Biochemical reaction method and reagent containing intrinsically denatured region

Abstract

A method for carrying out biochemical reactions in aqueous in vitro reaction systems and the like with improved efficiency is provided. [Solution] The method involves macromolecules, particularly polypeptides, containing one or more functional intrinsically disordered regions (IDRs). The invention also relates to IDR-macromolecules, including IDR-polypeptides, including macromolecules or polypeptides containing tagged amino acid sequences that contain or consist of one or more functional IDRs. Such functional IDRs can increase the efficiency of biochemical reactions. The invention relates to kits containing any such macromolecules and polypeptides. The invention further relates to methods for using any such macromolecules and polypeptides, including in combination with polyvalent metal ions, to provide reagents that can stimulate or enhance liquid-liquid demixing in solution, thereby increasing the efficiency of biochemical reactions.

Description

[Technical Field]

[0001] The present invention relates to methods for conducting biochemical reactions, such as in aqueous in vitro reaction systems. The methods involve macromolecules, particularly polypeptides, that contain one or more functional intrinsically disordered regions (IDRs). The present invention also relates to IDR-macromolecules, including IDR-polypeptides, that include macromolecules or polypeptides that contain a tagged amino acid sequence that includes or consists of one or more functional IDRs. Such functional IDRs can increase the efficiency of biochemical reactions. The present invention also relates to kits that include any such macromolecules and polypeptides. The present invention further relates to methods for using any such macromolecules and polypeptides, including in combination with polyvalent metal ions, to provide reagents that can stimulate or enhance liquid-liquid demixing in solution, thereby increasing the efficiency of biochemical reactions. [Background technology]

[0002] The performance of biochemical reactions, particularly in vitro biochemical reactions, is of fundamental importance in biological science. Many biochemical reactions may need to be performed outside of a laboratory, for example, at the point of care or in the field. In these situations, it may be impossible to control the biochemical reaction with the precision afforded by a laboratory environment. It would be valuable to improve the efficiency of biochemical reactions performed in these situations. Indeed, it may be desirable to increase the efficiency of biochemical reactions regardless of the precise setting, including in vitro and in vivo biochemical reactions. The present invention addresses these issues.

[0003] Many biochemical reactions require the use of cofactors to help increase their efficiency. One particular example of such a cofactor 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 amplifying nucleic acids. The use of crowding agents has been considered essential for increasing the efficiency of RPA performance. However, crowding agents can have drawbacks. Therefore, alternative means for increasing the efficiency of biochemical reactions, including RPA, and alternative means that eliminate the need for added / exogenous crowding agents would be useful. Furthermore, reagents that complement or synergize with the functional effects of crowding agents in increasing the efficiency of biochemical reactions would be useful. The present invention also addresses these issues. Summary of the Invention

[0004] The present invention provides a method for conducting a biochemical reaction in an aqueous in vitro reaction system, wherein the biochemical reaction depends on the function of at least one reactive macromolecule, optionally at least one reactive polypeptide, the method comprising introducing at least one IDR-macromolecule into the in vitro reaction system under conditions suitable for conducting the reaction, the at least one IDR-macromolecule comprising one or more functional intrinsically disordered regions (IDRs), and wherein upon introduction of the at least one IDR-macromolecule into the in vitro reaction system, the efficiency of the biochemical reaction is enhanced by the at least one IDR-macromolecule, preferably the at least one IDR-macromolecule is at least one type of IDR-polypeptide.

[0005] In the above methods, the biochemical reaction may depend on the function of at least one IDR-macromolecule, optionally at least one IDR-polypeptide, and when introduced into an in vitro reaction system, the at least one IDR-macromolecule or at least one IDR-polypeptide performs its reaction function in the biochemical reaction and increases the efficiency of the reaction.

[0006] Any of the methods described herein may further include maintaining an IDR-macromolecule or IDR-polypeptide in the system to cause liquid-liquid demixing and the formation of multiple phase-separated aqueous compartments within the system by the IDR-macromolecule or IDR-polypeptide, thereby increasing the efficiency of biochemical reactions within the system.

[0007] Any of the methods described herein may further include maintaining an IDR-macromolecule or IDR-polypeptide in the system to co-localize molecules necessary for carrying out the reaction with the IDR-macromolecule or IDR-polypeptide in the multiple phase-separated aqueous compartments, or to further stimulate or enhance co-localization of molecules necessary for carrying out the reaction with the IDR-macromolecule or IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby increasing the efficiency of the biochemical reaction in the system.

[0008] In any of the methods described herein, the plurality of phase-separated aqueous compartments can be a plurality of detectable phase-separated aqueous particles.

[0009] In a further aspect, the present invention provides a method for conducting a biochemical reaction in an aqueous in vitro reaction system, wherein the biochemical reaction depends on the function of at least one reaction macromolecule, optionally at least one reaction polypeptide, the method comprising introducing at least one polypeptide tagged with an amino acid sequence comprising or consisting of one or more functional intrinsically disordered regions (IDRs) (IDR-polypeptides) into the in vitro reaction system under conditions suitable for conducting the reaction, and maintaining the IDR-polypeptides in the system to cause liquid-liquid demixing and the formation of a plurality of phase-separated aqueous compartments, preferably detectable phase-separated aqueous particles, within the system, and to co-localize molecules necessary for carrying out the reaction with the IDR-polypeptides within the compartments, thereby increasing the efficiency of the biochemical reaction in the system.

[0010] Optionally, in the method according to this additional aspect, the biochemical reaction depends on the function of at least one reactive polypeptide, the reactive polypeptide being at least one IDR-polypeptide, and when introduced into the system, the at least one IDR-polypeptide performs its reactive function in the biochemical reaction and increases the efficiency of the reaction in the system.

[0011] In any of the methods according to this additional aspect, the conditions suitable for conducting the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing liquid-liquid demixing and the formation of multiple phase-separated aqueous compartments caused by the IDR-polypeptide, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, and preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0012] In any of the methods according to this additional aspect, the conditions suitable for conducting the reaction may include providing ATP to the IDR-polypeptide in an in vitro reaction system, thereby further simulating or enhancing IDR-polypeptide-induced liquid-liquid demixing and formation of multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0013] In any of the methods according to this additional embodiment, the conditions suitable for carrying out the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0014] In any of the methods according to this additional aspect, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0015] In any of the methods according to this additional aspect, the efficiency of the reaction in the system may be increased by the IDR-polypeptide compared to the efficiency of the reaction in the system after introduction of at least one polypeptide under the same reaction conditions, except that the at least one polypeptide is not tagged with an amino acid sequence comprising or consisting of one or more functional IDRs.

[0016] The present invention also provides a method for conducting a biochemical reaction in an aqueous in vitro reaction system, wherein the biochemical reaction depends on the function of at least one reactive macromolecule, optionally at least one reactive polypeptide, the method comprising: i. introducing a molecule comprising at least one IDR-macromolecule into a system under conditions suitable for carrying out a reaction, wherein the at least one IDR-macromolecule comprises one or more functional intrinsically disordered regions (IDRs), and preferably the at least one IDR-macromolecule is at least one IDR-polypeptide; ii. maintaining an IDR-macromolecule or IDR-polypeptide in the system to cause liquid-liquid demixing in the system, wherein the liquid-liquid demixing is caused by the IDR-macromolecule or IDR-polypeptide to form a plurality of phase-separated aqueous compartments in the system; iii. maintaining the IDR-macromolecule or IDR-polypeptide in the system so that molecules necessary for carrying out the reaction are co-localized with the IDR-macromolecule or IDR-polypeptide in the compartment; iv. Allowing a biochemical reaction to proceed within the compartment, wherein the efficiency of the biochemical reaction in the system is enhanced by the presence of at least one IDR-macromolecule.

[0017] In the above methods, the biochemical reaction may depend on the function of at least one IDR-macromolecule, optionally at least one IDR-polypeptide, and when introduced into an in vitro reaction system, the at least one IDR-macromolecule or at least one IDR-polypeptide performs its reaction function in the biochemical reaction and increases the efficiency of the reaction. The multiple phase-separated aqueous compartments may be a multiple detectable phase-separated aqueous particles.

[0018] In a further aspect, the present invention provides a method for conducting a biochemical reaction in an aqueous in vitro reaction system, wherein the biochemical reaction depends on the function of at least one reactive macromolecule, optionally at least one reactive polypeptide, the method comprising: i. introducing into the system, under conditions suitable for carrying out a reaction, a molecule comprising at least one polypeptide tagged with an amino acid sequence comprising or consisting of one or more functional intrinsically disordered regions (IDRs) (IDR-polypeptides); ii. maintaining an IDR-polypeptide in the system to cause liquid-liquid demixing and the formation of a plurality of phase-separated aqueous compartments, preferably detectable phase-separated aqueous particles, in the system, wherein the liquid-liquid demixing is caused by the IDR-polypeptide; and iii. maintaining the IDR-polypeptide in the system so that molecules necessary for carrying out the reaction are co-localized with the IDR-polypeptide in the compartment; iv. Allowing a biochemical reaction to proceed within the compartment, wherein the efficiency of the biochemical reaction in the system is increased by the presence of at least one IDR-polypeptide.

[0019] Optionally, in the method according to this further aspect, the biochemical reaction depends on the function of at least one reactive polypeptide, the reactive polypeptide being at least one IDR-polypeptide, and when introduced into the system, the at least one IDR-polypeptide performs its reactive function in the biochemical reaction and increases the efficiency of the reaction in the system.

[0020] In any of the methods according to this further aspect, the conditions suitable for conducting the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing liquid-liquid demixing and the formation of multiple phase-separated aqueous compartments caused by the IDR-polypeptide, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0021] In any of the methods according to this further aspect, the conditions suitable for carrying out the reaction may include providing ATP to the IDR-polypeptide in an in vitro reaction system, thereby further stimulating or enhancing liquid-liquid demixing and formation of multiple phase-separated aqueous compartments caused by the IDR-polypeptide, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0022] In any of the methods according to this further aspect, the conditions suitable for carrying out the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0023] In any of the methods according to this further aspect, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing the molecules required for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0024] In any of the methods according to this further aspect, the conditions suitable for carrying out the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-macromolecule or IDR-polypeptide within the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0025] In any of the methods according to this further aspect, the efficiency of the reaction in the system may be increased by the IDR-polypeptide compared to the efficiency of the reaction in the system after introduction of at least one polypeptide under the same reaction conditions, except that the at least one polypeptide is not tagged with an amino acid sequence comprising or consisting of one or more functional IDRs.

[0026] In any of the above methods, the method may be a biochemical reaction for synthesizing a nucleic acid molecule in an in vitro reaction system; (a) providing at least one nucleic acid primer; (b) providing a target nucleic acid molecule comprising at least one target strand and contacting at least one nucleic acid primer with the target strand, thereby forming a double-stranded structure; (c) providing the IDR-macromolecule as an IDR-polypeptide, wherein the IDR-polypeptide is a polymerase or one or more polypeptide cofactors; (d) allowing the reaction to proceed, thereby extending the 3' end of at least one nucleic acid primer using a polymerase and dNTPs, optionally in the presence of one or more polypeptide cofactors, to generate a double-stranded nucleic acid, wherein a first strand comprises the sequence of the target strand and a second strand comprises the sequence complementary thereto.

[0027] Alternatively, in any of the above methods, the method may be biochemical for amplifying a single-stranded or double-stranded target nucleic acid molecule in an in vitro reaction system, preferably wherein the target nucleic acid molecule is a DNA molecule.

[0028] The method may be a biochemical reaction for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, (a) providing first and second nucleic acid primers; (b) providing a double-stranded target nucleic acid molecule comprising a first strand and a second strand, and contacting first and second nucleic acid primers with the target nucleic acid molecule, thereby forming a first double-stranded structure having the first strand and a second double-stranded structure having the second strand; (c) providing the IDR-macromolecule as an IDR-polypeptide, wherein the IDR-polypeptide is a polymerase or one or more protein cofactors; (d) allowing the reaction to proceed, thereby extending the 3' ends of the first and second nucleic acid primers using a polymerase and dNTPs, optionally in the presence of one or more protein cofactors, to generate first and second double-stranded nucleic acids; (e) repeating steps (b) to (d) until a desired degree of amplification is achieved.

[0029] In the above method for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the method may be a recombinase polymerase amplification (RPA) process for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, (a) providing a recombinase agent, optionally a recombinase loading protein, a single-strand stabilizing agent, a polymerase, first and second nucleic acid primers, a double-stranded target nucleic acid comprising a first strand and a second strand, and optionally an exonuclease, e.g., exonuclease III; (b) contacting a recombinase agent with the first and second nucleic acid primers and, optionally, the recombinase loading protein to form first and second nucleoprotein primers comprising single-stranded regions at their 3' ends; (c) contacting first and second nucleoprotein primers with the target nucleic acid molecule, thereby forming a first double-stranded structure having a first strand and a second double-stranded structure having a second strand; (d) allowing the reaction to proceed, thereby extending the 3' ends of the first and second nucleoprotein primers using a polymerase and dNTPs to generate first and second double-stranded nucleic acids and first and second displaced nucleic acid strands, wherein the single-stranded stabilizing agent stabilizes the first and second displaced strands; (e) continuing the reaction by repeating steps (b) to (d) until a desired degree of amplification is reached; The recombinase agent, and / or the recombinase loading protein, and / or the single-strand stabilizing agent, and / or the polymerase are provided as IDR-polypeptides.

[0030] In the RPA process for amplifying double-stranded target nucleic acid molecules in an in vitro reaction system, the recombinase agents include 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, and Escherichia phage vB_EcoM_DalCa. Preferably, the recombinase agent is selected from the group consisting of UvsX, E. coli RecA, E. coli RadA, E. coli RadB, E. coli Rad51 or any functional analogue, homologue or derivative thereof, and any combination thereof, and is UvsX, more preferably Escherichia phage vB_EcoM_DalCa UvsX.

[0031] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the method can include a recombinase loading protein, wherein the recombinase loading protein is 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; preferably, the recombinase loading protein is UvsY, more preferably Escherichia phage STO UvsY.

[0032] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the polymerase can be a eukaryotic polymerase selected from the group consisting of pol-α, pol-β, pol-δ, pol-ε, or any functional analog, homolog, or derivative thereof, and any combination thereof, including Bacillus stearothermophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, Staphylococcus aureus DNA polymerase I (Saw polymerase), and E. coli DNA polymerase I. The polymerase may be a prokaryotic polymerase selected from the group consisting of 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, or any functional analog, homolog, or derivative thereof, and any combination thereof, and preferably, the polymerase may be Staphylococcus aureus DNA polymerase I (Saw polymerase) or Bacillus subtilis Pol I large fragment (Bsu polymerase). The polymerase may be a bacteriophage polymerase selected from the group consisting of bacteriophage T4 gp43 DNA polymerase, T7 DNA polymerase, and Phi-29 DNA polymerase, or any functional analog, homolog, or derivative thereof, and any combination thereof.

[0033] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the single-stranded stabilizing agent may be selected from the group consisting of Gp32, Escherichia coli (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, preferably the single-stranded stabilizing agent is Gp32 or phage vB_EcoM_NBG1 Gp32.

[0034] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, only the recombinase agent may be provided as an IDR-polypeptide, or only the recombinase loading protein may be provided as an IDR-polypeptide, or only the single-stranded stabilizing agent may be provided as an IDR-polypeptide, or only the polymerase may be provided as an IDR-polypeptide, or only the exonuclease may be provided as an IDR-polypeptide.

[0035] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, one or more functional IDRs of the IDR-polypeptide may be tagged to the IDR-polypeptide as an amino acid sequence comprising or consisting of one or more IDRs, such that the IDR-polypeptide is a genetically engineered fusion protein, and the one or more functional IDRs are located at the C-terminus of the IDR-polypeptide, the N-terminus of the IDR-polypeptide, or both the C-terminus of the IDR-polypeptide and the N-terminus of the IDR-polypeptide, or at any amino acid position along the length of the polypeptide.

[0036] In any one of the above methods, one or more functional IDRs of an IDR-macromolecule or IDR-polypeptide may be characterized as a sequence of amino acids that exhibits a score of greater than 0.5 when analyzed by the algorithm MetaDisorder.

[0037] In any one of the above methods, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of an amino acid sequence comprising one or more repeats of the tripeptide sequence RGG. In any such method, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of an amino acid sequence further comprising one or more repeats of the dipeptide sequence FG. In any such method, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of an amino acid sequence further comprising at least one aromatic amino acid residue consisting of tyrosine or phenylalanine.

[0038] In any one of the above methods, the one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide are i.(YNPQGGYQQ) n (SEQ ID NO: 19), wherein n is a positive integer between 1 and 10, and optionally, n=1, 2, or 3; or ii.(YSPTSPS) n (SEQ ID NO: 124), wherein n is a positive integer from 1 to 10, and optionally, n=1, 2, or 3; or iii.(FSPTSPT) n (SEQ ID NO: 125), wherein n is a positive integer from 1 to 10, and optionally, n=1, 2, or 3; or iv.(YSPTSP-A / N / G) n (SEQ ID NO: 126), wherein n is a positive integer from 1 to 10, and optionally, n=1, 2, or 3; or v.(YSPGSPA) n (SEQ ID NO: 127), where n is a positive integer from 1 to 10, and optionally, n=1, 2, or 3.

[0039] In any one of the above methods, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of a glutamine-rich amino acid sequence, optionally comprising at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive glutamine residues. In any such method, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of an amino acid sequence comprising one or more repeats of the tripeptide sequence QQ ... n (SEQ ID NO: 128), where n is a positive integer from 1 to 10, and optionally n=1, 2, or 3.

[0040] In any one of the above methods, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of a sequence of at least 5 consecutive amino acids of SEQ ID NO:1.

[0041] In any one of the above methods, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise or consist of an amino acid sequence of at least 5 consecutive amino acids of SEQ ID NO:9.

[0042] In any one of the above methods, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise an amino acid sequence comprising one or more aromatic tyrosine residues and one or more phenylalanine residues capable of participating in aromatic cation-π interactions with a polyvalent metal ion, preferably a divalent metal ion.

[0043] In any one of the above methods, one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide may comprise an amino acid sequence containing one or more arginine residues capable of participating in guanidine-metal interactions with polyvalent metal ions, preferably divalent metal ions.

[0044] In any one of the above methods, the IDR-macromolecule or IDR-polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 1 to 43, or may comprise or consist of a functional variant amino acid sequence of SEQ ID NOs: 1 to 43, for example a macromolecule or polypeptide tagged with an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 1 to 43.

[0045] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the IDR-polypeptide may be Gp32, a single-stranded stabilizer having the amino acid sequence of any one of SEQ ID NOs: 65 to 88, or the IDR-polypeptide is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 65 to 88.

[0046] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the IDR-polypeptide may be UvsX and may be a recombinase agent having the amino acid sequence of any one of SEQ ID NOs: 44 to 59, or the IDR-polypeptide is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 44 to 59.

[0047] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the IDR-polypeptide is UvsY, which may be a recombinase-loading protein having the amino acid sequence of any one of SEQ ID NOs: 60 to 64, or the IDR-polypeptide is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 60 to 64.

[0048] In any one of the above methods, the method may further comprise providing a polyvalent metal ion to the IDR-macromolecule or IDR-polypeptide in an in vitro reaction system, thereby further stimulating or enhancing liquid-liquid demixing in the in vitro reaction system, thereby further increasing the efficiency of the biochemical reaction in the system, wherein the polyvalent metal ion further stimulates or enhances the formation of multiple phase-separated aqueous compartments in the system, thereby further increasing the efficiency of the biochemical reaction in the system, preferably the polyvalent metal ion further stimulates or enhances the formation of multiple detectable phase-separated aqueous particles, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. In any such method, the polyvalent metal ion is a divalent metal ion, optionally Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ It could be.

[0049] In any one of the above methods, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing liquid-liquid demixing and formation of multiple phase-separated aqueous compartments caused by the IDR-macromolecule or IDR-polypeptide, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0050] In any one of the above methods, the conditions suitable for carrying out the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-macromolecule or IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0051] In any one of the above methods, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing the molecules required for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0052] In any one of the above methods, the biochemical reaction may be carried out in a solid-phase reaction system comprising a surface. In any such method, the biochemical reaction may be a method of amplifying a single-stranded or double-stranded target nucleic acid molecule in an in vitro reaction system as described above, wherein at least one nucleic acid primer and / or IDR-macromolecule and / or one or more polypeptide cofactors are bound to the surface.

[0053] In any one of the above RPA processes for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the reaction may be carried out in a solid-phase reaction system comprising a surface, wherein the recombinase agent and / or recombinase loading protein and / or single-stranded stabilizing agent and / or polymerase and / or exonuclease and / or first nucleic acid primer and / or second nucleic acid primer are bound to the surface, preferably, (i) the first nucleic acid primer or the second nucleic acid primer are bound to the surface, or (ii) both the first and second nucleic acid primers are bound to the surface.

[0054] In any of the above methods carried out in a solid-phase reaction system comprising a surface, the surface may be flat or may be a microbead, preferably the surface comprises silicon, glass, a gel-based material and / or a polymeric material such as polystyrene, more preferably the surface is a microbead comprising a polymeric material such as polystyrene. In any such method, the surface may be attached to a substrate, preferably the surface is flat and / or the substrate comprises glass. The surface, e.g., a flat surface and / or substrate, may be provided as a flow cell.

[0055] The present invention provides methods for conducting biochemical reactions in cells in culture by introducing at least one of any of the above IDR-macromolecules or at least one of any of the above IDR-polypeptides into the cultured host cells, or by expressing at least one of any of the above IDR-polypeptides in the cultured host cells, to increase the efficiency of the biochemical reactions in the cultured host cells.

[0056] Any of the above methods for conducting an in vitro biochemical reaction may include a biochemical reaction carried out within cells in culture, such as by introducing at least one IDR-macromolecule or at least one IDR-polypeptide into the cultured host cells, or by expressing at least one IDR-polypeptide in the cultured host cells, to increase the efficiency of the biochemical reaction within the cultured host cells.

[0057] The biochemical reaction can be any reaction that results in the manipulation of a nucleic acid molecule in a cultured host cell or that results in a change in a nucleic acid molecule in a cultured host cell, for example, a change in the structure of the nucleic acid molecule, for example, a change in the nucleotide sequence of the nucleic acid molecule. The biochemical reaction can be any reaction that results in the synthesis of a nucleic acid molecule in a cultured host cell. The biochemical reaction can be any reaction that results in the expression of a polypeptide from a nucleic acid molecule. The biochemical reaction can be any reaction that results in the editing of a nucleic acid sequence in a cultured host cell, for example, an IDR polypeptide is a CRISPR polypeptide, such as a Cas polypeptide, including a Cas9 polypeptide. The biochemical reaction can be any reaction that results in the cleavage of a nucleic acid in a cultured host cell. The biochemical reaction can be any reaction that results in the homologous recombination of a nucleic acid in a cultured host cell. The biochemical reaction can be a metabolic reaction in a cultured host cell to produce one or more biological products of interest in the cultured host cell, or to produce one or more biological products of interest that are secreted from the cultured host cell or otherwise released into the medium from the cultured host cell.

[0058] In any one of the above methods, increasing the efficiency of a biochemical reaction can include increasing the efficiency of a reaction using at least one IDR-macromolecule or at least one IDR-polypeptide compared to the efficiency of the reaction obtained by performing the reaction under the same conditions, but wherein the associated at least one macromolecule or at least one polypeptide does not comprise or is not tagged with one or more functional intrinsically disordered region polypeptide sequences, and optionally, the reaction is performed in the absence of exogenously added crowding agents.

[0059] In any one of the above RPA processes, increasing or enhancing the efficiency or performance of an RPA biochemical reaction can include increasing the amount of amplification product obtained in an RPA reaction using at least one IDR-polypeptide compared to the amount of amplification product obtained by performing the reaction under the same conditions, but wherein the at least one associated polypeptide is not tagged with one or more functional intrinsically disordered region polypeptide sequences, and optionally, the reaction is performed in the absence of exogenously added crowding agents.

[0060] In any one of the above methods comprising introducing at least one IDR-macromolecule or IDR-polypeptide into an in vitro reaction system, the efficiency of the reaction in the system is increased by the IDR-macromolecule or IDR-polypeptide compared to the efficiency of the reaction in the system after introduction of the at least one macromolecule or polypeptide under the same reaction conditions, except that the at least one macromolecule or polypeptide does not contain one or more functional intrinsically disordered regions (IDRs).

[0061] In any one of the above methods comprising introducing at least one polypeptide tagged with an amino acid sequence comprising or consisting of at least one functional intrinsically disordered region (IDR) (IDR-polypeptide) into an in vitro reaction system, the efficiency of the reaction in the system is increased by the IDR-polypeptide compared to the efficiency of the reaction in the system after introduction of the at least one polypeptide under the same reaction conditions, except that the at least one polypeptide is not tagged with an amino acid sequence comprising or consisting of one or more functional IDRs.

[0062] The present invention also provides non-naturally occurring IDR-macromolecules comprising a macromolecule and a tag amino acid sequence, wherein the tag amino acid sequence comprises or consists of one or more functional intrinsically disordered regions (IDRs), and the IDR-macromolecules are capable of causing liquid-liquid demixing in an aqueous in vitro reaction system. Any such IDR-macromolecules may be capable of causing liquid-liquid demixing and the formation of multiple phase-separated aqueous compartments within the system, preferably multiple detectable phase-separated aqueous particles. Any such liquid-liquid demixing caused by any such non-naturally occurring IDR-macromolecules in an in vitro reaction system may thereby enhance the efficiency of a biochemical reaction.

[0063] Any one of the above IDR-macromolecules can be a non-naturally occurring, artificial or engineered IDR-macromolecule or IDR-polypeptide comprising a macromolecule or polypeptide and a tag amino acid sequence. In the case of an IDR-polypeptide, the tag amino acid sequence can be located at the C-terminus of the polypeptide, the N-terminus of the polypeptide, or both the C-terminus of the polypeptide and the N-terminus of the polypeptide, or at any amino acid position along the length of the polypeptide.

[0064] In any one of the above IDR-macromolecules or IDR-polypeptides, one or more functional IDRs of the tag amino acid sequence are functional IDRs according to any one of the above methods.

[0065] In any one of the above IDR-macromolecules or IDR-polypeptides, the tag sequence is cation-reactive with a polyvalent metal cation, preferably a divalent metal cation, more preferably Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ ions, even more preferably Mg 2+ , Mn 2+ or Ca 2+ , and even more preferably Mg 2+ It contains amino acid residues that can participate in aromatic cation-π interactions with

[0066] In any one of the above IDR-macromolecules or IDR-polypeptides, the IDR-macromolecule or IDR-polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 43, or comprises or consists of a functional variant amino acid sequence of SEQ ID NOs: 1 to 43, for example, a macromolecule or polypeptide tagged with an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 1 to 43.

[0067] In any one of the above IDR-polypeptides, the polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs can be an enzyme, such as a helicase, a gyrase, a recombinase, e.g., an RPA recombinase agent, a nuclease, e.g., an exonuclease and an endonuclease, a ligase, a glycolyase, a methylase, a methyltransferase, a glucosyltransferase, a polymerase, a kinase, a phosphatase, a gene editing enzyme such as a CRISPR enzyme, e.g., a Cas9 enzyme; a cofactor, e.g., an RPA recombinase loading protein and an RPA single-strand stabilizer. The polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs can be a ligase, optionally RB69 ligase, e.g., RB69 ligase-His2 (SEQ ID NO: 112). The polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs may be an RPA single-stranded stabilizer, preferably Gp32, and optionally the IDR-polypeptide has the amino acid sequence of any one of SEQ ID NOs: 65 to 88 and 120, or the IDR-polypeptide may be a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 65 to 88 and 120. The polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs may be an RPA recombinase agent, preferably UvsX, and optionally the IDR-polypeptide has the amino acid sequence of any one of SEQ ID NOs: 44 to 59, or the IDR-polypeptide is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 44 to 59. The polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs may be an RPA recombinase loading protein, preferably UvsY, and optionally the IDR-polypeptide has the amino acid sequence of any one of SEQ ID NOs: 60 to 64, or is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 60 to 64.

