . An isolated nucleic acid molecule encoding a fusion protein as defined in any one of claim 1-3, 4 or 5-7 .
9. 9 . A kit comprising a fusion protein as defined in any one of claim 1-3, 4 or 5-7 .
10. 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. 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. 12 . The kit of claim 10 , wherein all components are provided in lyophilized form.

Description

RELATED APPLICATIONS This application is a continuation of International Patent Application No. PCT/GB2020/052866 filed on Nov. 11, 2020, which claims priority to United Kingdom application 1916379.9 filed on Nov. 11, 2019. The contents of the aforementioned applications are hereby incorporated by reference in their entireties. REFERENCE TO SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 23, 2021, is named BOM_001PCCN_SL.txt and is 203118 202841 bytes in size. FIELD OF THE INVENTION The invention relates to processes for performing biochemical reactions, such as in an aqueous in vitro reaction system. The processes involve macromolecules, particularly polypeptides, comprising one or more functional intrinsically disordered regions (IDRs). The invention also relates to IDR-macromolecules, including IDR-polypeptides, including macromolecules or polypeptides comprising a tagged amino acid sequence which comprises or consists of one or more functional IDRs. Such functional IDRs are capable of increasing the efficiency of the biochemical reaction. The invention relates to kits comprising any such macromolecules and polypeptides. The invention further relates to processes for stimulating or enhancing liquid-liquid demixing in a solution using any such macromolecules and polypeptides, including in combination with multivalent metal ions, thereby providing reagents capable of increasing the efficiency of a biochemical reaction. BACKGROUND TO THE INVENTION The performance of biochemical reactions, and in particular in vitro biochemical reactions, is of fundamental importance in the biological sciences. Many biochemical reactions may need to be performed outside of the laboratory, such as at the point of care or in the field. In these settings it may not be possible to control biochemical reactions in the precise manner afforded by the laboratory environment. Improving the efficiency of biochemical reactions performed in these settings would be of value. Indeed, it may be desirable to increase the efficiency of biochemical reactions, regardless of the exact setting, including in vitro and in vivo biochemical reactions. The present invention addresses these issues. Many biochemical reactions require the use of co-factors to aid in driving performance efficiency. One particular example of such a co-factor is a macromolecular crowding agent. Crowding agents are essential for the performance of many biochemical reactions. A notable example is the recombinase polymerase amplification (RPA) system for the amplification of nucleic acids. The use of a crowding agent has been considered essential in driving RPA performance efficiency. However, crowding agents may have drawbacks. Accordingly, alternative means for driving performance efficiency of biochemical reactions, including RPA, and that obviate the need for added/exogenous crowding agents would be of use. In addition, reagents that add to or synergise with the functional effects of crowding agents in increasing the performance efficiency of biochemical reactions would be of use. The present invention also addresses these issues. SUMMARY OF THE INVENTION The present invention provides a process of performing a biochemical reaction in an aqueous in vitro reaction system, wherein the biochemical reaction is dependent on the function of at least one reaction macromolecule, optionally at least one reaction polypeptide, the process comprising: introducing at least one IDR-macromolecule into the in vitro reaction system under conditions suitable for performing the reaction, wherein the at least one IDR-macromolecule comprises one or more functional intrinsically disordered regions (IDRs), wherein upon introduction of the at least one IDR-macromolecule into the in vitro reaction system the efficiency of the biochemical reaction is increased by the at least one IDR-macromolecule; preferably wherein the at least one IDR-macromolecule is at least one IDR-polypeptide. In the above-described process, the biochemical reaction may be dependent on the function of the at least one IDR-macromolecule, optionally the at least one IDR-polypeptide, wherein upon its introduction into the in vitro reaction system the at least one IDR-macromolecule or the at least one IDR-polypeptide performs its reaction function in the biochemical reaction and increases the efficiency of the reaction. Any of the herein-described processes may further comprise maintaining the IDR-macromolecule or the IDR-polypeptide in the system to cause liquid-liquid demixing and the formation of a plurality of phase-separated aqueous compartments within the system by the IDR-macromolecule or the IDR-polypeptide, thereby increasing the efficiency of the biochemical reaction in the system. Any of the herein-described processes may further comprise maintaining th