[0068] The present invention also provides isolated nucleic acid molecules comprising a first nucleic acid sequence encoding any of the above-described IDR-polypeptides, and optionally a second nucleic acid sequence encoding a promoter, wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence. The present invention also provides recombinant polynucleotide expression vectors comprising any such nucleic acid molecule. The present invention also provides host cells comprising any such nucleic acid molecule or any such recombinant polynucleotide expression vector. The present invention also provides cell cultures comprising a growth medium and a population of any such host cells.

[0069] The present invention also provides kits comprising any of the non-naturally occurring IDR macromolecules or IDR polypeptides described above. Any such kit may further comprise additional RPA components, including an RPA recombinase agent, and / or an RPA recombinase loading protein, and / or a polymerase, and / or first and second nucleic acid primers, and / or an exonuclease, and / or a buffer, and / or a source of polyvalent metal ions, preferably divalent metal cations. In any such kit, all components may be provided in lyophilized form.

[0070] The present invention also provides methods for stimulating or enhancing liquid-liquid demixing in a solution, the methods comprising providing a solution containing any of the above IDR-macromolecules or any of the above IDR-polypeptides, and contacting the IDR-macromolecules or IDR-polypeptides in the solution with a polyvalent metal ion, whereby liquid-liquid demixing in the solution is stimulated or enhanced. The present invention also provides additional methods for stimulating or enhancing liquid-liquid demixing caused by IDR-macromolecules or IDR-polypeptides in an aqueous in vitro reaction system, the methods comprising providing any of the above IDR-macromolecules or any of the above IDR-polypeptides to the system, providing a polyvalent metal ion to the system, and allowing the IDR-macromolecules or IDR-polypeptides to contact the polyvalent metal ion, whereby liquid-liquid demixing caused by the IDR-macromolecules or IDR-polypeptides in the solution is stimulated or enhanced. In any such method, liquid-liquid demixing can result in the formation of multiple phase-separated aqueous compartments in the solution, preferably multiple detectable phase-separated aqueous particles. In any such method, the polyvalent metal ion is a divalent metal ion, optionally Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ In any such further method, the polyvalent metal ion may engage in aromatic cation-π interactions with amino acid residues in one or more functional IDR amino acid sequences, thereby promoting liquid-liquid demixing.

[0071] In any such further method, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing liquid-liquid demixing and formation of multiple phase-separated aqueous compartments caused by the IDR-macromolecule or IDR-polypeptide, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0072] In any such further method, the conditions suitable for carrying out the reaction may further comprise providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-macromolecule or IDR-polypeptide within the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0073] In any such further method, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing the molecules necessary for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0074] The present invention also provides the use of a polyvalent metal ion in stimulating or enhancing liquid-liquid demixing in a solution, wherein the demixing is mediated by any one of the above IDR-macromolecules or any one of the above IDR-polypeptides. The present invention also provides the use of a polyvalent metal ion in stimulating or enhancing liquid-liquid demixing in an aqueous in vitro reaction system caused by an IDR-macromolecule or IDR-polypeptide introduced into the system, wherein the IDR-macromolecule or IDR-polypeptide is any one of the above IDR-macromolecules or any one of the above IDR-polypeptides. In any such use, the liquid-liquid demixing can result in the formation of multiple phase-separated aqueous compartments, preferably multiple detectable phase-separated aqueous particles in the solution, caused by the IDR-macromolecule or IDR-polypeptide. In any such use, the polyvalent metal ion can be a divalent metal ion, optionally Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ In any such use, the polyvalent metal ion may participate in aromatic cation-π interactions with amino acid residues in one or more functional IDR amino acid sequences, thereby promoting liquid-liquid demixing.

[0075] In any such use, the conditions suitable for carrying out the reaction may further include providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing liquid-liquid demixing and formation of multiple phase-separated aqueous compartments caused by the IDR-macromolecule or IDR-polypeptide, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0076] In any such use, the conditions suitable for carrying out the reaction may further include providing a polyvalent metal ion to the IDR-polypeptide, thereby further stimulating or enhancing molecules necessary for carrying out the reaction to co-localize with the IDR-macromolecule or IDR-polypeptide within the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, optionally wherein the polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein the polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM. The polyvalent metal ion may be a divalent metal ion, optionally including Mg. 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ , preferably Mg 2+ , Mn 2+ or Ca 2+ , more preferably Mg 2+ may be.

[0077] In any such use, the conditions suitable for carrying out the reaction may further comprise providing ATP to the IDR-macromolecule or IDR-polypeptide in the in vitro reaction system, thereby further stimulating or enhancing the molecules necessary for carrying out the reaction to co-localize with the IDR-polypeptide in the multiple phase-separated aqueous compartments, thereby further increasing the efficiency of the biochemical reaction in the system, wherein ATP is provided in the system at a concentration of 1 mM to 3.5 mM, optionally 1 mM to 2 mM, preferably 1 mM.

[0078] The present invention also provides any one of the above non-naturally occurring IDR-macromolecules or any one of the above IDR-polypeptides for use in therapy, for use as a drug, for use as a pharmaceutical, for use as a diagnostic method, or for use as a diagnostic agent.

[0079] The present invention also provides a method of making any one of the above non-naturally occurring IDR-macromolecules or any one of the above IDR-polypeptides, comprising providing a macromolecule, optionally a polypeptide, and tagging the macromolecule or polypeptide with one or more functional intrinsically disordered region polypeptide sequences. The tagging can be performed by any means described and defined herein. The one or more functional intrinsically disordered region polypeptide sequences can be any of the same as those described and defined herein. The macromolecule or polypeptide can be any suitable macromolecule or polypeptide, including any of the macromolecules or polypeptides described and defined herein.

[0080] Any one of the above IDR-macromolecules or IDR-polypeptides can increase the efficiency of a biochemical reaction. Increasing the efficiency of a biochemical reaction can include increasing the efficiency of a reaction using an IDR-macromolecule or IDR-polypeptide compared to the efficiency of the reaction obtained by performing the reaction under the same conditions, but where the related macromolecule or related polypeptide does not contain or is not tagged with one or more functional intrinsically disordered region polypeptide sequences, and optionally, the reaction is performed in the absence of exogenously added crowding agents.

[0081] Any one of the above IDR-macromolecules or IDR-polypeptides can increase the efficiency of a biochemical reaction in a recombinase polymerase amplification (RPA) reaction. Increasing the efficiency or performance of an RPA biochemical reaction can include increasing the amount of amplification product obtained in an RPA reaction using an IDR-polypeptide compared to the amount of amplification product obtained by performing the reaction under the same conditions, but the relevant polypeptide does not contain or is not tagged with one or more functional intrinsically disordered region polypeptide sequences, and optionally, the RPA reaction is performed in the absence of exogenously added crowding agents.

[0082] The present invention also provides a method for determining the nucleotide sequence of one or more target polynucleotide molecules, comprising: (i) performing the above method to amplify one or more target polynucleotide molecules, thereby obtaining a population comprising multiple copies of one or more target polynucleotide molecules; (ii) performing one or more nucleic acid sequencing reactions on the population comprising multiple copies of the target polynucleotide molecule; Preferably, the method is carried out in a solid phase reaction system comprising a surface.

[0083] The present invention also provides the use of any one of the above IDR-macromolecules or any one of the above IDR-polypeptides in a method for determining the nucleotide sequence of one or more target polynucleotide molecules, preferably the method is as described above.

[0084] The present invention also provides a polypeptide or isolated polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOS: 1-43, or a functional variant amino acid sequence of any one of SEQ ID NOS: 1-43, e.g., having 80% or greater identity to any one of SEQ ID NOS: 1-43. Any such polypeptide can be attached / tagged to a macromolecule or polypeptide to form an IDR-tagged macromolecule or IDR-tagged polypeptide, as further described herein. The tagged macromolecule or polypeptide can be a macromolecule or polypeptide necessary for carrying out a biochemical reaction in an aqueous reaction system. When maintained in an aqueous reaction system under conditions for carrying out a biochemical reaction, any such IDR-tagged macromolecule or IDR-tagged polypeptide can cause liquid-liquid demixing caused by the amino acid sequence of any one of SEQ ID NOS: 1-43 or any functional variant thereof, and the formation of multiple phase-separated aqueous compartments, preferably multiple detectable phase-separated aqueous particles, within the system, thereby increasing the efficiency of the biochemical reaction in the system. When maintained in an aqueous reaction system under conditions for carrying out a biochemical reaction, any such IDR-tagged macromolecule or IDR-tagged polypeptide will cause molecules necessary for carrying out the reaction to be co-localized with the IDR-macromolecule or IDR-polypeptide in multiple phase-separated aqueous compartments, thereby increasing the efficiency of the biochemical reaction in the system.

[0085] The aqueous reaction system may be an aqueous in vitro reaction system.

[0086] The present invention also provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having 80% or more identity to any one of SEQ ID NOs: 1 to 43, or comprising or consisting of an amino acid sequence of a functional variant of SEQ ID NOs: 1 to 43, for example.

[0087] The present invention also provides the use of an IDR moiety, which is a polypeptide comprising or consisting of one or more functional intrinsically disordered regions (IDRs), in producing an IDR-tagged macromolecule or IDR-tagged polypeptide in which the IDR moiety is attached / tagged to the macromolecule or polypeptide, wherein the tagged macromolecule or polypeptide is a macromolecule or polypeptide necessary for carrying out a biochemical reaction in an aqueous reaction system, and when maintained in the aqueous reaction system under conditions for carrying out a biochemical reaction, the IDR-tagged macromolecule or IDR-tagged polypeptide causes IDR moiety-induced liquid-liquid demixing, resulting in the formation of multiple phase-separated aqueous compartments, preferably multiple detectable phase-separated aqueous particles, within the system, thereby increasing the efficiency of the biochemical reaction in the system. When maintained in the aqueous reaction system under conditions for carrying out a biochemical reaction, any such IDR-tagged macromolecule or IDR-tagged polypeptide causes molecules necessary for carrying out the reaction to colocalize with the IDR-tagged macromolecule or IDR-tagged polypeptide within the multiple phase-separated aqueous compartments, thereby increasing the efficiency of the biochemical reaction in the system.

[0088] Preferably, the IDR portion is attached / tagged to the polypeptide, thereby producing an IDR-tagged polypeptide, which is preferably produced as a recombinant gene fusion protein.

[0089] Preferably, the IDR portion is a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 43, or a polypeptide comprising or consisting of a functional variant amino acid sequence of any one of SEQ ID NOs: 1 to 43, for example, a polypeptide having 80% or more identity to any one of SEQ ID NOs: 1 to 43.

[0090] Any of the above IDR-tagged macromolecules or IDR-tagged polypeptides may be defined as non-naturally occurring, artificial, or genetically engineered macromolecules or polypeptides.

[0091] Any of the above IDR-tagged macromolecules or IDR-tagged polypeptides may further possess any one or more characteristic properties of the IDR-macromolecules or IDR-polypeptides described and defined herein.

[0092] The aqueous reaction system may be an aqueous in vitro reaction system.

[0093] The present invention further provides an IDR-tagged macromolecule or an IDR-tagged polypeptide obtained according to any of the above uses.

[0094] The present invention also provides a method for producing an IDR-tagged macromolecule or IDR-tagged polypeptide, comprising providing a macromolecule or polypeptide and attaching / tagging it with an IDR moiety, where the IDR moiety is a polypeptide containing or consisting of one or more functional intrinsically disordered regions (IDRs), wherein the tagged macromolecule or polypeptide is a macromolecule or polypeptide necessary for carrying out a biochemical reaction in an aqueous reaction system, and wherein, when maintained in the aqueous reaction system under conditions for carrying out a biochemical reaction, the IDR-tagged macromolecule or IDR-tagged polypeptide causes liquid-liquid demixing induced by the IDR moiety, resulting in the formation of multiple phase-separated aqueous compartments, preferably multiple detectable phase-separated aqueous particles, within the system, thereby increasing the efficiency of the biochemical reaction in the system. When maintained in the aqueous reaction system under conditions for carrying out a biochemical reaction, any such IDR-tagged macromolecule or IDR-tagged polypeptide will cause molecules necessary for carrying out the reaction to colocalize with the IDR-tagged macromolecule or IDR-tagged polypeptide within the multiple phase-separated aqueous compartments, thereby increasing the efficiency of the biochemical reaction in the system.

[0095] Preferably, the method comprises providing a polypeptide and attaching / tagging it with an IDR moiety to produce an IDR-tagged polypeptide, which is preferably produced as a recombinant gene fusion protein.

[0096] Preferably, the IDR portion is a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 43, or a polypeptide comprising or consisting of a functional variant amino acid sequence of any one of SEQ ID NOs: 1 to 43, for example, a polypeptide having 80% or more identity to any one of SEQ ID NOs: 1 to 43.

[0097] Any of the above IDR-tagged macromolecules or IDR-tagged polypeptides may be defined as non-naturally occurring, artificial, or genetically engineered macromolecules or polypeptides.

[0098] Any of the above IDR-tagged macromolecules or IDR-tagged polypeptides may further possess any one or more characteristic properties of the IDR-macromolecules or IDR-polypeptides described and defined herein.

[0099] The aqueous reaction system may be an aqueous in vitro reaction system.

[0100] The present invention further provides an IDR-tagged macromolecule or an IDR-tagged polypeptide obtainable by any of the above methods. [Brief explanation of the drawings]

[0101] [Figure 1] 1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HIS2) at various template nucleic acid concentrations. [Figure 2] 1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HIS5) at various template nucleic acid concentrations. [Figure 3] 1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HRP1) at various template nucleic acid concentrations. [Figure 4]1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-Sup1) at various template nucleic acid concentrations. [Figure 5] Figure 5 shows real-time recombinase polymerase amplification traces using IDR-tagged Gp32 fusion proteins (Gp32-Sup2) at various template nucleic acid concentrations. The experiments shown in Figures 5A, 5B, 5C, and 5D use Gp32 fusion proteins with one, two, three, and four Sup2 IDR repeats, respectively. [Figure 6] Figure 1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HIS5) at various MgOAc concentrations. [Figure 7] Figure 1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HIS2) at various phosphocreatine concentrations. [Figure 8] Figure 1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HRP1) at various KOAc concentrations. [Figure 9] Figure 1 shows real-time recombinase polymerase amplification traces using Gp32 tagged with seven histidine residues (for protein purification purposes, i.e., no IDR tag) compared to an IDR-tagged Gp32 fusion protein (Gp32-Sup1) either in the presence or absence of a crowding agent (PEG). [Figure 10]Figure 10 shows the effect of multivalent metal cations on promoting phase separation (particle formation) mediated by IDR amino acid sequence tags in the absence of crowding agents. The IDR amino acid sequence was tagged onto Gp32 protein to generate Gp32-HIS2 fusion proteins (Figure 10A), Gp32-HRP1 fusion proteins (Figure 10B), Gp32-Sup1 fusion proteins (Figure 10C), and Gp32-Fib fusion proteins (Figure 10D). In each case, the effects of representative concentrations of divalent metal cations, namely magnesium (MgOAc), manganese (MgCl), and calcium (CaCl), were tested. [Figure 11] The effect of multivalent metal cations on promoting phase separation (particle formation) mediated by IDR amino acid sequence tags in the absence of crowding agents is shown. Gp32 proteins were tagged with IDR amino acid sequences to generate Gp32-Fib fusion proteins (Figure 11A), Gp32-Sup1 fusion proteins (Figure 11B), Gp32-HIS2 fusion proteins (Figure 11C), Gp32-HRP1 fusion proteins (Figure 11D), and Gp32-HIS5 fusion proteins (Figure 11E). In each case, the effects of representative concentrations of divalent metal cations, namely magnesium (MgOAc), manganese (MgCl), and calcium (CaCl), were tested. [Figure 12] FIG. 1 shows the effect of a divalent metal cation, namely magnesium (MgOAc), on its ability to promote phase separation (particle formation) of an IDR-tagged Gp32 fusion protein (Gp32-HRP1) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. [Figure 13] FIG. 1 shows the effect of a divalent metal cation, namely magnesium (MgOAc), on its ability to promote phase separation (particle formation) of an IDR-tagged Gp32 fusion protein (Gp32-HIS2) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. [Figure 14]We show the effect of varying concentrations of a divalent metal cation, namely magnesium (MgOAc), on the ability of an IDR-tagged Gp32 fusion protein (Gp32-HRP1) to promote phase separation (particle formation) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. [Figure 15] We show the effect of varying concentrations of a divalent metal cation, namely magnesium (MgOAc), on the ability of an IDR-tagged Gp32 fusion protein (Gp32-HIS2) to promote phase separation (particle formation) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. [Figure 16] Figure 16 shows the effect of adding a divalent metal cation, magnesium (MgOAc), on the ability of an IDR-tagged Gp32 fusion protein (Gp32-HRP1) to promote phase separation in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. Phase separation is demonstrated by the formation of an opaque solution after addition of MgOAc, resulting in particle formation (Figure 16A), which is further demonstrated by particle pelleting (Figure 16B). RPA protein components are demonstrated to associate with the particles, as revealed by SDS-PAGE analysis of the pelleted material. [Figure 17] Figure 1 shows real-time recombinase polymerase amplification traces using a native Gp32 fusion protein in either the presence or absence of a crowding agent. The experiment demonstrates that Gp32 not tagged with an amino acid sequence containing an intrinsically disordered region (IDR) is unable to mediate amplification in the absence of a crowding agent. [Figure 18A] FIG. 1 is a schematic depicting a reaction mixture set up for real-time amplification using dual primer beads. [Figure 18B] FIG. 1 is a schematic depicting amplification products in a real-time reaction. [Figure 18C] FIG. 1 is a schematic diagram illustrating the evaluation of amplification characteristics in an end-point reaction. [Figure 18D]1 shows real-time recombinase polymerase amplification traces using an IDR-tagged Gp32 fusion protein (Gp32-HIS2) with primers bound to a solid surface or free in solution. [Figure 18E] 1 shows an end-point recombinase polymerase amplification trace using an IDR-tagged Gp32 fusion protein (Gp32-HIS2) using primers bound to a solid surface. [Figure 18F] 1 shows an end-point recombinase polymerase amplification trace using an IDR-tagged Gp32 fusion protein (Gp32-HIS2) using primers bound to a solid surface. [Figure 19] Disorder profiles generated using the MetaDisorder software program for Gp32 (Figure 19A), UvsY (Figure 19B), and UvsX (Figure 19C) are shown. [Figure 20] We show the effect of varying concentrations of a divalent metal cation, i.e., magnesium (MgCl), on the ability of an IDR-tagged RB69 ligase fusion protein (RB69 ligase-HIS2) to promote phase separation (particle formation) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. [Figure 21] 1 shows the ligase activity performance of an IDR-tagged RB69 ligase fusion protein (RB69 ligase-HIS2) in an exemplary in vitro biochemical reaction environment in the absence of crowding agents. [Figure 22] FIG. 1 shows the ligase activity performance of an IDR-tagged RB69 ligase fusion protein (RB69 ligase-HIS2) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent, compared to untagged RB69 ligase and T4 DNA ligase. [Figure 23] FIG. 1 shows the ligase activity performance of an IDR-tagged RB69 ligase fusion protein (RB69 ligase-HIS2) in an exemplary in vitro biochemical reaction environment in the absence of crowding agents compared to NEBNext Ultra II Ligation Master Mix. [Figure 24] Figure 1 shows the effect of ATP on the ability of an IDR-tagged RB69 ligase fusion protein (RB69 ligase-HIS2) to promote phase separation (particle formation) in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent. [Figure 25] Brightfield and fluorescent images of representative sections of FlexWell™ chambers are shown in which 0, 5, 10, 20, 40, or 80 copies of single-stranded UP1-UP2'-TF1L template per bead were annealed for 1 hour at 50°C and then amplified by recombinase polymerase amplification using an IDR-tagged Gp32 fusion protein (Gp32-Hrp1) with primers bound to the solid surface in the absence of crowding agents such as PEG. Amplification was detected by nicking the amplicon with Nt and extension of the nick with BbvCI and aminoallyl-dUTP-XX-ATTO-594. No fluorescence was observed with beads to which no template had been added, and increasing amounts of fluorescence were observed with beads to which increasing amounts of template had been annealed. This indicates that amplification occurred on the solid surface of the beads in the absence of crowding agents. [Figure 26A] 1 shows bright-field and fluorescent images demonstrating the formation of phase-separated aqueous particles mediated by IDR-tagged Gp32 fusion protein (Gp32-Hrp1). [Figure 26B] 1 shows plots demonstrating the increased efficiency of the reaction (rate of nucleic acid cleavage by Cas12a) upon formation of phase-separated aqueous particles mediated by Gp32-Hrp1. DETAILED DESCRIPTION OF THE INVENTION

[0102] Recombinase polymerase amplification is a technique for amplifying nucleic acid molecules. This system utilizes, among other things, a recombinase enzyme, preferably a recombinase loading protein. These protein components form a complex with the amplification primers. After binding to the target nucleic acid molecule to be amplified, the complex "scans" the target nucleic acid molecule, "searching" for a region of complementarity between the target and primer sequences. If a complementary region is found, the complex promotes binding of the primer to the target sequence. A polymerase enzyme can then extend the primer to generate a copy of the target sequence. The use of a recombinase complex offers a key difference from other nucleic acid amplification methods, such as PCR. In RPA, the recombinase complex provides an entirely enzyme-based solution to the primer binding problem, eliminating the need for thermal cycling-driven melting and annealing steps. Therefore, RPA is an isothermal technique. The lack of the need for extreme thermal cycling means that RPA has many distinct advantages over techniques such as PCR.

[0103] A well-documented requirement in the RPA method is the presence of a "crowding agent," also referred to in the art as a "macromolecular crowding agent." These agents are well known in the art and have their art-understood meanings. Crowding agents are discussed in more detail herein. One of the most commonly used crowding agents in the RPA method is polyethylene glycol (PEG), although other crowding agents can also be used. Prior to the present invention, the use of a crowding agent was considered an essential requirement in the RPA method.

[0104] The present inventors have surprisingly discovered that it is possible to circumvent the previously recognized important requirement for crowding agents in RPA methods, and the present invention is based on this discovery.

[0105] The present inventors have surprisingly discovered that by "tagging" macromolecules, such as protein components required for RPA, with amino acid sequences containing or consisting of one or more functional "intrinsically disordered regions" (IDRs), the IDR amino acid sequence tags can promote efficient RPA in the complete absence of crowding agents. Thus, RPA systems can achieve efficient amplification without relying on crowding agents, thereby reducing the complexity of the RPA reaction.

[0106] The inventors have also surprisingly discovered that the efficiency of amplification in RPA involving IDR-tagged macromolecular components in the absence of crowding agents can be correlated with the functional ability of the IDR amino acid tag sequence to promote liquid-liquid demixing, resulting in phase separation in a biochemical reaction system. Phase separation can be assessed by the formation of phase-separated aqueous compartments, particularly sphere-like aqueous spherical foci or phase-separated aqueous particles, in the biochemical reaction environment, suitable for detection by standard methods, including microscopy, as further described herein.

[0107] Furthermore, the present inventors have also surprisingly discovered that the provision of an IDR-tagged macromolecular component and a crowding agent can provide additive and even synergistic effects on the efficiency of amplification in RPA methods.

[0108] Furthermore, the inventors surprisingly discovered that the efficiency of amplification in RPA involving IDR amino acid-tagged macromolecular components in the absence of crowding agents can be correlated with the concentration of polyvalent metal cations introduced into the biochemical reaction environment. Thus, polyvalent metal cations can further stimulate or enhance liquid-liquid demixing induced by IDR-macromolecules or IDR-polypeptides, thereby further increasing the reaction efficiency.

[0109] The inventors have also surprisingly discovered, as further described herein, that certain concentrations of ATP can further stimulate or enhance the liquid-liquid demixing caused by the IDR-macromolecule or IDR-polypeptide, thereby further increasing the reaction efficiency.

[0110] Based on these surprising discoveries, the present invention provides methods and reagents for increasing the efficiency of enzyme-based in vitro biochemical reactions, including RPA reactions, as further described herein.

[0111] The IDR amino acid sequences and IDR reagents described and defined herein have broad applicability as useful reagents applied to any suitable macromolecular component of a biochemical reaction, such as a polypeptide, thereby facilitating liquid-liquid demixing and promotion of phase separation in a biochemical reaction environment, without reliance on macromolecular crowding agents, particularly when the IDR amino acid sequences are used in conjunction with polyvalent metal cations. The present invention further encompasses the use of polyvalent metal cations, such as divalent metal cations, or any functional equivalent thereof, in promoting IDR amino acid sequence-mediated phase separation in a biochemical reaction environment, without reliance on macromolecular crowding agents.

[0112] Accordingly, the present invention provides IDR-based methods, macromolecules, polypeptides, nucleic acids, vectors, host cells and uses as described and defined herein.

[0113] The elements of the present invention are described in order below.

[0114] biochemical reactions As explained above, the present inventors have surprisingly discovered that it is possible to circumvent the need for crowding agents, previously thought to be essential components of RPA and other reactions. As described in detail herein, this can be achieved by attaching / tethering / tagging a protein component required for the RPA reaction with an amino acid sequence containing one or more functional intrinsically disordered regions (IDRs). The present inventors have also surprisingly shown that functional intrinsically disordered regions attached to a ligase enzyme can enhance the efficiency of the ligase reaction. The present inventors have shown that the degree of phase separation induced by IDR amino acid sequences can correlate with the efficiency of the reaction, e.g., amplification, in the absence of crowding agents and can be enhanced with multivalent metal cations. Based on these surprising observations, it is plausible to predict that such IDR amino acid sequences associated with macromolecular or protein components of biochemical reactions will plausibly improve the efficiency of the reaction in vitro or in vivo biochemical reaction environments, particularly in the absence of added / exogenous crowding agents.

[0115] Thus, the present invention encompasses the use of any of the IDR amino acid sequences described and defined herein applied to any suitable macromolecular or polypeptide component of an in vitro or in vivo biochemical reaction, thus providing IDR reagents that can promote liquid-liquid demixing in the biochemical reaction environment and increase the efficiency of the biochemical reaction. Such liquid-liquid demixing in the biochemical reaction environment can result in phase separation of the biochemical reaction environment. Such liquid-liquid demixing in the biochemical reaction environment can result in phase separation that results in, causes, or promotes the formation of phase-separated aqueous compartments containing detectable phase-separated aqueous particles in the biochemical reaction environment, as further described herein. Such IDR reagents or IDR-based reagents as further described and defined herein may be referred to interchangeably to describe any one or more of an IDR-macromolecule or IDR-tagged macromolecule, or an IDR-polypeptide or IDR-tagged polypeptide.

[0116] In any one of the methods, processes, and uses, or in any one of the non-naturally occurring IDR-macromolecules, IDR-fusion macromolecules, or isolated nucleic acid molecules, recombinant polynucleotide expression vectors, or host cells encoding same, as described and defined herein, increasing or enhancing the efficiency or performance of a biochemical reaction can include increasing the efficiency of the reaction using any one or more of the IDR-based macromolecules or polypeptides described herein compared to the efficiency obtained by performing the reaction under the same conditions, but where the relevant macromolecule or polypeptide does not comprise or is not tagged with one or more functional intrinsically disordered region polypeptide sequences, and optionally, the reaction is performed in the absence of exogenously added crowding agents.

[0117] Increasing or enhancing the efficiency or performance of a biochemical reaction should be understood in accordance with generally accepted concepts. For example, reaction efficiency in an RPA reaction or any other nucleic acid amplification reaction can be understood as providing an equivalent total population of amplicons using relatively less starting target nucleic acid, or providing a relatively faster detection time or a relatively faster amplification rate using the same amount of starting target nucleic acid.

[0118] Increasing or enhancing the efficiency or performance of an RPA biochemical reaction can include increasing the amount of amplification product obtained in an RPA reaction using any one or more of the IDR-based macromolecules or polypeptides described herein compared to the amount of amplification product obtained by performing the reaction under the same conditions, but where the relevant macromolecule or polypeptide does not comprise or is not tagged with one or more functional intrinsically disordered region polypeptide sequences, and optionally, the reaction is performed in the absence of exogenously added crowding agents.

[0119] Increasing the efficiency of a biochemical reaction in a reaction system, such as an in vitro reaction system, can include increasing any measurable parameter of the reaction in the system over a specified period of time, such as the rate of the reaction over a period of time, the amount of substrate consumed over a period of time, the amount of product produced over a period of time, etc.

[0120] Increasing the efficiency of a biochemical reaction in a reaction system, such as an in vitro reaction system, can include increasing a parameter of the reaction within a detectable phase-separated compartment, e.g., a detectable phase-separated aqueous particle, which can be inferred indirectly, for example, by measuring the parameter of the reaction and correlating the increase with the formation of a detectable phase-separated aqueous particle and / or detectable colocalization of reactant molecules to the detectable phase-separated aqueous particle.

[0121] Described herein are simple bioinformatics methods and phase separation assays that can be used to demonstrate whether any IDR amino acid sequence, when used with a given macromolecule or protein and contained in a desired in vitro biochemical reaction environment, can function in the requisite manner to promote liquid-liquid demixing and phase separation in the desired biochemical reaction environment. Furthermore, the suitability of any given cofactor, particularly multivalent, e.g., divalent, metal cations, can be demonstrated in these assays in a very straightforward manner.

[0122] Thus, the present invention provides IDR reagents as described and defined herein that can be usefully applied to any given desired in vitro or in vivo biochemical reaction environment.

[0123] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to carry out an in vitro or in vivo biochemical reaction, such as any of the reactions described herein.

[0124] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to carry out a nucleic acid synthesis reaction.

[0125] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to carry out a nucleic acid synthesis reaction in which a polymerase is used to synthesize a new nucleic acid molecule by extending a primer nucleic acid molecule.

[0126] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary for carrying out a nucleic acid amplification reaction. The nucleic acid amplification reaction can be a reaction involving thermal cycling. The nucleic acid amplification reaction can be an isothermal amplification reaction. The nucleic acid amplification reaction can be a polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), helicase-dependent amplification (HDA), divergent amplification (RAM), recombinase polymerase amplification (RPA), transcription-mediated amplification (TMA), or nicking enzyme amplification reaction (NEAR).

[0127] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to perform a gene editing reaction.

[0128] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component required to carry out a CRISPR reaction.

[0129] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to perform a prime editing gene editing reaction, wherein a Cas enzyme, e.g., a CRISPR enzyme such as Cas9, is provided in a complex with at least a reverse transcriptase, and optionally further provided in a complex with a prime editing guide RNA (pegRNA), and any component of the prime editing complex can be provided tagged with one or more functional intrinsically disordered region (IDR) polypeptide sequences, e.g., a CRISPR enzyme tagged with one or more functional IDR polypeptide sequences, or a reverse transcriptase tagged with one or more functional IDR polypeptide sequences.

[0130] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to perform a ligation reaction.

[0131] Any of the IDR amino acid sequences described and defined herein can be used in conjunction with any macromolecular or protein component necessary to perform an exonuclease reaction.

[0132] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to carry out an endonuclease reaction, a transcription reaction, a DNA methylation reaction, a DNA glycosylation reaction, an antibody-antigen reaction, or a drug-target reaction.

[0133] Any of the IDR amino acid sequences described and defined herein can be used with any macromolecular or protein component necessary to carry out a reaction involving a protein:protein interaction.

[0134] As used herein, methods for conducting in vitro biochemical reactions are intended to encompass biochemical reactions that are conducted directly in solution within a reaction vessel, such as RPA reactions, which are further described herein.

[0135] As used herein, methods for conducting in vitro biochemical reactions also include biochemical reactions conducted within cells in culture, such as by expressing an IDR reagent described herein in the cultured host cells to increase the efficiency of the biochemical reaction within the cultured host cells.

[0136] As used herein, a method for performing an in vitro biochemical reaction includes a biochemical reaction performed in a host cell in culture by introducing into or expressing in the cultured host cell an IDR reagent described herein to the cultured host cell to increase the efficiency of the biochemical reaction in the cultured host cell, where the biochemical reaction is any reaction that results in the manipulation of a nucleic acid molecule in the cultured host cell or that results in a change in the structure of the nucleic acid molecule, such as a change in the nucleotide sequence of the nucleic acid molecule.

[0137] As used herein, a method for conducting an in vitro biochemical reaction includes a biochemical reaction carried out within a cell in culture by introducing into or expressing in the cultured host cell an IDR reagent described herein to the cultured host cell to increase the efficiency of the biochemical reaction within the cultured host cell, where the biochemical reaction is any reaction that results in the synthesis of a nucleic acid molecule within the cultured host cell.

[0138] As used herein, a method for conducting an in vitro biochemical reaction includes a biochemical reaction conducted in a cell in culture by introducing into or expressing in a cultured host cell an IDR reagent described herein to the cultured host cell to increase the efficiency of the biochemical reaction in the cultured host cell, where the biochemical reaction is any reaction that results in the expression of a polypeptide from a nucleic acid molecule.

[0139] As used herein, a method for performing an in vitro biochemical reaction includes a biochemical reaction performed in a cell in culture by introducing an IDR reagent described herein into or expressing an IDR reagent described herein in a cultured host cell to increase the efficiency of the biochemical reaction in the cultured host cell, where the biochemical reaction is any reaction that results in editing of a nucleic acid sequence in the cultured host cell (e.g., the IDR-polypeptide is a CRISPR polypeptide, such as a Cas polypeptide including a Cas9 polypeptide, or the IDR-polypeptide is a polypeptide complexed with a CRISPR polypeptide, e.g., the IDR-polypeptide is a reverse transcriptase), cleavage of nucleic acid in the cultured host cell, and homologous recombination of nucleic acid in the cultured host cell.

[0140] As used herein, a method for conducting an in vitro biochemical reaction includes a biochemical reaction conducted in cells in culture by introducing into or expressing in the cultured host cells an IDR reagent described herein to the cultured host cells to increase the efficiency of the biochemical reaction in the cultured host cells, where the biochemical reaction is a metabolic reaction in the cultured host cells to produce one or more biological products of interest in the cultured host cells or to produce one or more biological products of interest that are secreted from the cultured host cells or otherwise released into the medium from the cultured host cells.

[0141] The present invention is also intended to encompass biochemical reactions performed ex vivo, for example, by expressing the IDR reagents defined herein in cells of tissue culture or any other suitable complex biological system developed outside the body. Thus, any reference to a method for performing a biochemical reaction in an aqueous in vitro reaction system as used herein using any of the IDR reagents described herein may alternatively be defined as a method for performing a biochemical reaction in an aqueous ex vivo reaction system using any of the IDR reagents described herein.

[0142] The present invention also provides methods, reagents, and methods for increasing the efficiency of biochemical reactions in vivo. Accordingly, any reference to a method for conducting a biochemical reaction in an aqueous in vitro reaction system as used herein using any of the IDR reagents described herein may alternatively be defined as a method for conducting a biochemical reaction in an aqueous in vivo reaction system using any of the IDR reagents described herein.

[0143] The present invention provides any non-naturally occurring IDR-macromolecule or IDR-polypeptide described or defined herein for use in therapy, for use as a therapeutic agent, for use as a drug, for use as a pharmaceutical, or for use as a diagnostic agent.

[0144] The present invention provides any non-naturally occurring IDR-macromolecule or IDR-polypeptide described or defined herein for use in a method of treatment of the human or animal body by therapy.

[0145] The present invention provides any non-naturally occurring IDR-macromolecule or IDR-polypeptide described or defined herein for use in a diagnostic method practiced on the human or animal body.

[0146] The present invention provides any non-naturally occurring IDR-macromolecule or IDR-polypeptide as described or defined herein for use in the manufacture of a medicament for treating the human or animal body by therapy.

[0147] The present invention provides any non-naturally occurring IDR-macromolecule or IDR-polypeptide as described or defined herein for use in the manufacture of a diagnostic agent for a diagnostic method performed on the human or animal body.

[0148] The present invention provides a method for treating a human or animal comprising administering to the human or animal in need thereof a therapeutically effective amount of any non-naturally occurring IDR-macromolecule or IDR-polypeptide described or defined herein.

[0149] In any one of the above methods, reagents, and methods for increasing the efficiency of a biochemical reaction, the non-naturally occurring IDR-macromolecule or IDR-polypeptide can promote liquid-liquid demixing. The liquid-liquid demixing can promote the formation of phase-separated aqueous compartments in the solution, including detectable phase-separated aqueous particles in the solution. The liquid-liquid demixing or the formation of the detectable phase-separated compartments or particles thereby increases the efficiency of the biochemical reaction caused by the IDR-macromolecule or IDR-polypeptide.

[0150] As used herein, a method for conducting an in vitro biochemical reaction includes any biochemical reaction carried out in vitro in solution in a reaction vessel or in cultured host cells by introducing an IDR reagent described herein into the solution or by introducing or expressing an IDR reagent in cultured host cells to promote liquid-liquid demixing in the solution or in the cultured host cells. In any such biochemical reaction, liquid-liquid demixing in the solution or in the cultured host cells promotes phase separation in the solution or in the cultured host cells, as described and defined herein.

[0151] Any such biochemical reaction can be performed to assess the effectiveness of any IDR amino acid sequence described and defined herein in promoting liquid-liquid demixing in solution or in cultured host cells and / or promoting phase separation in solution or in cultured host cells.

[0152] Any such biochemical reaction can be performed to evaluate the effectiveness of a test agent, such as a drug, polypeptide, or any other molecule, preferably one that interacts with an IDR amino acid sequence, in stimulating or enhancing liquid-liquid demixing mediated by the IDR amino acid sequence in solution or in cultured host cells, and / or stimulating or enhancing phase separation mediated by the IDR amino acid sequence in solution or in cultured host cells.

[0153] Any such biochemical reaction can be performed to assess the effectiveness of a test agent, such as a drug, polypeptide, or any other molecule, in inhibiting liquid-liquid demixing mediated by IDR amino acid sequences in solution or in cultured host cells and / or inhibiting phase separation mediated by IDR amino acid sequences in solution or in cultured host cells, preferably where the test agent interacts with an IDR amino acid sequence.

[0154] Any of the methods described herein for performing in vitro, in vivo, or ex vivo biochemical reactions may exclude methods for cloning humans.

[0155] Any of the methods described herein for conducting in vitro, in vivo, or ex vivo biochemical reactions may exclude methods for altering the genetic identity of a human germline.

[0156] Any of the methods described herein for conducting in vitro, in vivo, or ex vivo biochemical reactions may exclude methods that involve the use of human embryos or the use of totipotent human cells.

[0157] Any host cell described herein may exclude a human embryo, or a totipotent human cell, or a human germline cell.

[0158] Although some aspects include in vivo use, the present invention in some aspects includes the exclusion of in vivo use. Thus, any of the methods, uses or processes etc. described herein for conducting biochemical reactions in aqueous reaction systems may exclude in vivo aqueous reaction systems.

[0159] Although some aspects include ex vivo use, the present invention in some aspects encompasses the exclusion of ex vivo use. Thus, any of the methods, uses or processes etc. described herein for conducting biochemical reactions in aqueous reaction systems may exclude ex vivo aqueous reaction systems.

[0160] In any of the methods, methods, uses or IDR reagents described herein, the efficiency of a reaction in a system may be increased by the IDR-macromolecule or IDR-polypeptide compared to the efficiency of a reaction in the system after introduction of at least one macromolecule or polypeptide under the same reaction conditions, except that the at least one macromolecule or polypeptide does not contain one or more functional intrinsically disordered regions (IDRs).

[0161] In any of the methods, uses or IDR reagents described herein, wherein at least one macromolecule or at least one polypeptide is tagged with an amino acid sequence comprising or consisting of one or more functional intrinsically disordered regions (IDRs) (IDR-tagged macromolecule or IDR-tagged polypeptide), the efficiency of the reaction in the system may be increased by the IDR-tagged macromolecule or IDR-tagged polypeptide compared to the efficiency of the reaction in the system after introduction of the at least one macromolecule or polypeptide under the same reaction conditions, except that the at least one macromolecule or polypeptide is not tagged with an amino acid sequence comprising or consisting of one or more functional IDRs.

[0162] Therefore, whether an IDR-macromolecule or IDR-polypeptide, or an IDR-tagged macromolecule or IDR-tagged polypeptide, can increase the efficiency of a reaction in a system can be very easily established by comparing the reaction efficiency of the macromolecule or polypeptide with and without one or more functional IDRs. Those skilled in the art can perform simple comparative tests to establish the relevant functional capabilities. Exemplary test assays are further described herein.

[0163] Similarly, whether an IDR-macromolecule or IDR-polypeptide, or an IDR-tagged macromolecule or IDR-tagged polypeptide, can colocalize molecules required for reaction performance with the IDR-macromolecule or IDR-polypeptide, or IDR-tagged macromolecule or IDR-tagged polypeptide, in multiple phase-separated aqueous compartments, or can further stimulate or enhance the colocalization of molecules required for reaction performance in multiple phase-separated aqueous compartments, thereby increasing the efficiency of biochemical reactions in the system, can also be very easily demonstrated by comparing colocalization with and without one or more functional IDRs. Again, one skilled in the art can perform simple comparative tests to demonstrate the relevant functional capabilities. Exemplary test assays are further described herein.

[0164] Similarly, whether providing a polyvalent metal ion to an IDR-macromolecule or IDR-polypeptide, or to an IDR-tagged macromolecule or IDR-tagged polypeptide, thereby further stimulating or enhancing liquid-liquid demixing and the formation of multiple phase-separated aqueous compartments, thereby further increasing the efficiency of biochemical reactions in the system, can also be very simply demonstrated by comparing liquid-liquid demixing with and without the provision of polyvalent metal ions. Again, one skilled in the art can perform simple comparative tests to demonstrate the relevant functional capabilities. Exemplary test assays are described further herein.

[0165] Similarly, whether providing ATP to an IDR-macromolecule or IDR-polypeptide, or an IDR-tagged macromolecule or IDR-tagged polypeptide, may further stimulate or enhance liquid-liquid demixing and the formation of multiple phase-separated aqueous compartments, thereby further increasing the efficiency of biochemical reactions in the system, can be very easily demonstrated by comparing liquid-liquid demixing with and without ATP being provided. Whether providing ATP to an IDR-macromolecule or IDR-polypeptide, or an IDR-tagged macromolecule or IDR-tagged polypeptide, may further stimulate or enhance colocalization of molecules necessary for carrying out reactions within multiple phase-separated aqueous compartments, thereby increasing the efficiency of biochemical reactions in the system, can also be very easily demonstrated by comparing colocalization with and without ATP being provided. Again, those skilled in the art can perform simple comparative tests to demonstrate the relevant functional capabilities. Exemplary test assays are further described herein.

[0166] Assays for demonstrating the ability to cause liquid-liquid demixing are described herein with reference to their ability to cause the formation of phase-separated aqueous particles (see, e.g., "Phase Separation Assays" as described herein). The same assays can be used to demonstrate the ability to cause colocalization of molecules necessary for reaction conduct within phase-separated aqueous compartments (particles). Assays for demonstrating the ability to increase the efficiency of a reaction are described herein with reference to their ability to increase the efficiency of RPA (see, e.g., "RPA Assays" as described herein). Such assays can be used to evaluate the ability of an amino acid sequence consisting of or including one or more functional intrinsically disordered regions (IDRs) to increase the efficiency of a reaction, and / or to evaluate the ability of divalent metal ions to further increase the efficiency of a reaction, and / or to evaluate the ability of ATP to further increase the efficiency of a reaction.

[0167] Using simple assays such as those described herein, one of skill in the art can determine an increase in reaction efficiency of 5% or more, and the increase in reaction efficiency can be 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more.

[0168] Crowding Agents Crowding agents are typically high molecular weight macromolecules such as proteins or synthetic block polymers. Crowding agents are considered to be essentially biochemically inert, i.e., they do not participate in specific interactions or catalysis.

[0169] It is widely hypothesized that crowding agents affect biological / biochemical systems, whether in vitro or in vivo, through the effect of physically occupying volume in solution, thus causing steric hindrance and a reduction in available open solvent space. Through this excluded volume mechanism, crowding agents appear to increase the effective concentration of other macromolecules, particularly affecting changes in dissociation constants and promoting the association of multiple interacting macromolecules, such as proteins, into specific assembled complexes. The magnitude of the crowding effect depends, among other things, on the molecular weights of the molecules involved, generally being much stronger for larger molecules. Thus, in general, macromolecular crowding is the effect of large molecules on the properties of other large molecules.

[0170] Furthermore, crowding agents have been widely described as being able to promote the formation of biological / biochemical systems with favorable phases in which reactants segregate into micron-sized, phase-separated particles. This effect essentially arises from the volume exclusion effect on the dissociation constants of macromolecular complexes that become relatively "trapped" because they cannot readily diffuse into the bulk solvent, which occupies most of the volume. Additionally and / or alternatively, some crowding agents, such as block-chain polymers like polyethylene glycol, may exhibit covariant properties that result in global changes to the structure of bulk water, typically reducing its density. Such changes in bulk solvent properties can also have complex effects on other macromolecules and their aggregates whose surfaces must interact with water. This, in turn, can promote the separation of these other macromolecules into a separate phase, resulting in a significant enrichment of the biological components and, concomitantly, the depletion of the crowding agent that primarily occupies the bulk solvent phase.

[0171] In either scenario, the effect of individual or combined crowding agents in stimulating the condensation of macromolecules into distinct phase condensates, either through simple volume occupancy or solvent modification, appears to operate via a "repulsive" rather than an "attractive" mechanism from the perspective of the condensate components. In other words, from the perspective of the highly concentrated components of the condensate, the crowding agent acts to create a bulk phase environment that cannot be easily penetrated by diffusion, and / or its bulk solvent properties are altered so that it presents a net enthalpic disadvantage for dispersing. Thus, the effect of high concentrations of crowding agents, typically greater than 1% w / v, is referred to herein as stimulating phase separation by functioning via a "hindering" or "repulsive" mechanism only if this phenomenon occurs because the condensate components cannot readily disperse as they would in the absence of the crowding agent. At the same time, however, given their generally inert nature, crowding agents have little or no direct debilitating effect on other specific molecules in the system, e.g., because they do not directly significantly interact with or exert their effect in a direct manner on specific molecular side chains.

[0172] In standard RPA reactions, polyethylene glycol (PEG) can have a significant effect on recombination / DNA synthesis. PEG can, for example, affect the number of invasion / extension cycles that occur when RecA is combined with Gp32. PEG can stimulate amplification reactions configured in several different ways. PEG and other similar crowding agents can affect the cooperativity of Gp32 and recombinase, affect polymerase processivity, and influence the hybridization rate and behavior of oligonucleotides in solution. The chain length of the polyethylene glycol can also affect the outcome. PEG can also increase the stability of recombinase-loaded filaments, and improved persistence can enhance the efficacy of RPA.

[0173] To exert their effect in an in vitro biochemical reaction environment, added crowding agents are typically present at concentrations predicted to produce a steric exclusion / confinement effect, typically greater than about 1% by volume or weight of the reaction.

[0174] In a standard RPA reaction, the crowding agent is present at a concentration of about 1% to 12% by volume or weight of the reaction.

[0175] The term "macromolecular crowding agent" or more simply "crowding agent" is a very well-recognized and art-understood term. This is evident from the widespread use of these terms in the literature. For example, Kuznetsova, I., M. et al. (Macromolecular Crowding Can Do to a Protein, 2014, Int. J. Mol. Sci., 15, pp. 23090-23140) provides a review that claims to cover over 320 papers and is proposed to represent one of the most comprehensive summaries of current knowledge in the field. The term "crowding agent" is used extensively throughout the text, emphasizing its ubiquitous use (see also Mixed Macromolecular Crowding: A Protein and Solvent Perspective, Biswas, S. et al., 2018, ACS Omega, 3(4), pp 4316-4330 and Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions, Phillip Y. et al., 2009, Biophysical Journal, 97 pp 875-885 875).

[0176] A compound or macromolecule can be identified as a crowding agent by means known in the art. For example, a crowding agent can be identified as such by its experimentally determined and calculated hydrodynamic radius (Kuznetsova et al., supra). A crowding agent can be identified as such by sol-gel glass encapsulation analysis (Kuznetsova et al., supra).

[0177] The following compounds are examples of known crowding agents: synthetic block polymers, polyethylene glycol (PEG), PEG 1450, PEG 2050, PEG 3000, PEG 4600, PEG 6000, PEG 8000, PEG 10000, PEG 20000, PEG 35000, PEG compounds with molecular weights of 15,000 to 20,000 (also known as Carbowax 20M), dextran, dextran 6, dextran 40, dextran 70, dextran 670, dextran sulfate 10, dextran sulfate 500, Ficoll, Ficoll 70, Ficoll 400, poly(sodium 4-styrenesulfonate) (PSS), bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, β-lactoglobulin, hemoglobin, and bovine serum albumin (BSA).

[0178] In any one of the methods, processes and uses of the present invention (including any one of the RPA methods, methods and uses of the present invention), the method, process and use may be carried out in the absence of a crowding agent.

[0179] In any one of the methods, processes and uses of the present invention (including any one of the RPA methods, methods and uses of the present invention), the method, process and use may be carried out in the presence of a crowding agent.

[0180] In any one of the methods, processes and uses of the present invention (including any one of the RPA methods, methods and uses of the present invention), the method, process and use may be carried out in the presence of a crowding agent, wherein the crowding agent is provided at a concentration that provides an enhancement in the efficiency of the biochemical reaction provided by the IDR-macromolecule or IDR-polypeptide.

[0181] In any one of the methods, processes and uses of the present invention (including the RPA methods, methods and uses of the present invention), the method, process and use may be carried out in the presence of a crowding agent, wherein the crowding agent is provided at a concentration that provides an additive effect on the efficiency of the biochemical reaction provided by the IDR-macromolecule or IDR-polypeptide.

[0182] In any one of the methods, processes and uses of the present invention (including any one of the RPA methods, methods and uses of the present invention), the method, process and use may be carried out in the presence of a crowding agent, wherein the crowding agent is provided at a concentration that provides a synergistic effect on the efficiency of the biochemical reaction provided by the IDR-macromolecule or IDR-polypeptide.

[0183] In any one of the methods, processes, and uses of the present invention (including any one of the RPA methods, methods, and uses of the present invention), the method, process, and use may be carried out in the presence of a crowding agent, and the introduction of the IDR-macromolecule or IDR-polypeptide into the biochemical reaction system reduces the concentration of crowding agent that would be required to achieve the same improvement in the efficiency of the biochemical reaction in the absence of the introduction of the IDR-macromolecule or IDR-polypeptide into the biochemical reaction system.

[0184] In any one of the above methods, processes and uses which may be carried out in the presence of a crowding agent, the crowding agent may be present at a concentration lower than the concentration at which its normal biological effect (steric exclusion / confinement effect) occurs.

[0185] In any one of the above methods, processes and uses that may be carried out in the presence of a crowding agent, the crowding agent may be present at a concentration of about 3% by volume or weight of the reactants, about 2% by volume or weight of the reactants, about 1% by volume or weight of the reactants, about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% by volume or weight of the reactants.

[0186] When used in any one of the methods, processes and uses of the present invention, including the RPA reaction method, any suitable crowding agent can be used. Examples of suitable crowding agents are provided herein.

[0187] Macromolecules or polypeptides containing intrinsically disordered regions (IDRs) The methods, processes, and reagents of the invention include "IDR-macromolecules," including "IDR-tagged macromolecules," as described herein. The methods, processes, and reagents of the invention include "IDR-polypeptides," including "IDR-tagged polypeptides," as described herein. Any such IDR-macromolecules, IDR-tagged macromolecules, IDR-polypeptides, or IDR-tagged polypeptides may be referred to interchangeably herein as IDR reagents or IDR-based reagents.

[0188] As used herein, an IDR-macromolecule or IDR-polypeptide or IDR-tagged macromolecule or IDR-tagged polypeptide is any macromolecule or polypeptide or protein that contains one or more functional intrinsically disordered regions (IDRs).

[0189] As used herein, an IDR-macromolecule or IDR-polypeptide or IDR-tagged macromolecule or IDR-tagged polypeptide is any macromolecule or polypeptide or protein comprising an amino acid sequence consisting of or including one or more functional intrinsically disordered regions (IDRs).

[0190] Thus, an IDR-macromolecule or an IDR-polypeptide, as referred to herein, may refer to a macromolecule or polypeptide that consequently comprises an amino acid sequence consisting of one or more functionally intrinsically disordered regions, or may refer to a macromolecule or polypeptide that comprises an amino acid sequence that includes one or more functionally intrinsically disordered regions.

[0191] Furthermore, an IDR-macromolecule comprising one or more functionally intrinsically disordered regions (IDRs) can be a macromolecule of interest tagged with an amino acid sequence consisting of or comprising one or more functionally intrinsically disordered regions (IDRs), as referred to herein. Such an IDR-tagged macromolecule is also an IDR reagent, as described herein. An IDR-tagged polypeptide comprising one or more functionally intrinsically disordered regions (IDRs), can be a polypeptide of interest tagged with an amino acid sequence consisting of or comprising one or more functionally intrinsically disordered regions (IDRs), as referred to herein. Such an IDR-tagged polypeptide is also an IDR reagent, as described herein.

[0192] An IDR-tagged macromolecule or IDR-tagged polypeptide, as used herein, is any macromolecule or polypeptide or protein that is "tagged" with an amino acid sequence consisting of or including one or more functional intrinsically disordered regions (IDRs).

[0193] Thus, an IDR-tagged macromolecule or an IDR-tagged polypeptide, as referred to herein, may refer to a macromolecule or polypeptide that is consequently tagged with an amino acid sequence consisting of one or more functional intrinsically disordered regions, or may refer to a macromolecule or polypeptide that is tagged with an amino acid sequence that includes one or more functional intrinsically disordered regions.

[0194] A tagged amino acid sequence consisting of or including one or more functional intrinsically disordered regions (IDRs) is not naturally or normally found in the macromolecule, polypeptide, or protein that is tagged at the tagged position. Thus, a tagged amino acid sequence consisting of or including one or more functional intrinsically disordered regions (IDRs) can be considered to be an exogenous amino acid sequence compared to the macromolecule, polypeptide, or protein to which it is tagged. Thus, a tagged macromolecule, polypeptide, or protein can be considered to be an artificial or genetically engineered macromolecule, polypeptide, or protein that is not of natural origin.

[0195] The mechanisms by which amino acid sequences can be "tagged" to polypeptides and other macromolecules are further described herein.

[0196] Any one or more functional intrinsically disordered regions (IDRs), including any one or more of the specific IDR amino acid tag sequences disclosed herein, or functional variants, analogs, homologs, or derivatives of any one or more thereof, can be tagged onto a macromolecule or polypeptide or protein.

[0197] To be used in the present invention, both the intrinsically disordered region polypeptide sequence and its domains must be "functional." The term "functional" means that any IDR amino acid sequence must have one of the functional properties further outlined herein.

[0198] The term "intrinsically disordered region" is a technically understood term commonly used in the art. For a comprehensive review, see Classification of Intrinsically Disordered Regions and Proteins, van der Lee et al., 2014, Chem. Rev. 114, pp. 6589-6631.

[0199] The present invention provides, inter alia, methods for conducting a biochemical reaction in an aqueous in vitro reaction system, wherein the biochemical reaction depends on the function of at least one reactive macromolecule, optionally at least one reactive polypeptide, and the method comprises introducing at least one IDR-macromolecule into the in vitro reaction system under conditions suitable for conducting the reaction, wherein the at least one IDR-macromolecule comprises one or more functional intrinsically disordered regions (IDRs), and wherein upon introduction of the at least one IDR-macromolecule into the in vitro reaction system, the efficiency of the biochemical reaction is enhanced by the at least one IDR-macromolecule. The efficiency of the biochemical reaction is enhanced by the one or more functional IDRs of the IDR-macromolecule. In any such method, the at least one IDR-macromolecule or IDR-polypeptide comprising one or more functional intrinsically disordered regions (IDRs) need not be the "reactive macromolecule" or "reactive polypeptide" on whose function the biochemical reaction depends. Thus, in any such method, the IDR-macromolecule or IDR-polypeptide may not have an inherent biochemical role in the biochemical reaction itself, but its introduction into the reaction system nevertheless results in an improvement in the efficiency of the biochemical reaction.

[0200] Methods for conducting biochemical reactions in in vitro reaction systems can be methods in which the biochemical reaction depends on the function of at least one IDR-macromolecule, and upon its introduction into the in vitro reaction system, the at least one IDR-macromolecule performs its reaction function in the biochemical reaction, increasing the efficiency of the reaction. In any such methods, the at least one IDR-macromolecule can be at least one IDR-polypeptide. In any such methods, the IDR-macromolecule or IDR-polypeptide has an inherent biochemical role in the biochemical reaction itself. Thus, at least one IDR-macromolecule or IDR-polypeptide containing one or more functional intrinsically disordered regions (IDRs) is a "reaction macromolecule" or "reaction polypeptide" on whose function the biochemical reaction depends.

[0201] At least one IDR-macromolecule or IDR-polypeptide comprises an amino acid sequence that comprises or consists of one or more functional intrinsically disordered regions. The IDR-macromolecule or IDR-polypeptide is introduced into a biochemical reaction system under conditions suitable for carrying out a biochemical reaction. Due to the presence of one or more functional intrinsically disordered regions, the IDR-macromolecule or IDR-polypeptide increases the efficiency of the reaction.

[0202] By increasing the efficiency of the reaction, it is meant that the efficiency of the reaction is improved compared to the efficiency of the reaction that would be observed if the IDR-macromolecule or IDR-polypeptide were provided without the amino acid sequence comprising or consisting of one or more functional intrinsically disordered regions. Such an improvement can be readily demonstrated by comparative testing of the reacting macromolecule or polypeptide with and without the IDR amino acid sequence.

[0203] In any one of the methods of the present invention, one or more functionally intrinsically disordered regions of an IDR-macromolecule or IDR-polypeptide promote / cause liquid-liquid demixing in a reaction system, resulting in phase separation. The functional ability to promote liquid-liquid demixing, resulting in phase separation in a reaction system, can be readily demonstrated, for example, by performing a phase separation assay as described herein. Such liquid-liquid demixing promotes phase separation, which can result in the formation of phase-separated compartments within the reaction system, such as particles detectable under a microscope, as further detailed herein.

[0204] The IDR-macromolecule or IDR-polypeptide may or may not have catalytic activity. For example, the IDR-polypeptide may have catalytic activity, such as a polymerase enzyme used in a recombinase polymerase amplification reaction, as further described herein. The IDR-polypeptide may not have catalytic activity, such as a single-stranded stabilizer used in a recombinase polymerase amplification reaction, such as Gp32, as further described herein.

[0205] As discussed further herein, regardless of whether an IDR-macromolecule or IDR-polypeptide has catalytic activity, the IDR-macromolecule or IDR-polypeptide may have a function that is required for or affects a biochemical reaction such that, in the absence of the IDR-macromolecule or IDR-polypeptide in the biochemical reaction system, the biochemical reaction cannot proceed or proceeds with reduced efficiency. Alternatively, as discussed further herein, the IDR-macromolecule or IDR-polypeptide may not have a function that is required for or affects the biochemical reaction itself. Nevertheless, because of the IDR amino acid sequence, introduction of the IDR-macromolecule or IDR-polypeptide into a biochemical reaction system results in an increased efficiency of the biochemical reaction compared to the efficiency observed in the absence of the IDR-macromolecule or IDR-polypeptide or in the presence of the same macromolecule or polypeptide without the IDR amino acid sequence.

[0206] Structural characteristics of IDR polypeptides The presence of IDRs in amino acid sequences can be readily determined by structural analysis. Numerous bioinformatics-based platforms are available for predicting the presence of IDRs in polypeptides and proteins. These include ELM, MiniMotif, SLiMPrints, phylo-HMM, DiliMot, SLiMFinder, Phospho.ELM, PhosphoSite, PHOSIDA, ScanSite, NetPhorest, NetworKIN, PhosphoNET, IDEAL, MoRFpred, ANCHOR, Pfam, FFPred, DisProt, and D 2 P 2 Any of these methods can be used to identify IDR amino acid sequences. If necessary, such IDR amino acid sequences can be tested to assess their functional properties, as further described herein.

[0207] A preferred bioinformatics-based platform for IDR amino acid sequence identification is the MetaDisorder software program (MetaDisorder: a metaserver for predicting intrinsic disorder in proteins, Kozlowski, LP et al., BMC Bioinformatics, 2012, 13(1):111).

[0208] The MetaDisorder program is freely available online (http: / / genesilico.pl / metadisorder / ). Using this program, one simply pastes the amino acid sequence of interest into an internet browser window and the program is started. As the online documentation explains, any amino acid region with a score greater than 0.5 in the software package is considered to contain an intrinsically disordered region.

[0209] Using the MetaDisorder software platform, we identified several amino acid sequences containing one or more intrinsically disordered regions, which are shown in Table 1.

[0210] Thus, in any one of the methods, processes, and uses, or in any one of the non-naturally occurring IDR-macromolecules, IDR-fusion macromolecules, or isolated nucleic acid molecules, recombinant polynucleotide expression vectors, or host cells encoding same, one or more functional IDRs of the IDR-macromolecules or IDR-polypeptides can be characterized as a sequence of amino acids that has a score of greater than 0.5 when analyzed by the algorithm MetaDisorder. The sequence of amino acids can be a sequence of amino acids that shows a score of greater than 0.5 when analyzed by the algorithm MetaDisorder accordion according to the method of Kozlowski, LP et al., BMC Bioinformatics, 2012, 13(1):111.

[0211] The present invention provides and relates to preferred IDR amino acid sequences comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-43 (Table 1) and variants thereof. In all cases, the variants of the amino acid sequence of any one of SEQ ID NOs: 1-43 are functional variants that retain IDR functional properties, as further described herein.

[0212] Furthermore, as further described herein, an IDR-macromolecule or IDR-polypeptide may comprise or consist of an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-43, or a macromolecule or polypeptide tagged with an amino acid sequence comprising or consisting of a functional variant amino acid sequence of SEQ ID NOs: 1-43.

[0213] Functional variants may have at least 80% sequence identity compared to the IDR amino acid sequences set forth herein (Table 1). Functional variants may have at least 81% sequence identity, or 82% sequence identity, or 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to the IDR amino acid sequences set forth herein (Table 1).

[0214] For purposes of the present invention, to determine the percent identity between, for example, an amino acid sequence of any one of SEQ ID NOs: 1-43 and a functional variant of an amino acid sequence of any one of SEQ ID NOs: 1-43, the two respective amino acid sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for optimal alignment with the second sequence). The nucleotide residues at nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, the nucleotides are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence × 100).

[0215] Typically, sequence comparison is performed over the entire length of the reference sequence. For example, if one skilled in the art wants to determine whether a given ("variant") sequence is 80% identical to SEQ ID NO:2, SEQ ID NO:2 would be the reference sequence. For example, to assess whether a variant sequence is at least 80% identical to SEQ ID NO:2 (an example of a reference sequence), one skilled in the art would align the length of SEQ ID NO:2 and identify how many positions in the test sequence were identical to the positions in SEQ ID NO:2. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO:2. If the sequence is shorter than SEQ ID NO:2, gaps or missing positions should be considered non-identical positions.

[0216] Those skilled in the art are familiar with the various computer programs available for determining the homology or identity between two sequences.For example, the comparison of sequences and the determination of the percent identity between two sequences can be achieved using mathematical algorithms.The percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (1970) algorithm, which is incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), for example, using either Blosum 62 matrix or PAM 250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0217] A functional variant of the amino acid sequence of any one of SEQ ID NOs: 1-43 can be an amino acid sequence that differs from the amino acid sequence of any one of SEQ ID NOs: 1-43, respectively, by having fewer amino acids (i.e., the functional variant is shorter) or by having more amino acids (i.e., the functional variant is longer) compared to the amino acid sequence of any one of SEQ ID NOs: 1-43, respectively. Thus, a functional variant can include one or more amino acid deletions and / or one or more insertions compared to the reference amino acid sequence. The number of amino acids in a functional variant amino acid sequence by which the variant differs from the reference sequence can be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more.

[0218] A functional variant of the amino acid sequence of any one of SEQ ID NOs: 1 to 43 can include, for example, conservative amino acid substitutions of the amino acid residues shown in the sequences listed in Table 1. Conservative substitutions can be made, for example, according to the following table, which describes commonly accepted amino acid groupings. Thus, a functional variant can include conservative amino acid substitutions compared to a reference amino acid sequence. The number of amino acids in a functional variant amino acid sequence that are conservative amino acid substitutions compared to a reference sequence can be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more. [Table 1]

[0219] Whether a given variant retains IDR functional properties can be readily established, for example, by methods described further herein. Table 1 [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0220] Similar substitutions can be made for amino acids such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions can also be made, i.e., from one class of residue to another, or alternatively, with the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine ornithine, pyriylalanine, thienylalanine, naphthylalanine, and phenylglycine.

[0221] The specific IDR amino acid sequences disclosed herein (see Table 1) can be broadly divided into four groups. Some IDR sequences can be classified into more than one group. The RGG / RG group contains IDR sequences that are FG / YG-rich. This group includes fib, hnrpnA1, DDX, HRP1, and Sup. The Poly Q group contains IDR sequences that are Q / N-rich. This group includes PCF11, Ent-1, HRP1, Sup, His4, His8, and His10. The Poly P group contains P-rich sequences. This group includes His4, His9, and His10. The Poly H group contains H-rich sequences. This group includes His1-11. Some important features of IDR amino acid sequences are that they exhibit cation-π and π-π interactions and can form amide and salt bridges. Important features and important inter- and intramolecular interactions of preferred IDR amino acid sequences are shown in Tables 2 to 20 below. Table 2 [Table 3] Table 3 [Table 4] Table 4 [Table 5] Table 5 [Table 6] Table 6 [Table 7] Table 7 Table 8 Table 8 Table 9 Table 9 Table 10 Table 10 Table 11 Table 11 Table 12 Table 12 Table 13 Table 13 Table 14 Table 14 Table 15 Table 15 Table 16 Table 16 Table 17 Table 17 [Table 18] Table 18 [Table 19] Table 19 [Table 20] Table 20 [Table 21]

[0222] Functional properties of IDR-macromolecules or IDR-polypeptides The IDR-tagged macromolecules or polypeptides, or IDR-macromolecules or IDR-polypeptides described herein must have an amino acid sequence consisting of or including one or more functional IDRs for use in the methods of the invention. Whether such an IDR amino acid sequence or domain is functional can be established by routine methods, such as those described herein.

[0223] particle formation The present inventors have surprisingly discovered that IDR-tagged or IDR-polypeptides can form particles in suitable solutions, which is believed to occur through liquid-liquid demixing, which results in phase separation of the fluids within the solution mixture mediated by the IDR amino acid sequence.

[0224] Particle formation mediated by IDR amino acid sequences is further described in the examples below. The particles exhibit a spheroidal appearance and can be described as "globules," "spherical foci," or "particles."

[0225] The terms "particle," "globule," or "spherical focus" referred to herein are intended to be synonymous and can be used interchangeably. Conditions and methods that allow for the observation and detection of particles are described herein, including in the examples below.

[0226] In the examples described herein, particle formation was observed to occur in simple systems containing only a solution of an IDR-tagged polypeptide and a divalent metal cation. Particle formation was also found to occur in more complex mixtures, including mixtures containing components required for RPA in which one of the RPA protein components (Gp32) was IDR-tagged. In these situations, reaction components were found to strongly colocalize with the particles; for example, particles were found to be dense in oligonucleotides as detected by fluorescently labeled probes bound to them, and contained all other RPA reaction protein components.

[0227] Particle detection and monitoring can be performed using any suitable method, as well as those described in the examples below, exemplary methods including microscopy, light scattering, flow cytometry, and microfluidics.

[0228] Particles can be detected using microscopy, such as differential interference contrast or fluorescence microscopy, to directly observe the particles at high magnification. Microscopic images can be acquired and analyzed automatically with the aid of a computer. Furthermore, microscopy can allow for continuous or frequent monitoring of at least a portion of a mixture containing particles.

[0229] Particles can be detected using flow cytometry. In flow cytometry, one or more light beams, e.g., each of a single wavelength, are directed at a hydrodynamically focused stream of fluid. Suspended particles passing through the beam scatter the light, which can excite fluorescent chemicals found within or attached to the particles. The scattered light and / or fluorescence is analyzed by detectors within the instrument, from which information about particle size and fluorescence can be determined. Modern flow cytometers can analyze thousands of particles per second in "real time" and actively separate and isolate particles with specific properties.

[0230] Particles can be detected using cytometry methods, devices and systems, such as those disclosed in U.S. Patent Application Publication Nos. 2009 / 0079963 and 2010 / 0179068 and International Patent Application Publication No. WO2009 / 112594.

[0231] Particles can be detected using microfluidic methods, devices, and systems, such as lab-on-a-chip devices or systems (see, e.g., U.S. Patent Application Publication Nos. 2009 / 0326903 and 2009 / 0297733).

[0232] The particles can be between about any two sizes selected from about 0.5 to 20 μm in size, for example, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, and 20 μm (e.g., about 1 to 10 μm in size).

[0233] The particle concentration may be about 10 to 5000 particles / nL, for example, between any two numbers of particles selected from 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 particles / nL (e.g., about 100 to 500 particles / nL). The particle concentration may be about 200 to 400 particles per nanoliter.

[0234] Such phase-separated particles may be smaller than about 0.5 μm in size. Phase-separated particles, including those smaller than about 0.5 μm in size, can be detected by a change in the turbidity of the solution. The change in the turbidity of the solution can be measured by standard means and typically quantified according to formazin turbidity units (FTU) or formazin nephelometric units (FNU). Other methods include size-exclusion chromatography, including multi-angle light scattering (SEC-MALS).

[0235] Experimental determination of IDR function The IDR-macromolecules or IDR-polypeptides, or IDR-tagged macromolecules or IDR-tagged polypeptides described herein can be determined to have functional intrinsically disordered region (IDR) amino acid sequences and / or domains thereof, and thus can be used in the methods and reagents of the invention, for example, by using the phase separation assay or RPA assay described below.

[0236] Thus, an IDR-macromolecule, IDR-polypeptide, or IDR-tagged macromolecule or polypeptide is a macromolecule or polypeptide that comprises or is tagged with an amino acid sequence consisting of one or more functional intrinsically disordered regions, or is a macromolecule or polypeptide that is tagged with an amino acid sequence that comprises one or more functional intrinsically disordered regions. In all cases, the functional intrinsically disordered regions are those that can be determined to be functional in a phase separation assay described below and / or an RPA assay as described below.

[0237] Phase separation assay The phase separation assay method is 1. tagging a polypeptide with one or more intrinsically disordered region amino acid sequences to create an IDR-polypeptide fusion protein, preferably tagging a recombinant phage vB EcoM NBG1 Gp32 protein to create a Gp32-IDR fusion protein, and providing a purified IDR-polypeptide fusion protein; 2. Adding the IDR-polypeptide fusion protein to a volume of water to a final concentration of 1000 ng / μl, the final volume of the mixture being 50 μl, preferably adding a divalent metal cation to a final concentration of 2 mM or greater, more preferably the divalent metal cation is Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ and adding 3. Vortexing the mixture, followed by pulse centrifuging the mixture; 4. Transferring a 10 μl sample of the mixture supernatant to a hemocytometer slide; 5. View the hemocytometer slide under a microscope at 400x magnification and 6. Observing the formation of particles in the mixture; 7. If no particles are present in the mixture, repeat steps 1-6, incrementally increasing the concentration of divalent metal cations until the formation of particles in the mixture is observed; 8. Counting the number of particles formed in a magnified area of ​​218 μm × 175 μm at 400x magnification; 9. (i) demonstrating that an amino acid sequence consisting of or including one or more intrinsically disordered regions (IDRs) is functional when 10 or more particles are counted within the expanded region, preferably when 50 or more particles are counted within the expanded region, more preferably when 100 or more particles are counted within the expanded region; or (ii) demonstrating that an amino acid sequence consisting of or including one or more intrinsically disordered regions (IDRs) is non-functional when the concentration of the divalent metal cation is increased to 100 mM or greater and no particles are observed to form within the magnified area, or fewer than 10 particles are counted within the magnified area; The method includes:

[0238] In the above method, if it is desired to examine the effect of providing divalent metal cations on particle formation, step 2 can include adding divalent metal cations to any desired final concentration, thus allowing the effect of different concentrations of divalent metal cations to be examined.

[0239] In the above method, if it is desired to examine the effect of providing ATP on particle formation, step 2 can include adding ATP to any desired final concentration. Thus, the effect of different concentrations of ATP can be examined. ATP can be provided at a concentration of, for example, 1 mM to 3.5 mM, e.g., 1 mM to 2 mM.

[0240] Step 2 may include adding a detectable nucleic acid molecule, and step 8 may include counting the number of particles by detection means. For example, step 2 may include adding a probe having a nucleic acid sequence set to SEQ ID NO: 104 labeled with FAM (fluorescein), and step 8 may include detecting particles by fluorescence. The detectable nucleic acid molecule may be added to any suitable final concentration, such as 0.5 μM.

[0241] Thus, the above assays can be used to examine reaction efficiency, the ability to induce liquid-liquid demixing, and the ability to co-localize molecules into multiple phase-separated aqueous compartments (particles).

[0242] In the above method, the divalent metal cation is Mg 2+ When the divalent metal cation is Ca, the cation source is preferably MgOAc. 2+ When the divalent metal cation is Mn, the cation source is preferably CaCl. 2+ When the cation source is MnCl2, it is preferred that the cation source is MnCl2.

[0243] RPA assay The RPA assay method is 1. To generate a Gp32-IDR fusion protein, tag one or more intrinsically disordered region polypeptide sequences onto a Gp32 protein, preferably a recombinant phage vB EcoM NBG1 Gp32 protein, and provide a purified Gp32-IDR fusion protein; 2. Producing a reaction mixture comprising: a. Tris HCl pH 8.3, 25 mM, b.KOAc, 7.5mM; c.DTT, 1mM, d.ATP, 2.5 mM; e. Phosphocreatine, 20 mM; f. creatine kinase, 1 μM; g. dNTPs, 1 mM, h. purified Gp32-IDR fusion protein, 20 μM; i. Purified UvsX, 4.8 μM j. Purified UvsY, 8.6 μM k. Staphylococcus aureus DNA polymerase 1 (SAU), 0.135 μM l. Exonuclease III, 0.27 μM m. forward primer, 0.4 μM n. Reverse primer, 0.4 μM o. Probe, 0.12 μM 3. Initiating the recombinase polymerase amplification reaction by adding 33 mM MgOAc and 10 copies of template nucleic acid to the reaction mixture; 4. Incubating the reaction mixture at 39°C in a fluorometer with magnetic mixing using a bearing ball; 5. (i) demonstrating that one or more intrinsically disordered region polypeptide sequences are functional if a two-fold or greater increase in amplification product is detectable within 15 minutes by a measurable increase in fluorescence compared to baseline in a template-dependent manner; or (ii) if no amplification product is detected by fluorescence after 15 minutes, demonstrating that one or more of the intrinsically disordered region polypeptide sequences are non-functional; The method includes:

[0244] In the above method, the forward primer sequence is CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98), the reverse primer sequence is CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99), the probe sequence is CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, BHQ is black hole quencher, and the template is Listeria monocytogenes genomic DNA.

[0245] Alternatively, step 5 of the RPA assay described above may comprise verifying that the one or more intrinsically disordered region polypeptide sequences are functional if a 5-fold or greater increase in amplification product is detectable within 15 minutes by a measurable increase in fluorescence compared to baseline in a template-dependent manner, or if a 10-fold or greater increase is detectable, or if a 20-fold or greater increase, or a 30-fold or greater increase, or a 40-fold or greater increase, or a 50-fold or greater increase, or a 100-fold or greater increase, or a 150-fold or greater increase, or a 200-fold or greater increase, or a 250-fold or greater increase, or a 300-fold or greater increase, or a 350-fold or greater increase, or a 400-fold or greater increase, or a 450-fold or greater increase, or a 500-fold or greater increase, or a 1000-fold or greater increase, or a 2000-fold or greater increase, or a 3000-fold or greater increase, or a 40 ...5000-fold or greater increase, or a 5000-fold or greater increase, or a 5000-fold or greater increase, or a 5000-fold or greater increase, An increase in amplification product over baseline refers to an increase in amplification product compared to the amount of amplification product obtained by performing a reaction under the same conditions, except that the Gp32 protein is not tagged with one or more intrinsically disordered region polypeptide sequences.

[0246] In the above method, if it is desired to examine the effect of providing divalent metal cations on reaction efficiency, step 3 can include adding divalent metal cations to any desired final concentration, thus allowing the effect of different concentrations of divalent metal cations to be examined.

[0247] In the above method, if it is desired to examine the effect of providing ATP on reaction efficiency, step 2 can include adding ATP to any desired final concentration. Thus, the effect of different concentrations of ATP can be examined. ATP can be provided at concentrations of, for example, 1 mM to 3.5 mM, e.g., 1 mM to 2 mM.

[0248] Tagging macromolecules and polypeptides with IDR amino acid sequences The methods, methods and reagents of the invention include, inter alia, IDR-tagged macromolecules and IDR-tagged polypeptides, where an IDR-tagged macromolecule or IDR-tagged polypeptide is a macromolecule or polypeptide of interest tagged with an amino acid sequence consisting of or including one or more intrinsically disordered regions (IDRs) (which may be referred to herein as IDR portions).

[0249] The terms "tag" or "tagging" should be understood in their broadest sense to mean that an IDR portion, i.e., an amino acid sequence consisting of or including one or more functional IDRs, is bound, tethered, attached, or otherwise associated in any suitable manner with a macromolecule or polypeptide of interest.

[0250] The most preferred means of tagging a polypeptide of interest with an IDR portion is by creating a recombinant gene fusion protein, in which the polypeptide of interest is genetically engineered at the nucleotide level such that when transcribed and translated, the expressed protein contains the polypeptide of interest with the IDR portion.

[0251] If desired, a linker may be placed between the polypeptide of interest and the IDR portion. For example, flexible, strong, and cleavable linkers are well known in the art and are widely used in the production of fusion proteins (see, for example, Fusion Protein Linkers: Property, Design and Functionality, Chen, X., et al. 2013, Adv. Drug Deliv. Rev., 15, 65(10), pp1357-1369).

[0252] Standard methods for genetic manipulation, as well as methods for protein expression and purification, are well known in the art (see, e.g., Sambrook et al., 2001, Molecular Cloning: a Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press and Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (1995)).

[0253] Another means by which the IDR moiety can be tagged to a macromolecule or polypeptide of interest is through one or more covalent bonds or affinity interactions.

[0254] The IDR portion can be tagged to the polypeptide in any suitable orientation, such as at the N-terminus of the polypeptide of interest, at the C-terminus of the polypeptide of interest, or the polypeptide of interest can contain an IDR portion at both its N-terminus and C-terminus, or at any amino acid position along the length of the polypeptide.

[0255] Peptides / oligopeptides / polypeptides / proteins can be linked / tethered and conjugated to other macromolecules, including other peptides / oligopeptides / polypeptides / proteins, by using methods well known in the art.

[0256] One such method is "click chemistry." The term "click chemistry" is typically used to describe the reaction of azides with alkynes in the presence of a copper catalyst, resulting in 1,5-disubstituted 1,2,3 triazoles. Click chemistry allows peptides, oligopeptides, polypeptides, and proteins to be conjugated to other peptides, oligopeptides, polypeptides, and proteins, as well as a wide range of other macromolecules, including carbohydrates, nucleic acids, polymers, drugs, aptamers, hydrogels, and the like. This method is also referred to as "CuAAC" (Cu-catalyzed alkyne-azide cycloaddition) (see, e.g., "Click" Reaction: A Versatile Toolbox for the Synthesis of Peptide Conjugates, Tang, W. et al., 2014, Chem. Soc. Rev., 43, pp. 7013-7039).

[0257] Many other linker / crosslinker chemistries are available for conjugating peptides / oligopeptides / polypeptides / proteins to other macromolecules, such as crosslinkers containing maleimides that react with amines, sulfhydryl-reactive groups, or succinimidyl esters (often called NHS esters). For example, succinimides can be used to form covalent bonds between proteins or peptides and plastic materials.

[0258] Standard chemicals commonly used to prepare conjugates between polypeptides and non-polypeptide molecules, such as those for preparing antibody-drug conjugates, can be used, and many such techniques are well known in the art.

[0259] Affinity-based interactions can also be used, for example, an amino acid sequence consisting of or including one or more functional intrinsically disordered regions can be bound / tethered to a macromolecule or polypeptide of interest by affinity-based interactions such as streptavidin-biotin, receptor-ligand interactions, etc.

[0260] Polyvalent metal cations for IDR amino acid sequence function When an IDR-macromolecule or IDR-polypeptide as described and defined herein is used in an in vitro biochemical reaction, the in vitro biochemical reaction buffer preferably contains a polyvalent metal cation, preferably a divalent metal cation.

[0261] The presence of polyvalent / divalent metal cations in the reaction buffer serves to promote and enhance liquid-liquid demixing, which leads to phase separation in an in vitro biochemical reaction environment mediated / triggered by IDR-macromolecules or IDR-polypeptides.

[0262] The functional ability of divalent metal cations to enhance phase separation in an in vitro biochemical reaction environment mediated / triggered by an IDR-macromolecule or IDR-polypeptide can be readily demonstrated, such as by the techniques disclosed and defined herein. In particular, such functional ability can be demonstrated by the ability of polyvalent / divalent metal cations to induce the formation of spherical foci or particles in an in vitro biochemical reaction environment in an IDR-dependent manner, as further described and defined herein, e.g., as determined by the assays described herein.

[0263] The use of divalent metal cations in promoting / enhancing IDR-dependent liquid-liquid demixing leading to phase separation is preferred. However, functional equivalents of any polyvalent or any divalent metal cation are contemplated. A functional equivalent of the polyvalent / divalent metal cations described herein is any agent that can substitute for a divalent metal cation in promoting IDR-dependent liquid-liquid demixing leading to phase separation in an in vitro biochemical reaction environment, as determined, for example, by the assays described herein.

[0264] Any suitable polyvalent / divalent metal cation can be used as a single agent or combination of agents, optionally in the presence of a chelating agent, such as ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or nitroacetic acid (NTA).

[0265] The divalent metal cation is Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ , Ni 2+ or Cu 2+ These cations may be used alone or in combination. Preferably, they are used as single agents. The preferred divalent metal cations are Mg 2+ , Mn 2+ and Ca 2+ is.

[0266] The specific polyvalent / divalent metal cations and the specific concentrations of the polyvalent / divalent metal cations used that achieve optimal results in promoting IDR-mediated phase separation in an in vitro biochemical reaction environment may depend on the specific intrinsically disordered region amino acid sequence used to tag the macromolecule or polypeptide of interest. Optimal polyvalent / divalent metal cations and optimal concentrations can be empirically established using routine testing. The phase separation assays described further herein can be used for this purpose.

[0267] Preferred concentration ranges for polyvalent / divalent metal cations are about 300 μM to about 100 mM, about 300 μM to about 50 mM, about 400 μM to about 50 mM, about 400 μM to about 20 mM, about 400 μM to about 30 mM, about 500 μM to about 10 mM, about 500 μM to about 25 mM, and about 1 mM to about 35 mM.

[0268] In vitro biochemical reaction buffers contain Mg 2+ The buffer may contain MgOAc at the indicated concentration. A preferred concentration range is about 300 μM to about 100 mM, more preferably about 400 μM to about 50 mM, even more preferably about 500 μM to about 40 mM, and even more preferably about 25 mM to about 35 mM, for example, 33 mM. Preferably, the buffer contains MgOAc at the indicated concentration.

[0269] In vitro biochemical reaction buffers contain Ca 2+The buffer may contain CaCl ions. Preferred concentration ranges are about 300 μM to about 100 mM, more preferably about 400 μM to about 50 mM, even more preferably about 1 mM to about 40 mM, and even more preferably about 25 mM to about 35 mM, e.g., 33 mM. Preferably, the buffer contains CaCl at the indicated concentration.

[0270] In vitro biochemical reaction buffers are buffers containing Mn 2+ ions. A preferred concentration range is about 300 μM to about 50 mM, more preferably about 400 μM to about 50 mM, even more preferably about 500 μM to about 40 mM, and even more preferably about 25 mM to about 35 mM, e.g., 33 mM. Preferably, the buffer contains MnCl2 at the indicated concentration.

[0271] Recombinase polymerase amplification (RPA) Recombinase polymerase amplification (RPA) is a method for isothermal amplification of nucleic acids. Generally, in the first step of RPA, a recombinase agent is contacted with first and second nucleic acid primers and a recombinase loading protein to form first and second nucleoprotein primers. Generally, in the second step, the first and second nucleoprotein primers are contacted with a double-stranded template nucleic acid to form a first double-stranded structure in a first portion of the first strand of the template nucleic acid and a second double-stranded structure in a second portion of the second strand of the template nucleic acid, such that the 3' ends of the first and second nucleic acid primers are oriented toward each other on a given nucleic acid molecule. Generally, in the third step, the 3' ends of the first and second nucleoprotein primers are extended by a polymerase to generate first and second double-stranded nucleic acids and first and second displaced single strands of the nucleic acid. A single-strand stabilizing agent is used to stabilize the first and second displaced single strands of the nucleic acid. Generally, the second and third steps can be repeated until the desired degree of amplification is reached.

[0272] RPA methods are widely disclosed in, for example, U.S. Patent Nos. 7,270,981, 7,399,590, 7,666,598, 7,435,561, and International Patent Application Publication No. WO2010 / 141940. For a comprehensive recent review, see "Review: a comprehensive summary of a decade development of the recombinase polymerase amplification," Li, J. et al., 2019, Analyst, 144, pp. 31-67.

[0273] Recombinase agents RPA methods, including the methods of the present invention, use recombinase agents.

[0274] Any one or more IDR-polypeptides of the present invention may be conjugated / tethered / tagged to any recombinase agent.

[0275] Recombinase agents are molecules, typically enzymes, that can coat single-stranded nucleic acids, typically DNA (ssDNA), to form nucleoprotein filaments. Such filaments can then "scan" double-stranded nucleic acid molecules, typically DNA (dsDNA), for regions of sequence homology / complementarity. When a complementary sequence is located, the nucleoprotein filament strand (including the recombinase agent) invades the double-stranded nucleic acid molecule, forming a short hybrid and displaced strand bubble known as a D-loop.

[0276] Any suitable recombinase agent can be used in the RPA methods described herein and can be tagged with any of the IDR amino acid sequences described herein.

[0277] The recombinase agent may be derived from a prokaryotic, eukaryotic or viral organism.

[0278] The recombinase agent can be RecA, UvsX, RadA, RadB, Rad51, or any functional variant, analog, homolog or derivative of any of these proteins.

[0279] Any combination of these proteins may be used.

[0280] Suitable recombinase agents include the E. coli RecA protein, the T4 UvsX protein, or any homologous protein or protein complex from any phylum.

[0281] Eukaryotic RecA homologs are commonly named Rad51 after the first member of this group to be identified. Other heterologous recombinase agents, such as RecT or RecO, may be used in place of RecA.

[0282] Exemplary recombinase agents include RecA and UvsX, as well as fragments or variants thereof and combinations thereof. RecA and UvsX proteins can be obtained from any species. Available RecA and UvsX protein and nucleic acid sequences and molecular biology techniques can also be used to produce RecA and UvsX fragments or variant proteins. Exemplary UvsX proteins include those from Myoviridae phages such as T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, phage 31, phage 44RR2.81, RB49, phage Rb3, and phage LZ2. Further exemplary recombinase agents include archaeal RADA and RADB proteins and eukaryotic (e.g., plant, mammalian, and fungal) Rad51 proteins (e.g., RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCC2, XRCC3, and recA).

[0283] The recombinase agent is preferably 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, Escherichia phage vB_EcoM_G4507 UvsX, Shigella phage SHFML-11 UvsX, E. coli phage vB_EcoM_DalCa UvsX, E. coli RecA, E. coli RadA, E. coli RadB, E. coli Rad51, or any functional variant, analog, homolog, or derivative thereof, or any combination thereof. A particularly preferred recombinase agent is Escherichia phage vB_EcoM_DalCa UvsX.

[0284] The recombinase agent may also include a C-terminal deletion of acidic residues to improve its activity.

[0285] Any functional variants, analogs, homologs or derivatives of the above recombinase agents may themselves also function as recombinase agents, and these functional variants, analogs, homologs or derivatives are also contemplated as recombinase agents for use in the methods described and defined herein.

[0286] For example, a small peptide from RecA has been shown to retain some aspects of the recombination properties of RecA: this peptide, which contains residues 193-212 of E. coli RecA, can mediate the pairing of single-stranded oligonucleotides.

[0287] The recombinase agent (e.g., UvsX) can be a mutant recombinase agent or a hybrid recombinase agent. Mutant forms of UvsX are described in U.S. Pat. No. 8,071,308. The mutant UvsX can be an Rb69 UvsX containing at least one mutation in the Rb69 UvsX amino acid sequence, the mutation being selected from the group consisting of: (a) an amino acid other than histidine at position 64, a serine at position 64, the addition of one or more glutamic acid residues at the C-terminus, the addition of one or more aspartic acid residues at the C-terminus, and combinations thereof.

[0288] The mutant UvsX can be a T6 UvsX having at least one mutation in the T6 UvsX amino acid sequence, the mutation being selected from the group consisting of (a) an amino acid other than histidine at position 66, (b) a serine at position 66, (c) the addition of one or more glutamic acid residues at the C-terminus, (d) the addition of one or more aspartic acid residues at the C-terminus, and (e) a combination thereof. When a hybrid recombinase agent is used, the hybrid protein can be, for example, a UvsX protein containing at least one region comprising an amino acid sequence from a different UvsX species. The region can be, for example, the DNA-binding loop-2 region of UvsX.

[0289] If desired, the recombinase agent may be a temperature-sensitive (referred to herein as "ts") recombinase agent. When a ts recombinase agent is used, the RPA reaction can be initiated at one temperature (the permissive temperature) and terminated at another temperature (the non-permissive temperature). Permissive temperature combinations can be, for example, 25°C / 30°C, 30°C / 37°C, 37°C / 42°C, etc. The ts protein can be reversible. The activity of a reversible ts protein is restored upon shifting from the non-permissive temperature to the permissive temperature.

[0290] Any recombinase agent concentration can be used, but preferred recombinase concentrations can be in the ranges of, for example, 0.2 to 12 μM, 6 to 12 μM, 4 to 12 μM, and 4 to 6 μM, preferably about 5 μM, and more preferably about 4.8 μM.

[0291] Recombinase agents generally require the presence of ATP, ATPγS, or other nucleoside triphosphates or their analogs. Recombinase agents are preferably used in a reaction environment where regeneration of the target site can occur immediately after a round of D-loop-stimulated synthesis. The completed recombination event, accompanied by recombinase degradation, avoids stalled amplification or highly inefficient linear amplification of ssDNA caused by oscillatory one-sided synthesis from one end to the other.

[0292] Exemplary UvsX recombinase agents tagged with amino acid tag sequences containing intrinsically disordered regions are shown in Table 21 below. Table 21 [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 22-5] [Table 22-6] [Table 22-7] [Table 22-8]

[0293] Recombinase loading protein RPA methods, including those of the present invention, may further comprise / use a recombinase loading protein.

[0294] Any suitable recombinase loading protein may be used in the RPA methods described herein.

[0295] Any one or more IDR-polypeptides of the present invention may be attached / tethered / tagged to any recombinase-loading protein.

[0296] The recombinase loading protein can be derived from a prokaryotic, viral, or eukaryotic organism. Exemplary recombinase loading proteins include E. coli RecO, E. coli RecR, UvsY, and variants or fragments thereof, or combinations thereof. Exemplary UvsY proteins include those from Myoviridae phages such as T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, phage 31, phage 44RR2.8t, Rb49, phage Rb3, and phage LZ2.

[0297] A preferred recombinase loading protein is UvsY, E. coli RecO, E. coli RecR, or any functional variant, analog, homolog, or derivative of any of these proteins. A particularly preferred UvsY recombinase loading protein is Escherichia phage STO UvsY.

[0298] Any combination of any of these proteins may be used.

[0299] Preferred concentrations of these proteins are 0.1 to 24 μM, 6 to 24 μM, 4 to 24 μM, and 4 to 12 μM, preferably about 10 μM, and more preferably about 8.6 μM. The recombinase loading protein may be present at about 0.5 to about 2 times the micromolar concentration of the recombinase agent.

[0300] Exemplary UvsY recombinase loading proteins tagged with amino acid tag sequences containing intrinsically disordered regions are shown in Table 22 below. Table 22 [Table 23]

[0301] Single-stranded stabilizers RPA methods, including those of the present invention, use single-stranded stabilizing agents.

[0302] Any suitable single-stranded stabilizing agent (single-stranded DNA binding protein) may be used in the RPA methods described herein.

[0303] Any one or more IDR-polypeptides of the present invention may be attached / tethered / tagged to any single-stranded stabilizer.

[0304] Single-stranded stabilizing agents are used to stabilize nucleic acids during the various exchange reactions that occur during RPA reactions, particularly to stabilize the recombinase / ssDNA nucleoprotein filament.

[0305] The single-stranded stabilizer can be derived or obtained from any species, for example, a prokaryotic, viral, or eukaryotic species.

[0306] Single-stranded stabilizers include single-stranded DNA binding proteins from E. coli and those derived from Myoviridae phages such as T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, phage 31, phage 44RR2.81, Rb49, phage Rb3, and phage LZ2. Further examples of single-stranded stabilizers include A. denitrificans Alide_2047, Burkholderia thailandensis BthaB_33951, Prevotella pollens HMPREF 9144_0124, and the eukaryotic single-stranded DNA binding protein replication protein A.

[0307] Preferred single-stranded stabilizers are selected from the group consisting of Gp32, E. coli SSB protein, phage T4 Gp32 protein, phage Rb69 Gp32, phage vB_EcoM_NBG1 Gp32, or derivatives thereof, and any combination thereof. A particularly preferred single-stranded stabilizer is Gp32, especially phage vB_EcoM_NBG1 Gp32.

[0308] Any combination of any of these proteins may be used.

[0309] One preferred concentration of the single-stranded stabilizer is about 5 to 30 μM, for example, about 8.6 μM, preferably about 15 to 25 μM, more preferably about 20 μM.

[0310] Exemplary Gp32 single-stranded stabilizers tagged with amino acid tag sequences containing intrinsically disordered regions are shown in Table 23 below. Table 23 [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5] [Table 24-6] [Table 24-7] [Table 24-8] [Table 24-9] [Table 24-10]

[0311] polymerase RPA methods, including those of the present invention, use polymerases.

[0312] Any suitable polymerase can be used in the methods described herein.

[0313] Any one or more IDR-polypeptides of the present invention may be attached / tethered / tagged to any suitable polymerase.

[0314] For DNA synthesis or amplification, a DNA polymerase is preferably used.

[0315] One advantage of the RPA reaction is that there is no limitation on the type of polymerase that can be used: for example, eukaryotic, prokaryotic, and bacteriophage polymerases can be used.

[0316] The DNA polymerase may be a eukaryotic polymerase. Examples of eukaryotic polymerases that may be used include pol-α, pol-β, pol-δ, pol-ε, or any functional variant, analog, homolog, or derivative thereof, and any combination thereof.

[0317] The DNA polymerase may be a prokaryotic polymerase. Examples of prokaryotic polymerases that can be used include 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, Bacillus stearothennophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, Staphylococcus aureus DNA polymerase 1 (Sau), or any functional variant, analog, homolog, or derivative thereof, and any combination thereof.

[0318] The DNA polymerase may be a bacteriophage polymerase. Examples of bacteriophage polymerases that may be used in the methods described herein include Phi-29 DNA polymerase, T7 DNA polymerase, bacteriophage T4 gp43 DNA polymerase, or any functional variant, analog, homolog, or derivative thereof, and any combination thereof.

[0319] DNA polymerases typically contain strand displacement properties.

[0320] DNA polymerases can use the free 3'-hydroxyl of the invading strand to catalyze DNA synthesis by incorporating new nucleotides. Some polymerases can use the 3'-hydroxyl of the invading strand to catalyze synthesis, simultaneously displacing the other strand as synthesis occurs. For example, E. coli polymerase II or III can be used to extend the invaded D-loop. Additionally, E. coli polymerase V, commonly used in SOS lesion-targeted mutagenesis of E. coli, can be used. All of these polymerases can be made highly processive by interacting with and cooperating with the β-dimer clamp and single-stranded DNA-binding protein (SSB) and other components. Other polymerases from prokaryotes, viruses, and eukaryotes can also be used to extend the invading strand.

[0321] Many DNA polymerases have 3'-5' exonuclease activity, and some also have 5'-3' exonuclease activity, which is undesirable in RPA reactions because it leads to the gradual digestion of one DNA strand as the polymerase advances rather than displacing it.

[0322] 3'-5' exonuclease has potential advantages and obvious disadvantages. On the one hand, 3'-5' exonuclease activity can increase the fidelity of the replication reaction and prevent polymerase stalling at misincorporation points. High-fidelity amplification is desirable for many DNA applications. 3'-5' exonuclease activity may also be suitable for amplifying larger DNA fragments, where stalling due to misincorporation may inhibit effective amplification.

[0323] Despite these clear advantages of 3'-5' exonuclease activity, there are some drawbacks: Free oligonucleotides can undergo end-dependent degradation when polymerases with 3'-5' exonuclease activity are used.

[0324] Reaction noise can be reduced by utilizing a polymerase lacking 3'-5' exonuclease activity. This suggests that mispriming may result from oligonucleotides shortened by the 3'-5' exonuclease activity of the polymerase. Consequently, 3'-5' exonuclease editing activity, pyrophosphorylation, or any other similar editing activity may be a source of noise. This can be significantly suppressed by using saturating amounts of the relatively cooperative Gp32 protein with some polymerases, such as the Klenow fragment. Nevertheless, polymerases for use in the methods described herein lacking 3'-5' exonuclease activity can be provided.

[0325] The DNA polymerase may be present at a concentration of from 10,000 units / ml to 10 units / ml, for example, from 5000 units / ml to 500 units / ml.

[0326] adjuvants RPA reactions, including those of the present invention, may further utilize auxiliary agents.

[0327] Any one or more IDR-polypeptides of the present invention may be conjugated / tethered / tagged to any auxiliary agent.

[0328] These auxiliary agents include single-stranded binding proteins, helicases, topoisomerases, resolvases, and any combination thereof. Such agents can have unwinding, relaxing, and scission activity, respectively, on nucleic acids.

[0329] The auxiliary agents may also include RuvA, RuvB, RuvC, RecG, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, DnaX clamp loader, polymerase core complex, DNA ligase, and sliding clamp, and any combination thereof. The sliding clamp may be an E. coli β-dimer sliding clamp, a eukaryotic PCNA sliding clamp, or a T4 sliding clamp gp45, and combinations thereof. The auxiliary agents may further include a DNA polymerase III holoenzyme complex consisting of a β-clamp, a DnaX clamp loader, and a polymerase core complex. These latter auxiliary agents enable the preceding and following RPA to occur.

[0330] The RPA reaction can be performed with one or more additional enzymes that can promote efficient degradation of the recombinase agent / dsDNA complex after the initiation of DNA synthesis, including those that can stimulate 3' to 5' degradation and those that can support 5' to 3' degradation.

[0331] Such additional enzymes include several polymerases that can displace RecA in the 3' to 5' direction and stimulate 3' to 5' degradation of the recombinase agent / dsDNA complex. These DNA polymerases include E. coli PolV and homologous polymerases from other species. The inclusion of E. coli PolV or any functional variant, analog, homolog, or derivative thereof can improve amplification efficiency.

[0332] Other enzymes include a class of enzymes called helicases that can be used to promote the disassembly of RecA from dsDNA. These promote disassembly in both the 5' to 3' and 3' to 5' directions. An ideal helicase complex for stimulating the disassembly of RecA from intermediates consists of the E. coli (E. coli) proteins RuvA and RuvB. The RuvAB complex promotes branch migration, dissociating the RecA protein and allowing RecA to be recycled. Incorporation of RuvAB into the RPA mixture can promote the dissociation of RecA from dsDNA after strand exchange and displacement, allowing de novo synthesis of replication templates from the same site. Furthermore, the RuvAB complex can act in concert with RuvC to ultimately cleave and disassemble Holliday junctions. Adding RuvC to the RPA reaction mixture can resolve complex structures, such as Holliday junctions, formed at the invasion site.

[0333] Yet other enzymes include the E. coli RecG protein, which can stimulate the degradation of branched structures.

[0334] Other enzymes useful in RPA reaction mixtures are those that allow for the continuous generation of RecA nucleoprotein filaments in the presence of ATP and single-stranded stabilizers. Thus, RecO and RecR, and optionally RecF proteins, can be used.

[0335] Exonuclease enzymes are often included in RPA reaction mixtures. They are included for efficient manipulation of cleavable probes. One example of a commonly used exonuclease enzyme is exonuclease III. Any of the IDR polypeptides of the present invention can be conjugated / tethered / tagged to any exonuclease.

[0336] Primer RPA methods use a polymerase to generate copies of a template nucleic acid molecule, and therefore, RPA methods, including those of the present invention, use a primer to initiate extension by the polymerase.

[0337] Most nucleic acid polymerases require a free 3'-hydroxyl moiety on the terminal sugar of a short stretch of double-stranded nucleic acid adjacent to the new synthesis site for incorporation. This double-stranded nucleic acid stretch is typically formed on a template by a short oligonucleotide, called a primer, which typically has a complementary sequence and serves as the initiation site for the polymerase synthesis reaction. In some cases, 3' modifications such as sulfhydryls can be used to prime the synthesis reaction. The primer nucleic acid that base pairs with the template and is extended by the polymerase can be RNA or DNA. Typically, for in vitro reactions, primers are supplied as short, often chemically synthesized, single-stranded DNA (or modified DNA or RNA) and are commonly referred to as oligonucleotide primers. Primers are often sequence-specific, although random primers can also be used. Primers target complementary sequences through their specific base-pairing ability. Hybrid formation between the oligonucleotide primer and the target nucleic acid is typically achieved by incubation of the two in solution under conditions of salt, pH, and temperature that allow spontaneous annealing.

[0338] The primers used in RPA can have a single-stranded region for hybridization to target DNA in the presence of a recombinase agent. The single-stranded region can be, for example, about 10 bases, about 15 bases, about 20 bases, about 25 bases, about 30 bases, about 40 bases, and about 50 bases. Longer regions, such as about 75 bases, about 100 bases, about 150 bases, or more, can theoretically be used. The choice of single-stranded region depends on the complexity of the starting nucleic acid; for example, the human genome may require longer primers, while a plasmid may require much shorter primers.

[0339] Preferred primer lengths are about 30 to about 50 bases, such as 30 to 45 bases, 30 to 40 bases, 30 to 35 bases, 35 to 40 bases, 40 to 45 bases, and 45 to 50 bases. While the above indicated primer lengths are given, recombinases and / or single-stranded binding proteins with optimal primer lengths of less than 30 bases are possible and contemplated.

[0340] The primers used in RPA are preferably DNA, although PNA and RNA are also suitable for use as primers. Note that in natural DNA replication, DNA polymerases actually extend genomic DNA by extension from an RNA primer.

[0341] Primers can be synthesized according to standard techniques. Modified bases and / or linker backbone chemistries may be desirable and, in some cases, functional. Additionally, oligonucleotides can be modified at either the 5' or 3' end with groups that serve various purposes, such as fluorescent groups, quenchers, blocking groups (reversible or irreversible), magnetic tags, proteins, etc. In some cases, single-stranded oligonucleotides can be used for strand invasion, while in other cases, only partially single-stranded nucleic acids can be used, with the sequence of the 5' stretch of the invading nucleic acid already hybridized to the oligonucleotide.

[0342] Primers may contain a 5' region that is not homologous to the target nucleic acid. Note that amplification can be achieved even if the primer is not completely complementary to the target nucleic acid. Primers may be non-complementary by having additional sequences at their 5' ends. These additional sequences may be, for example, sequences for restriction endonuclease recognition sites or sequences complementary to sequencing primers. Restriction endonuclease recognition sites may be useful for subsequent cleavage of the amplified sequence. The use of restriction endonucleases that cleave nucleic acids outside of the restriction endonuclease recognition site is also contemplated. Sequences complementary to sequencing primers may allow for rapid DNA sequencing of the amplified products using commercially available primers or commercially available sequencing equipment.

[0343] Software for designing oligonucleotides for use in in vitro DNA synthesis reactions is well documented, particularly for use in PCR. Considerations for RPA are similar and include optimizing the melting temperature of the oligonucleotides, avoiding hairpin formation within the oligonucleotides, and selecting for complementarity with other oligonucleotides present in a given reaction. Therefore, it is important to design oligonucleotide primer pairs to avoid unwanted side reactions.

[0344] In addition to optimizing oligonucleotide sequence design, there are additional approaches to reducing or eliminating primer-dimer formation. As described elsewhere herein, reaction noise can be reduced by utilizing a polymerase lacking 3'-5' exonuclease activity. This suggests that mispriming may result from oligonucleotides shortened by the 3'-5' exonuclease activity of the polymerase. Consequently, 3'-5' exonuclease editing activity, pyrophosphorylation, or any other similar editing activity may be sources of noise. In addition to using a polymerase lacking exonuclease activity and removing pyrophosphate with pyrophosphatase, the use of synthetic oligonucleotides with non-hydrolyzable backbones at the final and / or penultimate linkages can be beneficial in reducing reaction noise. Alternative backbones can be selected from a wide range of available chemistries, such as phosphorothioates, morpholinos, locked nucleic acids, or peptide nucleic acids.

[0345] Reagents used in the RPA reaction Reagents for use in RPA methods, including those of the present invention, are outlined below.

[0346] dNTPs dNTPs, such as dATP, dGTP, dCTP, and dTTP, as well as their derivatives and analogs, can be added to the RPA reaction. In the leading and lagging strands, RPA, ATP, GTP, CTP, and UTP can also be included for the synthesis of RNA primers. In addition, ddNTPs (ddATP, ddTTP, ddGTP, and ddGTP, as well as their derivatives and analogs) can be used to generate fragment ladders.

[0347] dNTPs can be used at a concentration of 1 mM to 200 mM of each NTP species.

[0348] A mixture of dNTP and ddNTP may be used at a ddNTP concentration (1 mM to 200 mM) that is 1 / 100 to 1 / 1000 of the dNTP concentration.

[0349] RPA can be performed in the presence of ATP, a hydrolyzable ATP analog, or another nucleoside triphosphate. The ATP analog can be, for example, dATP, ddATP, or another nucleoside triphosphate analog such as UTP.

[0350] reducing agent An example of a reducing agent that can be used in the RPA reaction is DTT, whose concentration can be 1 mM to 10 mM, preferably 1 mM.

[0351] ATP ATP or an ATP analogue can be used in the RPA reaction.

[0352] The ATP or ATP analogue may be any of ATP, ATP-γ-S, ATP-β-S, ddATP, or a combination thereof. The preferred concentration of ATP or ATP analogue is 1 mM to 10 mM, preferably 2.5 mM.

[0353] Systems for ATP regeneration Other components of the RPA reaction may include systems for ATP regeneration (i.e., systems that convert ADP to ATP). Such systems may be, for example, phosphocreatine and creatine kinase.

[0354] Because recombinases have an extremely high rate of ATP hydrolysis when bound to nucleic acids, an ATP regeneration system enables sustained recombination reactions. In particular, the UvsX protein has a hydrolysis rate 10-20 times higher than RecA, consuming 200 molecules of ATP per minute per monomer. Numerous systems are available. The creatine kinase / phosphocreatine system is preferred. When UvsX is used, the AMP produced can be converted to ATP. Chicken myokinase may also be used, converting one molecule of AMP and one molecule of ATP into two molecules of ADP. The creatine kinase / phosphocreatine system can then be used to convert ADP to ATP. Poor ATP regeneration can slow the reaction rate.

[0355] In the RPA method described herein, phosphocreatine is preferably used at a concentration of 15 to 25 mM, more preferably 20 mM, and creatine kinase is preferably used at a concentration of about 0.25 to 5.0 μM, more preferably 1 μM.

[0356] Polyvalent metal cations Preferably, the buffer in the RPA reaction contains a polyvalent metal cation. The buffer may contain a functional equivalent of the polyvalent metal cation.

[0357] More preferably, the buffer in the RPA reaction comprises a divalent metal cation. The buffer may comprise a functional equivalent of a divalent metal cation.

[0358] Any suitable polyvalent or divalent metal cation or functional equivalent thereof can be used as a single agent or in combination of agents.

[0359] The particular polyvalent or divalent metal cation or functional equivalent thereof that achieves optimal results in promoting / enhancing IDR-mediated phase separation in an RPA reaction, and the particular concentration of the polyvalent / divalent metal cation used, may depend on the particular IDR polypeptide used. The optimal polyvalent / divalent metal cation or functional equivalent thereof, and its optimal concentration, can be established empirically using routine testing, including the RPA reaction itself and / or the phase separation assays described further herein.

[0360] The divalent metal cation is Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ , Ni 2+ or Cu 2+ These cations may be used alone or in combination. Preferably, they are used as single agents. The preferred divalent metal cations are Mg 2+ , Mn 2+ and Ca 2+A particularly preferred divalent metal cation is Mg 2+ is.

[0361] The preferred concentration range is 30 to 40 mM, more preferably 33 to 39 mM.

[0362] The buffer solution is preferably Mg-free at the concentrations indicated. 2+ ions. More preferably, the buffer contains MgOAc at the concentrations indicated.

[0363] The buffer solution preferably contains Ca at the concentrations indicated. 2+ ions. More preferably, the buffer contains CaCl2 at the concentrations indicated.

[0364] The buffer preferably contains Mn at the concentrations indicated. 2+ ions. More preferably, the buffer contains MnCl2 at the indicated concentration.

[0365] buffer solution The buffer in the RPA reaction can be a Tris-HCl buffer, a Tris-acetate buffer, or a combination thereof. The buffer can be present at a concentration of about 10 mM to about 100 mM. A preferred buffer is a Tris-HCl buffer used at a concentration of about 20 mM to about 30 mM, most preferably 25 mM. The buffered pH can be between 6.5 and 9.0, preferably pH 8.3.

[0366] The buffer solution may contain about 5 mM to about 50 mM, preferably about 10 mM to about 40 mM, of potassium acetate.

[0367] Reaction components A preferred, but non-limiting set of reaction components for an RPA reaction is as follows: Tris HCl pH 8.3 25mM KOAc 7.5mM DTT 1mM ATP 2.5mM Phosphocreatine 20mM Creatine kinase 1 μM dNTPs 1mM Gp32 20 μM UvsX 4.8 μM UvsY 8.6 μM Staphylococcus aureus (S. aureus) DNA polymerase 1 (Sau) 0.135 μM or B. subtilis DNA polymerase 1 (Bsu) Exonuclease III 0.27 μM MgOAc 33mM Forward primer 0.4 μM Reverse primer 0.4 μM Exoprobe 0.12 μM

[0368] RPA reaction conditions RPA reactions, including reactants of the present invention, can be incubated for any suitable length of time.

[0369] Any of the RPA reactions can be incubated for 5 minutes to 16 hours or more, for example, 15 minutes to 3 hours or 30 minutes to 2 hours.

[0370] Incubation can be carried out until the desired degree of amplification is achieved, which can be 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, or 1,000,000-fold amplification.

[0371] One advantage of RPA is that the reaction can be performed at low temperatures compared to techniques that require thermal cycling, such as PCR. An additional advantage of RPA is that temperature is not critical, and while precise control is preferred, it is not absolutely necessary. For example, in a field environment, it is sufficient to incubate the RPA reaction at room temperature or temperatures close to body temperature (35°C–38°C), for example, by placing the sample in a body cavity. Furthermore, the RPA reaction may be performed without temperature-induced melting of the template nucleic acid.

[0372] Thus, any of the RPA reactions can be carried out at any suitable temperature.

[0373] The RPA reaction may be carried out at less than 45° C. The RPA reaction may be carried out at less than 40° C. The RPA reaction may be carried out at less than 35° C. The RPA reaction may be carried out at less than 30° C.

[0374] The RPA reaction can be carried out at 20°C to 50°C, 20°C to 40°C, for example, 20°C to 30°C.

[0375] Lyophilization of RPA reaction components One advantage of the RPA reaction is that the reagents, with the exception of the crowding agent (if used) and buffer, can be lyophilized (i.e., freeze-dried) before use. Lyophilized reagents offer the advantage of not requiring refrigeration to maintain activity. For example, tubes of RPA reagent can be stored at room temperature. This advantage is particularly useful in field conditions where access to refrigeration is limited.

[0376] The RPA reagents can be lyophilized to the bottom of a tube, or onto beads or any other suitable type of solid support. To perform an RPA reaction, the lyophilized reagents are reconstituted in a buffered solution and crowding agent (if used), or simply with buffer or water, depending on the composition of the lyophilized reagent. The target nucleic acid, or a sample suspected of containing the target nucleic acid, is then added. The reconstitution solution may also include sample nucleic acid. The reconstituted reaction is incubated for a period of time, and the amplified nucleic acid, if present, is detected.

[0377] In any one of the RPA methods described herein, the reagents that may be lyophilized prior to use include at least the recombinase agent, recombinase loading protein, single-strand stabilizing agent, DNA polymerase, dNTPs or a mixture of dNTPs and ddNTPs, reducing agent, ATP or an ATP analog, primers, and probes.

[0378] The lyophilization mixture may contain a stabilizer, such as trehalose sugar, at 20 mM to 200 mM, optimally 40 mM to 80 mM in the reconstitution reaction, to improve lyophilization performance and shelf life. If desired, the lyophilized reagent may be stored for 1 day, 1 week, 1 month, or 1 year or more before use.

[0379] Biochemical reaction reagents, such as RPA reagents, may be lyophilized with crowding agents. However, complex interrelated issues may exist that may justify omitting the crowding agent from the lyophilization mixture. For example, users may experience difficulty effectively rehydrating the lyophilized crowding agent, or they may experience other adverse effects, including the need for larger lyophilized pellets. Therefore, there may be advantages to omitting some or all of the crowding agent from the lyophilized material, including reduced pellet size, shorter cycle times, and easier rehydration, among others. However, this has the resulting disadvantage of requiring the addition of fresh crowding agent before use, once the biochemical reaction mixture has been rehydrated and prepared for use. This may be problematic in certain situations, such as point-of-care or field use. An advantage of the IDR-based reagents of the present invention is that they are not expected to exhibit the same drawbacks as crowding agents in a lyophilization setting and can therefore be easily lyophilized with other biochemical reaction components, thus eliminating the need to add fresh additional reagents before use.

[0380] Detection of RPA reaction products Detection of the RPA reaction products can be carried out using any suitable method.

[0381] For example, detection can be performed using electrophoresis on an agarose or PAGE gel followed by ethidium bromide staining.

[0382] Monitoring the RPA reaction can include, for example, removing a fraction of the RPA, reacting, isolating the unincorporated fraction, and detecting unincorporated primers. The size of the unincorporated primers can be less than 50 bp, less than 40 bp, less than 30 bp, or less than 25 bp, and the size of the amplification product can be more than 1 Kb, more than 2 Kb, more than 5 Kb, or more than 10 Kb, so there is a large size difference between the incorporated and unincorporated primers. Isolation of the unincorporated primers can be performed quickly using size exclusion chromatography, such as a spin column. If the primers are labeled, the monitoring procedure, including spin column and measurement (e.g., fluorescence or radioactivity), can be performed in less than 1 minute.

[0383] Another alternative for separating extended from unextended primers involves the use of PAGE, for example, extended primers can be separated from unextended primers by gel electrophoresis in less than 5 minutes.

[0384] Another alternative method for separating the extended primer involves the use of immobilized oligonucleotides. For example, oligonucleotides homologous to sequences uniquely found in the amplified DNA sequence can be used to specifically capture the nucleic acids generated by primer extension. These capture oligonucleotides can be immobilized on a chip or other substrate. The capture of the extended oligonucleotides by the capture oligonucleotides can be performed by RecA protein-mediated methods, or by conventional solution hybridization as needed.

[0385] The use of fluorescent probes is the most commonly used and preferred for detecting RPA amplification products and has the advantage of providing real-time detection.

[0386] These probes are labeled with a fluorophore (e.g., fluorescein (FAM)) and a quencher (e.g., a black hole quencher) in close proximity to the fluorophore. The probes have a blocking group at the 3' end to prevent polymerase extension from the probe. A fluorescent signal is detected when the probe is cleaved, separating the quencher and fluorophore, allowing for real-time detection. The probes contain an abasic site, typically a tetrahydrofuran (THF) or dR group, and cleavage occurs at the abasic site, typically by E. coli exonuclease III (cleaving with THF) or E. coli fpg (glycolase / lyase) (cleaving at the dR group).

[0387] Kit containing RPA reaction components The present invention also provides a kit for performing an RPA reaction.

[0388] The kit may contain any of the reagents described herein in any one of the RPA concentrations described above.

[0389] The kit may comprise any of the IDR-tagged macromolecules and / or IDR-tagged polypeptides described and defined herein. Preferably, the kit comprises an RPA recombinase agent, and / or an RPA recombinase loading protein, and / or a polymerase, and / or first and second nucleic acid primers, and / or an exonuclease, and / or a buffer, and / or a polyvalent metal ion, preferably Mg 2+ , Mn 2+ , Ca 2+ , Co 2+ or Ni 2+ and the like.

[0390] The reagents of the kit may be lyophilized, in which case they may be provided in any suitable amount such that the appropriate reagent concentrations are achieved upon reconstitution.

[0391] polymerase As noted above, any of the IDR amino acid sequences described and defined herein can be tagged to any protein component necessary for carrying out a nucleic acid synthesis reaction.

[0392] Any of the IDR amino acid sequences described and defined herein can be tagged to any protein component necessary for carrying out a nucleic acid synthesis reaction in which a polymerase is used to synthesize a new nucleic acid molecule by extending a primer nucleic acid molecule.

[0393] Thus, any suitable polymerase can be tagged with an IDR amino acid sequence as described and defined herein. The polymerase can be compatible with and can be used in any reaction used to synthesize new nucleic acid molecules by extending primer nucleic acid molecules.

[0394] The polymerase may be compatible with any nucleic acid amplification reaction and may be used for any nucleic acid amplification reaction. The nucleic acid amplification reaction may be a reaction involving thermal cycling. The nucleic acid amplification reaction may be an isothermal amplification reaction. The nucleic acid amplification reaction may be a polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), helicase-dependent amplification (HDA), divergent amplification (RAM), recombinase polymerase amplification (RPA), transcription-mediated amplification (TMA), or nicking enzyme amplification reaction (NEAR).

[0395] Array Tags Any of the IDR-macromolecules or IDR-polypeptides involved in the biochemical reactions described herein, including those involved in the RPA reaction, can contain one or more sequence tags. When used, any such sequence tags are preferably attached to the polypeptide as a fusion protein, as described herein. Sequence tags and means for adding sequence tags to polypeptides are well known in the art.

[0396] Sequence tags can be short amino acid sequences or larger polypeptides, including proteins.

[0397] The sequence tag can be attached to the C-terminus of the polypeptide, the N-terminus of the polypeptide tag, both the C-terminus and N-terminus of the polypeptide, or any amino acid position along the length of the polypeptide in any combination.

[0398] Non-limiting examples of suitable amino acid sequence tags include 6-histidine (6X-His, HHHHHH, SEQ ID NO: 89), c-myc epitope (EQKLISEEDL, SEQ ID NO: 90), FLAG® octapeptide (DYKDDDDK, SEQ ID NO: 91), protein C (EDQVDPRLIDGK, SEQ ID NO: 92), Tag-100 (EETARFQPGYRS, SEQ ID NO: 93), V5 epitope (GKPIPNPLLGLDST, SEQ ID NO: 94), VSV-G (YTDIEMNRLGK, SEQ ID NO: 95), Xpress (DLYDDDDK, SEQ ID NO: 96), and hemagglutinin (YPY-DVPDYA, SEQ ID NO: 97).

[0399] Non-limiting examples of suitable protein tags include β-galactosidase, thioredoxin, His-patch thioredoxin, IgG binding domain, intein chitin binding domain, T7 gene 10, glutathione-S-transferase (GST), green fluorescent protein (GFP), and maltose binding protein (MBP).

[0400] It will be understood by those skilled in the art that sequence tags and protein tags can be used interchangeably, for example, for purification and / or identification purposes.

[0401] Solid-phase biochemical reactions The biochemical reactions carried out in the methods according to the invention may be carried out using solid phase or reversible solid phase techniques. Solid phase reaction systems suitable for carrying out the processes, uses and methods described herein may comprise a surface. Any suitable surface may be used.

[0402] The data described herein demonstrate that biochemical reactions can be performed using the IDR-based reagents according to the present invention using solid-phase techniques in the absence of crowding agents. One specific example is recombinase polymerase amplification of nucleic acids in which primers are bound to a solid surface. Any suitable biochemical reaction suitable for performing using solid-phase methods can be performed using methods according to the present invention that include any of the IDR-based reagents described and defined herein.

[0403] A variety of such solid phase techniques are known in the art and may be used.

[0404] Macromolecules such as polynucleotides, including nucleic acid amplification primers, peptides, haptens, hormones, drugs, etc., can be immobilized on the surface.

[0405] Any suitable macromolecular component of a biochemical reaction can be immobilized on a surface, including the IDR-based reagents described and defined herein.

[0406] Macromolecules such as polynucleotides, e.g., primers for use in amplification reactions, can be immobilized to a surface directly or indirectly. For example, they may be directly attached to the surface by chemical bonding. They may also be indirectly attached to the surface through an intermediate interface.

[0407] The surface may be, for example, a flat surface such as glass, a gel-based material, or the surface of a particulate such as a bead or functionalized quantum dot. The material comprising the surface may itself be attached to a substrate. The substrate may comprise any suitable material, such as glass, plastic, or a polymeric material.

[0408] The macromolecules involved in the biochemical reaction according to the method of the invention may be immobilized on a gel-based material, such as polyacrylamide or a hydrogel, which is itself bound to a support substrate, such as glass or a plastic or polymeric material.

[0409] For example, preformed polynucleotides can be immobilized on a surface by methods commonly used to prepare nucleic acid microarrays. For example, polynucleotides can be synthesized and then spotted or printed on a surface, typically a flat surface. Polynucleotides can be deposited on a surface using contact printing techniques. For example, a solid or hollow tip or pin can be immersed in a solution containing preformed polynucleotides and brought into contact with the surface. Alternatively, polynucleotides can be adsorbed onto a microstamp and then transferred to the surface by physical contact. Non-contact printing techniques include thermal printing or piezoelectric printing, which can eject sub-nanoliter droplets containing preformed polynucleotides from a printing tip using methods similar to those used in inkjet and bubble jet printing.

[0410] Polynucleotides can be synthesized directly on a surface, such as by using the so-called "on-chip" method used to create nucleic acid microarrays. On-chip techniques for creating polynucleotides include photolithography, which involves the use of UV light directed through a photolithographic mask to selectively activate protected nucleotides and allow the subsequent incorporation of new protected nucleotides. Cycles of UV-mediated deprotection and coupling of predetermined nucleotides allow for the in situ generation of polynucleotides with desired sequences. Instead of using photolithographic masks, polynucleotides can be created on a surface by sequentially depositing nucleic acid bases using inkjet printing technology and using cycles of coupling, oxidation, and deprotection to generate oligonucleotides with the desired sequence (for a review, see Kosuri and Church, Nature Methods, 2014, 11, 499-507).

[0411] Surfaces for attachment of macromolecules, including polynucleotides, peptides, haptens, hormones, drugs, and the like, can be made of any suitable material. Typically, the surface can comprise any suitable polymeric material, such as silicon, glass, or polystyrene. The surface can comprise a gel surface, e.g., a polyacrylamide surface, or a hydrogel surface. The gel surface can then be attached or bound to a solid support or substrate, which can comprise any suitable material, such as silicon, glass, or any suitable polymeric material. The surface can comprise a hydrogel material bound to a polystyrene material.

[0412] The surface may be the surface of a microparticle, often called a microsphere or microbead, or simply a bead.

[0413] The surface may comprise a hydrogel material bound to a polystyrene material in the form of microbeads.

[0414] A variety of surface attachment methods and chemistries are available for immobilizing macromolecules, such as polynucleotides, to surfaces such as microbeads. Surfaces can be functionalized or derivatized to facilitate attachment. Such functionalization is known in the art. For example, surfaces can be functionalized with polyhistidine tags (hexahistidine tags, 6xHis-tags, His6-tags, or His-tags®), Ni-NTA, streptavidin, biotin, oligonucleotides, polynucleotides (e.g., DNA, RNA, PNA, GNA, TNA, or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipids, FLAG tags (FLAG octapeptides), polynucleotide-binding proteins, peptides, proteins, antibodies, or antibody fragments. Surfaces can also be functionalized with molecules or groups that specifically bind to the macromolecule to be immobilized or another moiety attached to the macromolecule to be immobilized. Covalent immobilization of macromolecules to surfaces is commonly used. Purely by way of example, carboxylate-modified polystyrene latex surfaces are suitable for covalent attachment of, for example, amine-terminated proteins, DNA, or other molecules, for example, by EDAC-mediated coupling. Other techniques are available. Macromolecules will typically be chemically coupled, but can also be attached to the surface by indirect means, such as affinity interactions. For example, immobilized macromolecules can be functionalized with biotin and attached to an avidin- or streptavidin-coated surface, or vice versa.

[0415] In any of the processes, uses, and methods described and defined herein, the macromolecule can be attached to the surface via one or more covalent bonds. One or more covalent bonds can be formed between a functional group on the surface and a functional group on the macromolecule. The functional group on the macromolecule can be, for example, an amine group, a thiol group, a thiophosphate group, or a thioamide group. The functional group on the surface can be, for example, a bromoacetyl group, optionally provided on a polyacrylamide surface derivatized with N-(5-bromoacetamidylpentyl)acrylamide (BRAPA).

[0416] In any of the processes, uses and methods described and defined herein, the macromolecule may be attached to the surface directly or indirectly via a linker. Any suitable linker that is biocompatible in nature may be used.

[0417] The linker can be a linear linker or a branched linker.

[0418] The linker may comprise a hydrocarbon chain. The hydrocarbon chain may comprise from 2 to about 2000 or more carbon atoms. The hydrocarbon chain may comprise an alkylene group, for example, an alkylene group of from C2 to about 2000 or more. The hydrocarbon chain may have the general formula -(CH2) n - (where n is 2 to about 2000 or more). The hydrocarbon chain may optionally be interrupted by one or more ester groups (i.e., -C(O)-O-) or one or more amide groups (i.e., -C(O)-N(H)-).

[0419] The optional linker may be selected from the group including polyacrylamide, poly(2-hydroxyethyl methacrylate), poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers such as poly(carboxybetaine methacrylate) (PCBMA), poly[N-(3-sulfopropyl)-N-methacryloxyethyl-N,N-dimethylammonium betaine] (PSBMA), glycopolymers, and polypeptides.

[0420] The linker has the general formula -[(CH2-CH2-O) n -PO2 - -O] m - (where n can be from 1 to about 600 or more, and m can be from 1 to 200 or more).

[0421] Any of the above-described linkers may be attached to a macromolecule described herein at one end and to a first functional group at the other end, which may provide a covalent bond to a surface. The first functional group may be, for example, an amine group, a thiol group, a thiophosphate group, or a thioamide group, as further described herein. The surface may be functionalized with an additional functional group to provide a covalent bond to the first functional group. The additional functional group may be, for example, a 2-bromoacetamide group, as further described herein. Optionally, a polyacrylamide surface derivatized using N-(5-bromoacetamidylpentyl)acrylamide (BRAPA) may be provided with a bromoacetyl group. The additional functional group on the surface may be a bromoacetyl group; optionally, a bromoacetyl group may be provided on a polyacrylamide surface derivatized using N-(5-bromoacetamidylpentyl)acrylamide (BRAPA), and the first functional group may be, for example, an amine group, a thiol group, a thiophosphate group, or a thioamide group, as desired. The surface to which the polynucleotide is attached can comprise a gel. The surface may comprise a polyacrylamide surface, such as about 2% polyacrylamide, preferably the polyacrylamide surface is attached to a solid support, such as glass.

[0422] Microparticles and beads that facilitate reversible immobilization can be used. Solid-phase reversible immobilization (SPRI) methods or modified methods are known in the art and can be used (see, e.g., DeAngelis MM et al. (1995) Solid-Phase Reversible Immobilization for the Isolation of PCR Products, Nucleic Acids Research, 23(22):4742-4743).

[0423] The surface can be provided, for example, in the form of paramagnetic beads. Paramagnetic beads can aggregate under the influence of a magnetic field. For example, the paramagnetic surface can be provided with chemical groups, such as carboxyl groups, that act as binding moieties for macromolecules, including nucleic acids, under appropriate binding conditions. The macromolecules can be eluted from such surfaces under appropriate elution conditions. The surfaces of microparticles and beads can be provided with UV-sensitive polycarbonate. Nucleic acids can be bound to the activated surface, for example, in the presence of an appropriate immobilization buffer.

[0424] Microparticles and beads can be allowed to move freely within the reaction solution and then reversibly immobilized, for example, by retaining the beads in microwells or pits etched into the surface. The beads can be localized as part of an array, for example, by the use of unique nucleic acid "barcodes" attached to the beads or by the use of color coding.

[0425] The surface may be part of an electrowetting-on-dielectric (EWOD) system. EWOD systems provide dielectric-coated surfaces that facilitate microfluidic manipulation of very small liquid volumes in the form of microdroplets (see, e.g., Chou, W.L. et al. (2015) Recent Advances in Applications of Droplet Microfluidics, Micromachines, 6:1249-1271). Droplet volumes can be programmably created, moved, dispensed, and bound on-chip by electrowetting techniques. Thus, electrowetting systems offer an alternative means for reversibly immobilizing macromolecules on surfaces and / or manipulating macromolecules immobilized on surfaces.

[0426] Thus, in any one of the methods or uses according to the invention as described or defined herein, the biochemical reaction may be carried out in a solid phase reaction system comprising a surface.

[0427] In any one of the methods or uses according to the invention described or defined herein, in which the biochemical reaction is carried out in a solid-phase reaction system comprising a surface, any macromolecule necessary for carrying out the reaction may be bound to the surface. For example, in one such method, in which the biochemical reaction is a method for amplifying a single-stranded or double-stranded target nucleic acid molecule in an in vitro reaction system described herein, at least one nucleic acid primer and / or reaction macromolecule, and / or IDR-macromolecule and / or one or more polypeptide cofactors may be bound to the surface.

[0428] In any one of the methods or uses according to the invention as described or defined herein, wherein the biochemical reaction is carried out in a solid phase reaction system comprising a surface, the IDR-macromolecules necessary for carrying out the reaction may be bound to the surface.

[0429] In any one of the methods or uses according to the present invention described or defined herein, the biochemical reaction is a recombinase polymerase amplification process that amplifies a double-stranded target nucleic acid molecule in an in vitro reaction system, and the reaction is carried out in a solid-phase reaction system comprising a surface, and the recombinase agent and / or recombinase loading protein and / or single-strand stabilizing agent and / or polymerase and / or exonuclease and / or first nucleic acid primer and / or second nucleic acid primer can be bound to the surface. In one such method or use, the first nucleic acid primer or the second nucleic acid primer can be bound to the surface. Alternatively, in another such method or use, both the first nucleic acid primer and the second nucleic acid primer can be bound to the surface.

[0430] In any one of the methods or uses according to the invention as described or defined herein, wherein the biochemical reaction is carried out in a solid phase reaction system, the surface to which the macromolecule is attached may be a microbead, preferably the microbead comprises silicon, glass, gel, or a polymeric material such as polystyrene, or any combination thereof.

[0431] In any one of the methods or uses described herein, in which a biochemical reaction is carried out in a solid-phase reaction system comprising a surface and / or a substrate, the surface and / or substrate may be provided as a flow cell. Any suitable flow cell compatible with the biochemical reaction to be carried out may be used. A suitable flow cell may include multiple fluid channels through which reagents used to carry out the biochemical reaction can flow. Any one or more macromolecules used to carry out the biochemical reaction may be bound to a surface lining the fluid channels. A suitable flow cell may be used to carry out a biochemical reaction for amplification of single-stranded or double-stranded target nucleic acid molecules. Sequencing reactions carried out using the processes, uses, and methods described herein may also be carried out using a suitable flow cell.

[0432] example The following examples are offered to illustrate, but not to limit, the present invention.

[0433] Example 1. Recombinase polymerase amplification of the Listeria monocytogenes gene hly using Gp32 with an IDR tag from human Otx1. Experimental objectives and overview This experiment was performed to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing a histidine-rich amino acid domain sequence found in the intrinsically disordered region (IDR) of the human homeobox protein Otx1.

[0434] This example demonstrates recombinase polymerase amplification (RPA) of the Listeria monocytogenes gene hly over a range of template concentrations using Gp32 tagged at the C-terminus with a histidine-rich intrinsically disordered region (IDR) domain (Otx1) in the absence of crowding agents.

[0435] Materials and Methods The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS2. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 82 (Table 23).

[0436] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents, using the indicated copies of a DNA template derived from Listeria monocytogenes genomic DNA. The test template was titrated by copy number, as shown in Figure 1.

[0437] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template at a given concentration and 33 mM MgOAc.

[0438] The relevant primers and probes are shown below.

[0439] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0440] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0441] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0442] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0443] Results and Conclusions As shown in Figure 1, the test template was readily detected with high sensitivity within 7 minutes of initiating the RPA reaction. Amplicons were detected with as few as 10 copies of the target.

[0444] Thus, it was found that using this Gp32 IDR-tagged fusion protein, amplification occurred efficiently in the absence of crowding agents such as PEG.

[0445] Example 2. Recombinase polymerase amplification of the Listeria monocytogenes gene hly using Gp32 with an IDR tag from human MafA. Experimental objectives and overview This experiment was performed to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing a histidine-rich domain sequence found in the intrinsically disordered region (IDR) of the human transcription factor MafA.

[0446] This example demonstrates recombinase polymerase amplification (RPA) of the Listeria monocytogenes gene hly over a range of template concentrations using Gp32 tagged at the C-terminus with a histidine-rich intrinsically disordered region (IDR) domain (MafA) in the absence of crowding agents.

[0447] Materials and Methods The specific amino acid sequence of the IDR domain tag used was SGHHGAHHGAHHPAAAAAYEAFRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHGGAGHGGGAGHH (SEQ ID NO: 27). This was ligated to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS5. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 85 (Table 23).

[0448] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents, using the indicated copies of a DNA template derived from Listeria monocytogenes genomic DNA. The test template was titrated by copy number, as shown in Figure 2.

[0449] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template at a given concentration and 33 mM MgOAc.

[0450] The relevant primers and probes are shown below.

[0451] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0452] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0453] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0454] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0455] Results and Conclusions As shown in Figure 2, the test template was readily detected with high sensitivity within 10 minutes of initiating the RPA reaction. Amplicons were detected with as few as 10 copies of the target.

[0456] Thus, it was found that using this Gp32 IDR-tagged fusion protein, amplification occurred efficiently in the absence of crowding agents such as PEG.

[0457] Example 3. Recombinase polymerase amplification of the Listeria monocytogenes gene hly using Gp32 with an IDR tag from Saccharomyces cerevisiae Hrp1. Experimental objectives and overview This experiment was performed to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) of the Saccharomyces cerevisiae Hrp1 protein.

[0458] This example demonstrates recombinase polymerase amplification (RPA) of the Listeria monocytogenes gene hly over a range of template concentrations using Gp32 tagged at the C-terminus with a sequence containing the intrinsically disordered region (IDR) of the yeast Hrp1 protein in the absence of crowding agents.

[0459] Materials and Methods The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-HRP1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 79 (Table 23).

[0460] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents, using the indicated copies of a DNA template derived from Listeria monocytogenes genomic DNA. The test template was titrated by copy number, as shown in Figure 3.

[0461] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template at a given concentration and 33 mM MgOAc.

[0462] The relevant primers and probes are shown below.

[0463] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0464] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0465] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0466] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0467] Results and Conclusions As shown in Figure 3, the test template was readily detected with high sensitivity within 7 minutes of initiating the RPA reaction. Amplicons were detected with as few as 10 copies of the target.

[0468] Thus, it was found that using this Gp32 IDR-tagged fusion protein, amplification occurred efficiently in the absence of crowding agents such as PEG.

[0469] Example 4. Recombinase polymerase amplification of the Listeria monocytogenes gene hly using IDR-tagged Gp32 from Saccharomyces cerevisiae Sup2. Experimental objectives and overview This experiment was performed to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) domain of the Saccharomyces cerevisiae Sup2 protein.

[0470] This example demonstrates recombinase polymerase amplification (RPA) of the Listeria monocytogenes gene hly over a range of template concentrations using Gp32 tagged at the C-terminus with a sequence containing the intrinsically disordered region (IDR) domain of the yeast Sup2 protein in the absence of crowding agents.

[0471] Materials and Methods The specific amino acid sequence of the IDR domain tag used was YNPQGGYQQ (SEQ ID NO: 19). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-Sup1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 72 (Table 23).

[0472] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents, using the indicated copies of a DNA template derived from Listeria monocytogenes genomic DNA. The test template was titrated by copy number, as shown in Figure C.

[0473] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template at a given concentration and 33 mM MgOAc.

[0474] The relevant primers and probes are shown below.

[0475] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0476] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0477] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0478] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0479] Results and Conclusions As shown in Figure 4, the test template was readily detected with high sensitivity within 7 minutes of initiating the RPA reaction. Amplicons were detected with as few as 10 copies of the target.

[0480] Thus, it was found that using this Gp32 IDR-tagged fusion protein, amplification occurred efficiently in the absence of crowding agents such as PEG.

[0481] Example 5. Recombinase polymerase amplification of the human apoB gene using Gp32 with an IDR tag from Saccharomyces cerevisiae Sup2. Experimental objectives and overview This experiment was conducted to evaluate the performance of a number of Gp32 fusion protein preparations containing tags containing the amino acid sequence of the intrinsically disordered region (IDR) domain of the Saccharomyces cerevisiae Sup2 protein. Variable numbers of IDR domain repeat units were evaluated, and a range of fusion protein concentrations was examined.

[0482] This example demonstrates recombinase polymerase amplification (RPA) of the human apolipoprotein B (apoB) gene using Gp32 tagged at the C-terminus with a sequence containing the intrinsically disordered region (IDR) domain of the yeast Sup2 protein in the absence of crowding agents.

[0483] Materials and Methods The specific amino acid sequence of the IDR domain tag used was YNPQGGYQQ (SEQ ID NO: 19). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. Either a single YNPQGGYQQ unit or two, three, or four repeats were attached. The recombinant fusion proteins were purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The fusion proteins were designated Gp32-Sup2 (two repeats, SEQ ID NO: 20), Gp32-Sup3 (three repeats, SEQ ID NO: 21), and Gp32-Sup4 (four repeats, SEQ ID NO: 22). The complete amino acid sequences of the fusion proteins are shown as SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively (Table 23).

[0484] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested together with Gp32-Sup1 in PEG-free amplification, i.e., in the absence of crowding agents, using a DNA template derived from human genomic DNA.

[0485] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, Gp32 fusion protein at the concentrations indicated in Figures 5A-5D, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template and 33 mM MgOAc. The test template copy number used in each case was 10,000.

[0486] The relevant primers and probes are shown below.

[0487] Forward primer: GCAGCTGTATAGCAAATTCCTGTTGAAAGCAG (SEQ ID NO: 101).

[0488] Reverse primer: TCCTGGCTGTATTCATTGTTGTTAAATTGG (SEQ ID NO: 102).

[0489] Probe: CACTGATGCT TTTCCTAGACACGAGATGA[FAM-dT]G[THF]C[BHQ1-dT]TGTGGAGCCTTTGT (SEQ ID NO: 103), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0490] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0491] Results and Conclusions The results are shown in Figure 5. Figure 5A shows the results using a single IDR domain tag unit. Figures 5B-5D show the results using two, three, and four IDR domain tag unit repeats, respectively. The test template was detected approximately 10 minutes after the initiation of the RPA reaction.

[0492] Amplification in the absence of crowding agents such as PEG was found to occur efficiently using these Gp32-IDR tagged fusion proteins. The best performance was observed with a single IDR domain tag unit and two IDR domain tag units. Three IDR domain tag units also gave good performance.

[0493] Example 6. Recombinase polymerase amplification of the Listeria monocytogenes gene hly using Gp32 with an IDR tag from human MafA - Comparison of magnesium ion concentrations. Purpose of the experiment This experiment was conducted to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing the histidine-rich domain sequence found in the intrinsically disordered region (IDR) of the human transcription factor MafA over a range of magnesium concentrations.

[0494] This example demonstrates recombinase polymerase amplification (RPA) of the Listeria monocytogenes gene hly over a range of magnesium concentrations using Gp32 tagged at the C-terminus with a histidine-rich intrinsically disordered region (IDR) domain (MafA) in the absence of crowding agents.

[0495] Materials and Methods The specific amino acid sequence of the IDR domain tag used was SGHHGAHHGAHHPAAAAAYEAFRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHGGAGHGGGAGHH (SEQ ID NO: 27). This was ligated to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS5. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 85 (Table 23).

[0496] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents, using the indicated copies of a DNA template derived from Listeria monocytogenes genomic DNA. The test template was provided at 10,000 copies per reaction, and the magnesium ion concentration was varied from 5.6 mM to 44.8 mM.

[0497] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template and the indicated concentrations of MgOAc ranging from 5.6 mM to 44.8 mM.

[0498] The relevant primers and probes are shown below.

[0499] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0500] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0501] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0502] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0503] Results and Conclusions Amplification in the absence of crowding agents such as PEG was found to occur efficiently using this Gp32 IDR-tagged fusion protein.

[0504] As shown in Figure 6, good amplification was found to occur using this Gp32 IDR-tagged fusion protein in the presence of 28 mM or more magnesium. The optimal concentration in this experiment appeared to be 33.6 mM, and a further increase to 44.8 mM resulted in similar times to detection.

[0505] Example 7. Effect of phosphocreatine levels on recombinase polymerase amplification of human ApoB gene fragments. Experimental objectives and overview This experiment was performed to evaluate the effect of varying phosphocreatine levels on the performance of Gp32 fusion protein preparations containing tags containing the histidine-rich domain sequence found in the intrinsically disordered region (IDR) of the human homeobox protein Otx1.

[0506] This example demonstrates recombinase polymerase amplification (RPA) of a fragment of the human apolipoprotein B (apoB) gene using Gp32 tagged at the C-terminus with a histidine-rich intrinsically disordered region (IDR) domain (Otx1) in the absence of crowding agents.

[0507] Materials and Methods The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS2. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 82 (Table 23).

[0508] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents. Phosphocreatine titration was performed using the human apoB assay. The test template was 10 4 Provided in copy density.

[0509] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, the level of phosphocreatine indicated, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. 10 per reaction. 4 Copy template was added and the reaction was initiated with 33 mM MgOAc.

[0510] Forward primer: GCAGCTGTATAGCAAATTCCTGTTGAAAGCAG (SEQ ID NO: 101).

[0511] Reverse primer: TCCTGGCTGTATTCATTGTTGTTAAATTGG (SEQ ID NO: 102).

[0512] Probe: CACTGATGCT TTTCCTAGACACGAGATGA[FAM-dT]G[THF]C[BHQ1-dT]TGTGGAGCCTTTGT (SEQ ID NO: 103), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0513] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0514] Results and Conclusions Amplification was found to occur using this Gp32 IDR-tagged fusion protein in the absence of crowding agents such as PEG. As shown in Figures 7A, 7B, and 7C, at the standard phosphocreatine concentration used in PEG-based RPA (50 mM), little amplification activity was observed within 20 minutes. Optimal performance was obtained when phosphocreatine was reduced to 20 mM, but good performance was also observed between 15 and 25 mM, and lower levels of amplification were observed within 20 minutes at 30 and 35 mM.

[0515] Example 8. Recombinase polymerase amplification of the Listeria monocytogenes gene hly using IDR-tagged Gp32 from Saccharomyces cerevisiae Hrp1 - Comparison of salt concentrations. Experimental objectives and overview This experiment was conducted to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) of the Saccharomyces cerevisiae Hrp1 protein. Potassium acetate was used in this experiment to evaluate performance over a range of salt concentrations.

[0516] This example demonstrates that recombinase polymerase amplification (RPA) of the Listeria monocytogenes gene hly can be optimized over a range of salt concentrations using Gp32 tagged at the C-terminus with the intrinsically disordered region (IDR) of the Saccharomyces cerevisiae Hrp1 protein in the absence of crowding agents.

[0517] Materials and Methods The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-HRP1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 79 (Table 23).

[0518] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested in PEG-free amplification, i.e., in the absence of crowding agents, using 100 copies of a DNA template derived from Listeria monocytogenes genomic DNA. Potassium acetate concentrations were varied from 10 mM to 100 mM.

[0519] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. Reactions were initiated by the addition of template and 33 mM MgOAc.

[0520] The relevant primers and probes are shown below.

[0521] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0522] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0523] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0524] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0525] Results and Conclusions Amplification in the absence of crowding agents such as PEG was found to occur efficiently using this Gp32 IDR-tagged fusion protein.

[0526] It was also found that amplification in the absence of crowding agents using this Gp32 IDR-tagged fusion protein could be optimized over a range of salt concentrations, with potassium acetate being a representative example.

[0527] As shown in Figure 8, good amplification was observed using this Gp32 IDR-tagged fusion protein in the presence of 10 mM or more potassium acetate. The optimal concentration range in this experiment appeared to be between 10 and 40 mM. At concentrations above 40 mM, less efficient amplification was observed.

[0528] Example 9. Recombinase polymerase amplification of human ApoB gene fragment using IDR-tagged Gp32 from Saccharomyces cerevisiae Sup2 - Synergistic effect with crowding agents. Experimental objectives and overview This experiment was conducted to evaluate the effect of low concentrations of a crowding agent, in this case PEG, on the reaction efficiency of a Gp32 fusion protein preparation containing a histidine-rich sequence found in the intrinsically disordered region of the yeast SUP2 gene, specifically the Sup1 sequence YNPQGGYQQ (SEQ ID NO: 19), attached to the C-terminus of phage vB EcoM NBG1 Gp32. The performance of this fusion protein was compared to that of a Gp32 protein lacking the Sup1 IDR tag in recombinase polymerase amplification of a fragment of the human apolipoprotein B (apoB) gene.

[0529] It was found that low concentrations of clowning agents can increase the reaction efficiency of Sup1 IDR-tagged Gp32, achieving conditions under which a synergistic effect can be observed.

[0530] Materials and Methods Gp32-Sup1 The specific amino acid sequence of the IDR domain tag used was YNPQGGYQQ (SEQ ID NO: 19). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-Sup1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 72 (Table 23).

[0531] Gp32(7His) Phage vB EcoM NBG1 Gp32 was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the heptahistidine tag placed at the very C-terminus of the protein under test. The fusion protein was designated Gp32(7His). The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 65 (Table 23).

[0532] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was tested in RPA reactions using a DNA template containing a fragment of the human apolipoprotein B (apoB) gene, either in the presence or absence of crowding agents.

[0533] Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 50 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM Gp32 fusion protein, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase, and 0.27 μM exonuclease III. The reaction was initiated by the addition of template and 33 mM MgOAc. The test template copy number used in each case was 10,000. PEG was added to the final concentration indicated in the relevant figures. The PEG species used had a molecular weight of 35,000.

[0534] The relevant primers and probes are shown below.

[0535] Forward primer: GCAGCTGTATAGCAAATTCCTGTTGAAAGCAG (SEQ ID NO: 101).

[0536] Reverse primer: TCCTGGCTGTATTCATTGTTGTTAAATTGG (SEQ ID NO: 102).

[0537] Probe: CACTGATGCT TTTCCTAGACACGAGATGA[FAM-dT]G[THF]C[BHQ1-dT]TGTGGAGCCTTTGT (SEQ ID NO: 103), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0538] The reactions were then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0539] Results and Conclusions The results are shown in Figure 9. Figure 9 shows that when the Gp32-Sup1 IDR-tagged fusion protein was tested in the absence of the crowding agent PEG, the test template was efficiently detected.

[0540] When the Gp32-7His fusion protein without the Sup1 IDR tag was tested in the presence of the crowding agent PEG between 0.5% and 2%, a small but detectable amount of amplification product was observed.

[0541] When the Gp32-Sup1 IDR-tagged fusion protein was tested in the presence of the crowding agent PEG, the test template was efficiently detected. In this case, a synergistic effect could be observed in the amount of amplification product exceeding the total amount when comparing the amounts observed for (i) the Gp32-Sup1 IDR-tagged fusion protein in the absence of PEG and (ii) the Gp32-7His fusion protein without the Sup1 IDR tag in the absence of PEG (see, e.g., Figure 9, comparing Sup1 1% PEG with Sup1 0% PEG + normal GP32 1% PEG).

[0542] These results demonstrate that when IDR-tagged macromolecular components of a reaction are combined with low concentrations of crowding agents, an enhanced effect on the efficiency with which a biochemical reaction is carried out can be observed, and that when IDR-tagged macromolecular components of a reaction are combined with low concentrations of crowding agents, conditions can be achieved that promote a synergistic effect on reaction efficiency.

[0543] Example 10. Enhancement of phase separation by IDR tags in the presence of multivalent metal cations. Experimental objectives and overview This experiment was performed to evaluate the effect of multivalent metal cations in promoting phase separation driven / triggered by several Gp32 fusion proteins, each bearing a tag containing an intrinsically disordered region (IDR) domain amino acid sequence, in an aqueous in vitro biochemical system.

[0544] The examples demonstrate that tags containing IDR domain amino acid sequences can surprisingly promote phase separation, and even more surprisingly, this effect is enhanced by the presence of polyvalent metal cations.

[0545] Materials and Methods Gp32-HIS2 fusion protein The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The complete amino acid sequence of the Gp32-HIS2 fusion protein is provided as SEQ ID NO: 82 (Table 23).

[0546] Gp32-HRP1 fusion protein The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The complete amino acid sequence of the Gp32-HRP1 fusion protein is provided as SEQ ID NO: 79 (Table 23).

[0547] Gp32-Sup1 fusion protein The specific amino acid sequence of the IDR domain tag used was YNPQGGYQQ (SEQ ID NO: 19). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The complete amino acid sequence of the Gp32-Sup1 fusion protein is provided as SEQ ID NO: 72 (Table 23).

[0548] Gp32-Fib fusion protein The specific amino acid sequence of the IDR domain tag used was PGFSPRGGGFGGRGGFGDRGGRGGRGGFGGGRGRGGGFRGRGRGR (SEQ ID NO: 1). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The complete amino acid sequence of the Gp32-Fib fusion protein is provided as SEQ ID NO: 69 (Table 23).

[0549] Phase separation assay The method outlined below applies to all fusion proteins tested. The volume of fusion protein solution used depended on the protein concentration after purification.

[0550] In each case, a 50 μl solution was made containing tagged fusion protein at a final concentration of 1000 ng / μl and metal ions in either acetate or chloride form at the target concentrations shown below and in the relevant figures presented herein.

[0551] For the Gp32-HIS2 fusion, the protein concentration after purification was 48 mg / ml. 1.04 μl of this fusion protein was used in each 50 μl reaction to achieve a final concentration of 1000 ng / μl in solution. For the Gp32-HRP1 fusion, the protein concentration after purification was 39 mg / ml. 1.28 μl of this fusion protein was used in each 50 μl reaction to achieve a final concentration of 1000 ng / μl in solution. For the Gp32-Sup1 fusion, the protein concentration after purification was 36 mg / ml. 1.4 μl of this fusion protein was used in each 50 μl reaction to achieve a final concentration of 1000 ng / μl in solution. For the Gp32-Fib fusion, the protein concentration after purification was 20.2 mg / ml. 2.48 μl of this fusion protein was used in each 50 μl reaction to achieve a final concentration of 1000 ng / μl in solution.

[0552] In these experiments, the divalent metal cation concentrations required to cause detectable enhanced phase separation were tested for representative divalent metal cations: magnesium (MgOAc), manganese (MgCl), and calcium (CaCl). The acetate forms of manganese and calcium were not used simply because of their known instability in solution. Manganese oxidizes over time in acetate solutions, and calcium acetate appears to support the growth of some bacteria in solution, whereas calcium chloride does not.

[0553] After formation of the mixture containing water, IDR-tagged protein, and divalent metal cations, the mixture was vortexed and spun down, and a 10 μl sample of the mixture was transferred to a DHC-B01 C-Chip hemocytometer slide. The slide was then imaged under a microscope at 400x magnification. Detection of phase separation was assessed by the formation of sphere-like spherical foci / particles, which could be visually identified through magnification and counted using a hemocytometer. Spherical foci counts per unit volume could then be performed. Spherical foci counts were performed by counting the number of spherical foci formed in a magnified area of ​​218 μm x 175 μm at 400x magnification. This was done by dividing the magnified image into 20 square segments (4 x 5 of the image), counting the spherical foci in one of these segments, and then multiplying this number by 20.

[0554] Results and Conclusions The transition between just below and just above the minimum detectable phase-separated concentration (MPSC) in this assay was observed to occur very suddenly in all reactions performed. Just below the MPSC, no detectable phase-separated aqueous particles were observed, and the solution was found to be empty of visually detectable particles (spherical foci). Above the MPSC, the transition was very clear, with the sudden formation of hundreds of visually detectable particles (spherical foci).

[0555] The size of the spherical foci was found to vary and correlate with the IDR tag and divalent metal cation used. The specific size of the spherical foci was not determined to be critical.

[0556] The spherical foci existed as particulate structures that were broadly spherical in shape. For any given IDR tag and any given divalent metal ion combination, the average diameter of the population of spherical foci can be readily determined using standard methods.

[0557] Results using individual fusion proteins are summarized below.

[0558] Gp32-HIS2 fusion protein The minimum concentration of magnesium required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 10 mM, with approximately 600 particles (spherical foci) counted within the field of view.

[0559] The minimum concentration of calcium ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 12 mM, with approximately 500 particles (spherical foci) counted within the field of view.

[0560] The minimum concentration of manganese ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 2 mM, with approximately 180 particles (spherical foci) counted within the field of view.

[0561] A representative magnified image is shown in Figure 10A.

[0562] Gp32-HRP1 fusion protein Under these conditions, the minimum concentration of magnesium ions required to enhance the formation of detectable phase-separated aqueous particles was determined to be 16 mM, at which approximately 580 particles (spherical foci) were counted within the field of view.

[0563] The minimum concentration of calcium ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 24 mM, at which approximately 240 particles (spherical foci) were counted within the field of view.

[0564] The minimum concentration of manganese ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 6 mM, at which approximately 260 particles (spherical foci) were counted within the field of view.

[0565] A representative magnified image is shown in Figure 10B.

[0566] Gp32-Sup1 fusion protein Under these conditions, the minimum concentration of magnesium ions required to enhance the formation of detectable phase-separated aqueous particles was determined to be 24 mM, at which approximately 280 particles (spherical foci) were counted within the field of view.

[0567] The minimum concentration of calcium ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 32 mM, at which approximately 460 particles (spherical foci) were counted within the field of view.

[0568] The minimum concentration of manganese ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 4 mM, at which approximately 220 particles (spherical foci) were counted within the field of view.

[0569] A representative magnified image is shown in Figure 10C.

[0570] Gp32-Fib fusion protein The minimum concentration of magnesium ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 500 μM, at which approximately 340 particles (spherical foci) were counted within the field of view.

[0571] The minimum concentration of calcium ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 1 mM, at which approximately 500 particles (spherical foci) were counted within the field of view.

[0572] The minimum concentration of manganese ions required to enhance the formation of detectable phase-separated aqueous particles under these conditions was determined to be 500 μM, at which approximately 360 particles (spherical foci) were counted within the field of view.

[0573] A representative magnified image is shown in Figure 10D.

[0574] Using these assays, it was determined that the functional ability of an IDR or IDR domain to enhance the formation of detectable phase-separated aqueous particles in an in vitro biochemical environment when tagged to a protein can be demonstrated when 10 or more particles (spherical foci) are formed in a magnified area of ​​218 μm × 175 μm at 400× magnification. The functional ability of an IDR or IDR domain to induce phase separation in an in vitro biochemical environment when tagged to a protein can be demonstrated when preferably 50 or more particles (spherical foci) are formed in a magnified area of ​​218 μm × 175 μm at 400× magnification, more preferably 100 or more particles (spherical foci) are formed.

[0575] As used herein, the term "spherical focus" is synonymous with "globule," "particle," or "spherical particle," and these terms can be used interchangeably.

[0576] Example 11. Formation of spherical foci by IDR tags in the presence of polyvalent metal cations. Experimental objectives and overview This experiment was conducted to evaluate the effect of polyvalent metal cations on the promotion of phase separation driven / triggered by several Gp32 fusion proteins, each bearing a tag containing an intrinsically disordered region (IDR) domain amino acid sequence, in an in vitro biochemical reaction system.

[0577] The examples demonstrate that tags containing IDR domain amino acid sequences can promote / enhance phase separation, and that this effect occurs in the presence of various multivalent metal cations.

[0578] Materials and Methods Gp32-Fib fusion protein The specific amino acid sequence of the IDR domain tag used was PGFSPRGGGFGGRGGFGDRGGRGGRGGFGGGRGRGGGFRGRGRGR (SEQ ID NO: 1). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The complete amino acid sequence of the Gp32-Fib fusion protein is provided as SEQ ID NO: 69 (Table 23).

[0579] Gp32-Sup1 fusion protein The specific amino acid sequence of the IDR domain tag used was YNPQGGYQQ (SEQ ID NO: 19). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR domain tag at the C-terminus of the fusion protein. The complete amino acid sequence of the Gp32-Sup1 fusion protein is provided as SEQ ID NO: 72 (Table 23).

[0580] Gp32-HIS2 fusion protein The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The complete amino acid sequence of the Gp32-HIS2 fusion protein is provided as SEQ ID NO: 82 (Table 23).

[0581] Gp32-HRP1 fusion protein The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The complete amino acid sequence of the Gp32-HRP1 fusion protein is provided as SEQ ID NO: 79 (Table 23).

[0582] Gp32-HIS5 fusion protein The specific amino acid sequence of the IDR domain tag used was SGHHGAHHGAHHPAAAAAYEAFRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHGGAGHGGGAGHH (SEQ ID NO: 27). This was ligated to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The complete amino acid sequence of the Gp32-HIS5 fusion protein is shown as SEQ ID NO: 85.

[0583] Phase separation assay The method outlined below applies to all fusion proteins tested. The volume of fusion protein solution used depended on the protein concentration after purification.

[0584] In each case, 50 μl of solution was prepared containing the tagged fusion protein at a final concentration of 1000 ng / μl (29.4 μM) and a divalent metal cation. The metal ions tested were Mg 2+ (MgOAc), Mn 2+ (MnCl2) and Ca 2+(CaCl2), which were used at a final concentration of 20 mM in each case.

[0585] After formation of the mixture containing water, IDR-tagged protein, and polyvalent metal cations, the mixture was vortexed, spun down, and a 10 μl sample of the mixture was transferred to a DHC-B01 C-Chip hemacytometer slide. The slide was then imaged at 400x magnification using a bright-field microscope. Phase separation was assessed by the formation of sphere-like spherical foci (particles), which could be visually identified by magnification and counted using a hemacytometer.

[0586] Results and Conclusions Representative enlarged images are shown in Figures 11A to 11E.

[0587] For each of the IDR-tagged Gp32 fusion proteins tested, detectable phase separation was observed as determined by the formation of detectable globular-like phase-separated particles (spherical foci). 2+ , Mn 2+ and Ca 2+ The effect was observed in the presence of divalent metal ions.

[0588] Therefore, the ability of polyvalent metal ions to induce / enhance phase separation appears to be a general property applicable to a wide range of different IDR tags with quite different amino acid sequences.

[0589] Example 12. Formation of spherical foci by IDR-tagged Gp32 from Saccharomyces cerevisiae Hrp1. Experimental objectives and overview This experiment was performed to evaluate the ability of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) of the Saccharomyces cerevisiae Hrp1 protein to form spherical foci in an exemplary in vitro biochemical reaction environment in the absence of crowding agents.

[0590] The examples demonstrate that tags comprising IDR domain amino acid sequences were able to promote / enhance phase separation, as determined by the formation of detectable phase-separated aqueous particles, in exemplary in vitro biochemical reaction environments and in the absence of crowding agents.

[0591] Materials and Methods The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-HRP1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 79 (Table 23).

[0592] To test the effect of IDR domain sequence tags, an exemplary in vitro biochemical reaction environment was created, in this case featuring a recombinase polymerase amplification reaction.

[0593] The reaction was set up according to the following protocol. The reaction mixture was made with the following components: 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.2 μM forward primer, 0.2 μM reverse primer, 0.516 μM probe, 22.6 μM Gp32-HRP fusion, 8.4 μM UvsX, 15.3 μM UvsY, 0.135 μM S. aureus DNA polymerase (large subunit), and 0.27 μM exonuclease III. Gp32, UvsX, UvsY, polymerase, and exonuclease III were prepared as a premix and then added to the mixture of primers, buffer, nucleotides, and creatine kinase in one step. The total volume was 44 μl. Once combined, 6 μl of 280 mM MgOAc was added to the mixture to achieve a final concentration of 33 mM. 10 μl of the reaction mixture was then transferred to a C-chip hemocytometer slide placed on a heating stage set at 39°C, after which it was observed under a microscope and images were taken under brightfield and fluorescent conditions.

[0594] The relevant primers and probes are shown below.

[0595] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0596] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0597] Probe: CCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATG (SEQ ID NO: 104) labeled with FAM (fluorescein).

[0598] Results and Conclusions As shown in Figure 12, the HRP tag attached to Gp32 promoted the formation of many detectable phase-separated aqueous particles (spherical foci) that appeared densely packed in the oligonucleotide (as detected by the fluorescently labeled probe).

[0599] Separate experiments were performed using identical materials and conditions, except that the Gp32 protein was tagged only with the heptahistidine sequence and not with the HRP IDR tag. In these experiments, no globular foci formed (data not shown), indicating that the formation of globular foci was specifically driven by the IDR tag and, consequently, that the heptahistidine sequence is not a functional IDR as described herein.

[0600] The results demonstrate the functional ability of IDR domain tags, in this case represented by the Saccharomyces cerevisiae HRP1 amino acid sequence tag described above, to promote detectable phase separation in an in vitro biochemical reaction environment, in this case a reaction mixture environment characterized by a recombinase polymerase amplification reaction, and in the absence of crowding agents.

[0601] Example 13. Formation of spherical foci by Gp32 carrying an IDR tag from human Otx1. Experimental objectives and overview This experiment was performed to evaluate the ability of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) of the human Otx1 protein to form spherical foci in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent.

[0602] The examples demonstrate that tags comprising IDR domain amino acid sequences were able to promote detectable phase separation in exemplary in vitro biochemical reaction environments and in the absence of crowding agents.

[0603] Materials and Methods The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS2. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 82 (Table 23).

[0604] To test the effect of IDR domain sequence tags, an exemplary in vitro biochemical reaction environment was created, in this case featuring a recombinase polymerase amplification reaction.

[0605] The reaction was set up according to the following protocol. The reaction mixture was made with the following components: 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.2 μM forward primer, 0.2 μM reverse primer, 0.516 μM probe, 22.6 μM Gp32-HIS2 fusion, 8.4 μM UvsX, 15.3 μM UvsY, 0.135 μM S. aureus DNA polymerase (large subunit), and 0.27 μM exonuclease III. Gp32-His2, UvsX, UvsY, polymerase, and exonuclease III were prepared as a premix and then added to the mixture of primers, buffer, nucleotides, and creatine kinase in one step. The total volume was 44 μl. Once combined, 6 μl of 280 mM MgOAc was added to the mixture to achieve a final concentration of 33 mM. 10 μl of the reaction mixture was then transferred to a C-chip hemocytometer slide placed on a heating stage set at 39°C, after which it was observed under a microscope and images were taken under brightfield and fluorescent conditions.

[0606] The relevant primers and probes are shown below.

[0607] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0608] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0609] Probe: CCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATG (SEQ ID NO: 104) labeled with FAM (fluorescein).

[0610] Results and Conclusions As shown in Figure 13, the HIS2 IDR tag attached to Gp32 promoted the formation of many detectable phase-separated aqueous particles (spherical foci) that appeared densely packed in the oligonucleotide (as detected by the fluorescently labeled probe). Note that the globules appear smaller in size compared to those formed when the HRP IDR tag was attached to Gp32, as described further herein.

[0611] The results demonstrate the functional ability of IDR domain tags, in this case represented by the HIS2 amino acid sequence tag described above, to promote detectable phase separation in an in vitro biochemical reaction environment, in this case a reaction mixture environment characterized by a recombinase polymerase amplification reaction, and in the absence of crowding agents.

[0612] Example 14. Effect of polyvalent metal cations on the formation of spherical foci by IDR-tagged Gp32 from Saccharomyces cerevisiae Hrp1. Experimental objectives and overview This experiment was performed to evaluate the effect of polyvalent metal cations on the ability of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) of the Saccharomyces cerevisiae Hrp1 protein to form spherical foci in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent.

[0613] The examples demonstrate that tags containing IDR domain amino acid sequences can promote / enhance phase separation in the absence of crowding agents, as determined by the formation of detectable phase-separated aqueous particles, and that phase separation is enhanced in the presence of multivalent metal cations, allowing the determination of optimized concentrations for promoting phase separation.

[0614] Materials and Methods The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-HRP1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 79 (Table 23).

[0615] To test the effect of IDR domain sequence tags in the presence of varying concentrations of divalent metal cations, an exemplary in vitro biochemical reaction environment was created, in this case featuring a recombinase polymerase amplification reaction.

[0616] The reaction was set up according to the following protocol. The reaction mixture was made with the following components: 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.26 μM forward primer, 0.26 μM reverse primer, 0.4 μM probe, 22.6 μM Gp32-HRP fusion, 8.4 μM UvsX, and 15.3 μM UvsY. Gp32, UvsX, and UvsY were prepared as premixes and then added to the primer, buffer, nucleotide, and creatine kinase mixture in one step. MgOAc was added to the mixture to achieve the final concentrations shown in the relevant figures. 10 μl of the reaction mixture was then transferred to a C-chip hemacytometer slide placed on a heating stage set at 39°C, followed by observation under a microscope. Images were taken under brightfield and fluorescent conditions.

[0617] The relevant primers and probes are shown below.

[0618] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0619] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0620] Probe: CCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATG (SEQ ID NO: 104) labeled with FAM (fluorescein).

[0621] Results and Conclusions As shown in Figures 14A and 14B, the HRP IDR tag attached to Gp32 promoted the formation of many detectable phase-separated aqueous particles (spherical foci) that appeared densely packed in the oligonucleotide (as detected by a fluorescently labeled probe).

[0622] Spherical foci were clearly visible at 22.4 mM MgOAc. Optimal formation of spherical foci occurred at 33 mM MgOAc. At concentrations above 33 mM, some clumps of globules began to be observed.

[0623] Notably, 33 mM MgOAc is the magnesium concentration at which optimal amplification efficiency is observed in recombinase polymerase amplification (RPA) reactions using IDR-tagged Gp32 in the absence of crowding agents, as described herein. Thus, the efficiency of IDR-tag-mediated formation of spherical foci surprisingly correlates with the efficiency of amplification in an exemplary biochemical reaction in an in vitro system in the absence of crowding agents, in this case, an RPA reaction using an IDR-tagged protein as an example of a test biochemical system.

[0624] The results support the surprising conclusion that the performance of IDR domain sequence tags in driving / increasing the efficiency of biochemical reactions in the absence of crowding agents can be correlated with the efficiency of phase separation, which in turn appears to be enhanced by the concentration of polyvalent metal cations or their functional equivalents included in the system, which affects the function of intrinsically disordered regions or domains.

[0625] Example 15. Effect of polyvalent metal cations on the formation of spherical foci by IDR-tagged Gp32 from human Otx1. Experimental objectives and overview This experiment was performed to evaluate the effect of polyvalent metal cations on the ability of a Gp32 fusion protein bearing a tag containing the intrinsically disordered region (IDR) of the human Otx1 protein to form particles / spherical foci in an exemplary in vitro biochemical reaction environment in the absence of crowding agents.

[0626] The examples demonstrate that tags comprising IDR domain amino acid sequences can promote detectable phase separation in exemplary in vitro biochemical reaction environments and in the absence of crowding agents, that detectable phase separation is enhanced by the presence of polyvalent metal cations, and that optimized concentrations for promoting detectable phase separation can be determined.

[0627] Materials and Methods The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS2. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 82 (Table 23).

[0628] To test the effect of IDR domain sequence tags in the presence of varying concentrations of divalent metal cations, an exemplary in vitro biochemical reaction environment was created, in this case featuring a recombinase polymerase amplification reaction.

[0629] The reaction was set up according to the following protocol. The reaction mixture was made with the following components: 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.26 μM forward primer, 0.26 μM reverse primer, 0.4 μM probe, 20 μM Gp32-HIS2 fusion, 8.4 μM UvsX, and 8.6 μM UvsY. Gp32, UvsX, and UvsY were prepared as premixes and then added to the primer, buffer, nucleotide, and creatine kinase mixture in one step. MgOAc was added to the mixture to achieve the final concentrations shown in the relevant figures. 10 μl of the reaction mixture was then transferred to a C-chip hemacytometer slide placed on a heating stage set at 39°C and observed under a microscope. Images were taken under brightfield and fluorescent conditions.

[0630] The relevant primers and probes are shown below.

[0631] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0632] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0633] Probe: CCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATG (SEQ ID NO: 104) labeled with FAM (fluorescein).

[0634] Results and Conclusions As shown in Figures 15A and 15B, the HIS2 tag attached to Gp32 promoted the formation of many spherical foci that appeared dense in the oligonucleotide (as detected by a fluorescently labeled probe).

[0635] Spherical foci were clearly visible at 22.4 mM MgOAc. Optimal formation of spherical foci occurred between 33 and 39 mM MgOAc. At concentrations above 39 mM, some clumps of globules began to be observed.

[0636] Notably, 33-39 mM MgOAc is the magnesium concentration at which optimal amplification efficiency is observed in recombinase polymerase amplification (RPA) reactions using IDR-tagged Gp32 in the absence of crowding agents, as described herein. Thus, the efficiency of IDR-tag-mediated formation of spherical foci surprisingly correlates with the efficiency of amplification in an exemplary in vitro biochemical reaction in the absence of crowding agents, in this case, an RPA reaction using an IDR-tagged protein as an example of a test biochemical system.

[0637] The results support the surprising conclusion that the performance of IDR domain sequence tags in driving / increasing the efficiency of biochemical reactions in the absence of crowding agents can be correlated with the efficiency of phase separation, which in turn appears to be enhanced by the concentration of polyvalent metal cations or their functional equivalents included in the system, which affects the function of intrinsically disordered regions or domains.

[0638] Example 16. Effect of magnesium concentration on the formation of spherical foci by IDR-tagged Gp32 from Saccharomyces cerevisiae Hrp1. Experimental objectives and overview This experiment was performed to evaluate the effect of magnesium ions on the ability of a Gp32 fusion protein preparation containing a tag containing the intrinsically disordered region (IDR) of the Saccharomyces cerevisiae Hrp1 protein to form spherical foci in an exemplary in vitro biochemical reaction environment in the absence of a crowding agent.

[0639] The examples demonstrate that tags containing IDR domain amino acid sequences were able to promote / enhance phase separation in an exemplary in vitro biochemical reaction environment and in the absence of crowding agents, as determined by the formation of detectable phase-separated aqueous particles, that phase separation was dependent on the presence of magnesium ions, and that all protein components of the reaction mixture were found to be associated with the phase-separated particles rather than the bulk phase.

[0640] Materials and Methods The specific amino acid sequence of the IDR domain tag used was GGNNGGNNMNRRGGNFGNQGDFNQMYQNPMMGGYNPMMNPQAMTDYYQKMQEYYQQMQ (SEQ ID NO: 9). This was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on an additional heptahistidine tag placed at the very C-terminus of the fusion protein under test, i.e., after the IDR tag at the C-terminus of the fusion protein. The fusion protein was designated Gp32-HRP1. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 79 (Table 23).

[0641] An exemplary in vitro biochemical reaction environment was created to test the effect of the IDR domain sequence tag either in the presence or absence of magnesium ions.

[0642] Reactions were set up according to the following protocol: A 1 ml reaction mixture was made containing the following components: 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 0.4 μM forward primer, 0.4 μM reverse primer, 0.4 μM probe, 20.26 μM Gp32-HRP fusion, 5 μM UvsX, 8.67 μM UvsY, 0.127 μM S. aureus DNA polymerase (large subunit), and either 0 mM or 33.6 mM MgOAc.

[0643] The relevant primers and probes are shown below.

[0644] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0645] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0646] Probe: CCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATG (SEQ ID NO: 104) labeled with FAM (fluorescein).

[0647] A photograph of the finished mixture was taken.

[0648] The mixture was spun at 2,000 rcf for 1 minute. The supernatant was removed from the MgOAc mixture. A small pellet, likely composed of phase-separated globules / particles, remained in the mixture with 33.6 mM MgOAc. No similar pellet was observed in the mixture without MgOAc. 10 μl of 1% SDS solution was added to the pellet for solubilization. The pellet volume was estimated to be 4.5 μl, for an estimated total volume of 14.5 μl. 1 μl of each sample was analyzed by SDS-PAGE.

[0649] Results and Conclusions As shown in Figure 16A, adding magnesium acetate to 1 ml of RPA mixture caused the mixture to become opaque. This was not observed in the absence of magnesium acetate. This opaque effect was the same as that seen in comparable, smaller reactions, where microscopy was observed to form spherical focus / phase-separated particles with typical diameters estimated to be in the 2-3 micron range, typically about 200-400 particles per nanoliter. When subjected to centrifugation, these opaque mixtures clarified, and a pellet or lower phase was seen to form at the bottom of the tube, which was assumed to be a clump of particles that had coalesced into a single volume (Figure 16B). The estimated volume of this pellet fraction was approximately 4 μl, which is the predicted total volume of particles expected to form assuming a mean particle size of 3 μm (thus a volume of approximately 13 femtoliters) and an abundance of approximately 400 particles per nanoliter, which is 400,000 particles / microliter based on hemocytometer / microscope field calculations, generating approximately 5 nl of particles per microliter of mixture and therefore an estimated volume of approximately 5 μl per ml of mixture.

[0650] Analysis of 1 microliter of the bulk mixture (or clear phase) before and after the addition of magnesium acetate showed that various proteins could be identified as expected in the clear liquid before addition, with Gp32 being the most prominent protein by mass. After condensation and clarification, only trace amounts of protein could be found in the supernatant, while the pellet is highly enriched in all proteins added to the RPA mixture (Figure 16C). By extrapolation, we can assume that an approximately 200-fold concentration of the reactants was achieved, resulting in a total protein concentration of approximately 200 μg / μl.

[0651] The results demonstrate that all protein components of the RPA reaction mixture, namely creatine kinase, Gp32-HRP fusion, UvsX, UvsY and polymerase, associate with phase-separated particles rather than with the bulk phase.

[0652] Example 17. Demonstration of the essential properties of amino acid sequences containing intrinsically disordered regions in increasing the efficiency of biochemical reactions. Experimental objectives and overview This experiment was conducted to evaluate the performance of Gp32 protein lacking a tag containing an amino acid sequence containing an intrinsically disordered region (IDR) in an exemplary in vitro biochemical reaction environment either in the presence or absence of a crowding agent.

[0653] This example demonstrates that in the absence of a tag containing an IDR domain amino acid sequence, Gp32 was unable to mediate recombinase polymerase amplification efficiently in the absence of a crowding agent, and to the point where detection occurred in this assay system within the analysis period. Compared to other examples above, such as Examples 1-5, these data demonstrate that a tag containing an IDR domain amino acid sequence is essential for increasing the efficiency of biochemical reactions in the absence of a crowding agent.

[0654] Materials and Methods Phage vB EcoM NBG1 Gp32 protein was purified in its native form, lacking either an exogenous IDR tag or a histidine tag. The protein was purified using heparin resin and eluted with a NaCl step gradient. Native Gp32 protein from the 400 mM NaCl fraction was used for testing.

[0655] An exemplary in vitro biochemical reaction environment was created to test the effect of native Gp32 protein either in the presence or absence of crowding agents.

[0656] The reaction was set up according to the following protocol.

[0657] A PEG-free reaction mixture was made containing the following components: 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM native Gp32 protein, 4.8 μM UvsX, 8.6 μM UvsY, 0.135 μM S. aureus DNA polymerase (large subunit), and 0.27 μM exonuclease III.

[0658] A PEG-based reaction mixture was prepared using the following components: 50 mM Tris-HCl pH 8.3, 100 mM KOAc, 1 mM DTT, 2.5 mM ATP, 50 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 0.4 μM forward primer, 0.4 μM reverse primer, 0.12 μM probe, 20 μM native Gp32 protein, 4.8 μM UvsX, 8.6 μM UvsY, 0.27 μM S. aureus DNA polymerase (large subunit), 0.27 μM exonuclease III, and the final concentrations of PEG shown in the relevant figures. The PEG species used had a molecular weight of 35,000.

[0659] In all reactions, the relevant primers and probes are shown below.

[0660] Forward primer: CGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAAC (SEQ ID NO: 98).

[0661] Reverse primer: CTGCATCTCCGTGGTATACTAATACATTGTTTTTA (SEQ ID NO: 99).

[0662] Probe: CGAAAAGAAACACGCGGATGAAATCGATAAG[FAM][THF][BHQ-1]ATACAAGGATTGGA (SEQ ID NO: 100), where FAM is fluorescein, THF is tetrahydrofuran, and BHQ is a black hole quencher.

[0663] All reactions were initiated by the addition of 33 mM MgOAc and 100 copies of DNA template derived from Listeria genomic DNA, then incubated at 39°C and placed in a fluorometer with magnetic mixing using a bearing ball.

[0664] Results and Conclusions As shown in Figure 17, rapid amplification was observed with native Gp32 protein in the presence of 5.5% PEG, but no amplification was observed with native Gp32 protein in the absence of PEG.

[0665] In other examples above, such as Examples 1 to 5, Gp32-mediated amplification was observed in the absence of PEG only when the Gp32 protein was tagged with an amino acid sequence containing an intrinsically disordered region (IDR).

[0666] Thus, together with data presented in other examples described herein, these data demonstrate that tags comprising amino acid sequences containing intrinsically disordered regions (IDRs) applied to protein components essential for the function of in vitro biochemical reactions can circumvent the need for crowding agents in the reaction, increasing the efficiency of the biochemical reaction compared to the efficiency observed in the absence of the IDR tag sequence.

[0667] Example 18. Recombinase polymerase amplification on solid surfaces using Gp32 with an IDR tag from human Otx1. Experimental objectives and overview This experiment was performed to evaluate the performance of a Gp32 fusion protein preparation containing a tag containing a histidine-rich amino acid domain sequence found in the intrinsically disordered region (IDR) of the human homeobox protein Otx1.

[0668] This example demonstrates recombinase polymerase amplification (RPA) of artificial nucleic acid templates on solid surfaces using Gp32 tagged at the C-terminus with a histidine-rich intrinsically disordered region (IDR) domain (Otx1) in the absence of crowding agents, in both real-time and endpoint assays.

[0669] Materials and Methods The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24). It was attached to the C-terminus of phage vB EcoM NBG1 Gp32. The recombinant fusion protein was purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the histidine naturally present in the IDR domain tag of the fusion protein under test. The fusion protein was designated Gp32-HIS2. The complete amino acid sequence of the fusion protein is shown as SEQ ID NO: 82 (Table 23).

[0670] The recombinant phage vB EcoM NBG1 Gp32 fusion protein was then tested on a solid surface in PEG-free amplification, i.e., in the absence of crowding agents. Tests were performed using two oligonucleotide primers bound to the surface of beads in different ratios. Amplification was detected fluorescently in real time using a quenched cleavable fluorescent probe or by endpoint detection of an annealed fluorescent probe. The beads were the same for both real-time and endpoint RPA reactions. The beads were supplied by Bangs Laboratories, Inc. (https: / / www.bangslabs.com / ) and had a carboxylated polystyrene core onto which a hydrogel was grown and to which oligonucleotides were covalently attached.

[0671] Real-time RPA reaction Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 120 nM probe, 20 μM Gp32 fusion, 4.9 μM UvsX, 7.6 μM UvsY, 0.146 μM S. aureus DNA polymerase, and 0.34 μM exonuclease III. The reaction mixture also contained 800,000 beads / μl, each with approximately 750,000 oligonucleotide primers per bead consisting of a mixture of PA30 forward and PB30 reverse primers.

[0672] The reaction was initiated by adding 33.6 mM MgOAc and an artificial DNA template called TF1L at a final concentration of 0.8 million template copies per μl of reaction mixture.

[0673] The relevant primers, probes and templates are shown below.

[0674] PA30 forward primer: CCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ ID NO: 105).

[0675] PB30 reverse primer: CCTATCCCCTGTGTGCCTTGGCAGTCTCAG (SEQ ID NO: 106).

[0676] Probe: AGCAGAAGCAATACCGCCAGCAATAGCA[dT-FAM]G[THF]G[dT-quencher]AGAGCGAGCTGCC (SEQ ID NO: 107), where FAM is fluorescein, THF is tetrahydrofuran, and quencher is a black hole quencher.

[0677] TF1L template sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGTGTTTTAGGGTCCCCGGGGTTAAAAGGTTTCGAACTCAACAGCTGTCTGGCAGCTCGCTCTACGCATGCTATTGCTGGCGGTATTGCTTCTGCTCTTGCTGGTGGCGCCATGTCTAAATTGTTTGGAGCTGAGACTGCCAAGGCACACAGGGGATAGG (SEQ ID NO: 108).

[0678] The reaction was then incubated for 30 minutes at 39°C in a T8 fluorometer and the fluorescence recorded in the FAM channel.

[0679] Figure 18A is a schematic depicting a reaction mixture set up for real-time amplification using dual primer beads, and Figure 18B is a schematic depicting the amplification products in a real-time reaction.

[0680] End point RPA reaction Reactions were set up by mixing 25 mM Tris-HCl pH 8.3, 7.5 mM KOAc, 1 mM DTT, 2.5 mM ATP, 20 mM phosphocreatine, 1 μM creatine kinase, 1 mM dNTPs, 20 μM Gp32 fusion, 4.9 μM UvsX, 7.6 μM UvsY, and 0.146 μM S. aureus DNA polymerase. The reaction mixture also contained 800,000 beads / μl, each with approximately 750,000 oligonucleotide primers per bead consisting of a mixture of PA30 forward and PB30 reverse primers.

[0681] The reaction was initiated by adding 33.6 mM MgOAc and an artificial DNA template called TF1L at a final concentration of 0.8 million template copies per μl of reaction mixture.

[0682] The relevant primers and templates are shown below.

[0683] PA30 forward primer: CCATCTCATCCCTGCGTGTCTCCGACTCAG (SEQ ID NO: 105).

[0684] PB30 reverse primer: CCTATCCCCTGTGTGCCTTGGCAGTCTCAG (SEQ ID NO: 106).

[0685] TF1L template sequence: CCATCTCATCCCTGCGTGTCTCCGACTCAGTGTTTTAGGGTCCCCGGGGTTAAAAGGTTTCGAACTCAACAGCTGTCTGGCAGCTCGCTCTACGCATGCTATTGCTGGCGGTATTGCTTCTGCTCTTGCTGGTGGCGCCATGTCTAAATTGTTTGGAGCTGAGACTGCCAAGGCACACAGGGGATAGG (SEQ ID NO: 108).

[0686] The reaction was then incubated at 39°C for 30 min and then stopped by adding sodium dodecyl sulfate (SDS) to a final concentration of 1% and heating to 65°C for 10 min to denature the proteins.

[0687] SDS was removed by diluting 10-fold with water, vortexing, and centrifuging at approximately 18,000 g for 15 minutes, followed by removal of the supernatant. The beads were resuspended in TE pH 8.0, 0.05% Triton X-100 buffer to obtain approximately 0.8 million beads / μl.

[0688] Two fluorescent oligonucleotide probes (PB30' probe (ROX-5'-CTGAGACTGCCAAGGCACACAGGGGATAGG; SEQ ID NO: 109), where ROX is carboxyrhodamine and FAM is fluorescein, and TF1L probe (FAM-5'-GGTTTCGAACTCAACAGCTG; SEQ ID NO: 110)) were hybridized to beads in TE pH 8.0, 0.05% Triton X-100, 100 mM NaCl buffer at 1 μM for both probes and a final concentration of 80,000 beads / μl. Hybridization was performed by heating to 95°C for 2 minutes followed by cooling to 25°C at 0.1°C / s. A positive control was performed using beads already containing the TF1L amplicon. The beads were then washed, and unhybridized probe was removed by diluting the hybridization mixture 6-fold with TE pH 8.0, 0.05% Triton X-100 buffer and centrifuging at approximately 18,000 g for 15 minutes, followed by removing as much of the supernatant as possible. The beads were resuspended in TE pH 8.0, 0.05% Triton X-100 buffer. The reaction was then incubated at 39°C for 5 minutes in a T8 fluorometer (FAM level set to 17% and ROX level set to 8%), and fluorescence was recorded in the FAM and ROX channels.

[0689] FIG. 18C is a schematic depicting amplification characterization in an end-point reaction.

[0690] result Real-time RPA reaction Figure 18D shows real-time fluorescent detection of the TF1L amplicon using a specific exonuclease cleavage probe. The percentage of PA30 primers identified in the figure indicates the percentage of bead-bound oligonucleotides that are PA30 oligonucleotides, with the remainder of the bead-bound oligonucleotides being PB30 oligonucleotides. Amplification is detected when all PA30 and PB30 oligonucleotide primers are bound to the beads, and when PA30 and PB30 oligonucleotide primers are in the liquid phase, or when PB30 is bound to the beads and PA30 is in the liquid phase. No amplification was detected when only PB30 was present on the beads, or when no PA30 was present.

[0691] End point RPA reaction End-point fluorescence detection of the TF1L amplicon was observed using a PB30 oligonucleotide primer (ROX-labeled, Figure 18E) and a probe specific for the TF1L amplicon (FAM-labeled, Figure 18F). The percentages specified in the figure indicate the percentage of bead-bound oligonucleotides that are PA30 oligonucleotides, with the remainder of the bead-bound oligonucleotides being PB30 oligonucleotides. The table below shows the fluorescence level for each bead type, the ratio of TF1L probe fluorescence to PB30' probe fluorescence, and the same ratio normalized to unamplified control beads with directly bound TF1L amplicon to account for background fluorescence caused by incomplete washing. [Table 25]

[0692] conclusion In both real-time and endpoint assays, nucleic acid amplification was found to occur efficiently using the Gp32-HIS2 fusion protein in the absence of crowding agents such as PEG.

[0693] Example 19. Identification of amino acid sequences containing intrinsically disordered regions. The amino acid sequences of phage vB EcoM NBG1 Gp32, T4 UvsY, and T4 UvsX were examined using the MetaDisorder software program (MetaDisorder: a metaserver for predicting intrinsic protein disorder. Kozlowshi, LP et al., BMC Bioinformatics, 2012, 13(1):111).

[0694] As shown in Figures 19A, 19B, and 19C, respectively, the full-length amino acid sequences of phage vB EcoM NBG1 Gp32, T4 UvsY, and T4 UvsX contain amino acid sequence stretches that have a score greater than 0.5 when analyzed by the algorithm and therefore contain intrinsically disordered region sequences.

[0695] This example demonstrates that intrinsically disordered region sequences or domains thereof can be readily identified using standard analytical methods.

[0696] Example 20. Comparison of the phase separation promoting activity of RB69 ligase with an IDR tag derived from human Otx1 and RB69 ligase Experimental objectives and overview This experiment was performed to evaluate the phase separation-promoting activity of a ligase enzyme fusion protein preparation containing a tag containing a histidine-rich amino acid domain sequence found in the intrinsically disordered region (IDR) of the human homeobox protein Otx1 (His2 tag).

[0697] The experiment showed that the formation of phase-separated aqueous particles (spherical foci) by RB69 ligase-His2 in the absence of crowding agents was significantly suppressed by Mg 2+ The formation of spherical foci by RB69 ligase was enhanced by Mg concentration. 2+ demonstrated little or no correlation with concentration.

[0698] Materials and Methods The specific amino acid sequence of the IDR domain tag used was AGHHHHHPHAHHPLSQSSGHHHHHHHHHHQGYGGSG (SEQ ID NO: 24, Table 1). This was attached to the C-terminus of RB69 DNA ligase. The recombinant fusion protein and the IDR-free protein were purified using standard one-step immobilized metal (nickel) affinity chromatography, which relies on the naturally occurring histidine in the IDR domain tag of the fusion protein under test and the polyhistidine tag at the C-terminus of the IDR-free protein. The fusion protein was designated RB69 ligase-His2, and the IDR-free protein was designated RB69 ligase. The complete amino acid sequences of the proteins are shown below in Table 24 as SEQ ID NO: 111 and SEQ ID NO: 112, respectively. Table 24 [Table 26]

[0699] The probe oligo used in the experiment was CCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATG (SEQ ID NO: 104) labeled with FAM (fluorescein).

[0700] A 50 μl solution was made containing a final concentration of 1 mg / ml ligase, 50 mM NaCl, 0.4 μM FAM-oligo, and the target concentrations of MgCl2 indicated in the relevant figures. 10 μl of the reaction mixture was then transfer...

Claims

1. 1. A method for conducting a biochemical reaction in an aqueous in vitro reaction system, said biochemical reaction depending on at least one reactive macromolecule, optionally depending on the function of at least one reactive polypeptide, comprising: introducing at least one IDR-macromolecule into said in vitro reaction system under conditions suitable for carrying out said reaction; with the proviso that said at least one IDR-macromolecule comprises one or more functional intrinsically disordered regions (IDRs); and when the at least one IDR-macromolecule is introduced into the in vitro reaction system, the efficiency of the biochemical reaction is increased by the at least one IDR-macromolecule; Said at least one IDR-macromolecule is preferably at least one IDR-polypeptide.

2. 2. The method of claim 1, wherein a biochemical reaction depends on the function of at least one IDR macromolecule, and optionally on the function of at least one IDR polypeptide, and when introduced into a 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 in the system.

3. The method of claim 1 or 2, further comprising maintaining an IDR-macromolecule or IDR-polypeptide in the system to cause liquid-liquid demixing and formation of multiple phase-separated aqueous compartments in the system by the IDR-macromolecule or IDR-polypeptide, thereby increasing the efficiency of biochemical reactions in the system.

4. 4. The method of claim 3, further comprising maintaining the IDR-macromolecule or IDR-polypeptide in a system so that molecules necessary for carrying out the reaction are co-localized with the IDR-macromolecule or IDR-polypeptide in a plurality of phase-separated aqueous compartments, thereby increasing the efficiency of the biochemical reaction in the system.

5. 5. The method of claim 3 or claim 4, wherein the plurality of phase-separated aqueous compartments is a plurality of detectable phase-separated aqueous particles.

6. A method according to any one of claims 1 to 5, which is a biochemical reaction for synthesizing a nucleic acid molecule in an in vitro reaction system, comprising: (a) providing at least one nucleic acid primer; (b) providing a target nucleic acid molecule comprising at least one target strand and contacting said at least one nucleic acid primer with said target strand, thereby forming a double-stranded structure; (c) providing the IDR-macromolecule as an IDR-polypeptide, wherein the IDR-polypeptide is a polymerase or one or more polypeptide cofactors; (d) allowing the reaction to proceed, thereby extending the 3' end of said at least one nucleic acid primer with a polymerase and dNTPs, optionally in the presence of one or more polypeptide cofactors, to generate a double-stranded nucleic acid, wherein a first strand comprises the sequence of said target strand and a second strand comprises the sequence complementary thereto; 。

7. 7. The method according to claim 6, which is a biochemical reaction for amplifying a single-stranded or double-stranded target nucleic acid molecule in an in vitro reaction system, wherein the target nucleic acid molecule is preferably a DNA molecule.

8. A method according to any one of claims 1 to 5, which is a biochemical reaction for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, comprising: (a) providing first and second nucleic acid primers; (b) providing a double-stranded target nucleic acid molecule comprising a first strand and a second strand, and contacting said first and second nucleic acid primers with said target nucleic acid molecule, thereby forming a first double-stranded structure with said first strand and a second double-stranded structure with said second strand; (c) providing the IDR-macromolecule as an IDR-polypeptide, wherein said IDR-polypeptide is a polymerase or one or more protein cofactors; (d) allowing the reaction to proceed, thereby extending the 3' ends of the first and second nucleic acid primers using a polymerase and dNTPs, optionally in the presence of the one or more protein cofactors, to generate first and second double-stranded nucleic acids; (e) Repeating steps (b) through (d) until the desired degree of amplification is reached.

9. 9. The method of claim 8, wherein the recombinase polymerase amplification method amplifies a double-stranded target nucleic acid molecule in an in vitro reaction system, the method comprising: (a) providing a recombinase agent, optionally a recombinase loading protein, a single-strand stabilizing agent, a polymerase, a first and a second nucleic acid primer, a double-stranded target nucleic acid comprising a first strand and a second strand, and optionally an exonuclease, e.g., exonuclease III; (b) contacting the recombinase agent with the first and second nucleic acid primers and, optionally, the recombinase loading protein to form first and second nucleoprotein primers comprising single-stranded regions at their 3' ends; (c) contacting said first and second nucleoprotein primers with said target nucleic acid molecule, thereby forming a first double-stranded structure with said first strand and a second double-stranded structure with said second strand; (d) allowing the reaction to proceed, thereby extending the 3' ends of the first and second nucleoprotein primers using a polymerase and dNTPs to generate first and second double-stranded nucleic acids and first and second displaced nucleic acid strands, wherein the single-stranded stabilizing agent stabilizes the first and second displaced strands; (e) continuing the reaction by repeating steps (b) through (d) until a desired degree of amplification is achieved; However, said recombinase agent, and / or said recombinase loading protein, and / or said single-strand stabilizing agent, and / or said polymerase are provided as IDR-polypeptides.

10. The recombinase agent is selected from 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, 10. The method of claim 9, wherein the recombinase agent is selected from the group consisting of UvsX, E. coli RecA, E. coli RadA, E. coli RadB, E. coli Rad 51 or any functional analogue, homologue or derivative thereof, and any combination thereof, preferably said recombinase agent is UvsX, more preferably Escherichia phage vB_EcoM_DalCa UvsX.

11. 11. The method of claim 9 or claim 10, comprising a recombinase loading protein, wherein the recombinase loading protein is selected from the group consisting of UvsY, E. coli RecO, E. coli RecR or any functional analogue, homologue or derivative thereof, and any combination thereof, preferably the recombinase loading protein is UvsY, more preferably Escherichia phage STO UvsY.

12. The method of any one of claims 6 to 11, wherein the polymerase is a eukaryotic polymerase selected from the group consisting of pol-α, pol-β, pol-δ, pol-ε, or any functional analog, homolog, or derivative thereof, and any combination thereof.

13. The polymerase may be Bacillus stearothermophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, Staphylococcus aureus (S. aureus) DNA polymerase I (Saw polymerase), or E. coli DNA polymerase I.

12. The method of any of claims 6 to 11, wherein the prokaryotic polymerase is selected from the group consisting of 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, or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the polymerase is S. aureus DNA polymerase I (Saw polymerase) or Bacillus subtilis Pol I large fragment (Bsu polymerase).

14. 12. The method of any one of claims 6 to 11, wherein the polymerase is a bacteriophage polymerase selected from the group consisting of bacteriophage T4 gp43 DNA polymerase, T7 DNA polymerase and Phi-29 DNA polymerase, or any functional analog, homolog or derivative thereof, and any combination thereof.

15. 15. The method of any of claims 9 to 14, wherein the single-stranded stabilizer 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 analogue, homologue or derivative thereof, and any combination thereof, preferably wherein said single-stranded stabilizer is Gp32 or phage vB_EcoM_NBG1 Gp32.

16. The method according to any one of claims 9 to 15, wherein only a recombinase agent is provided as the IDR-polypeptide, or only a recombinase loading protein is provided as the IDR-polypeptide, or only a single-stranded stabilizing agent is provided as the IDR-polypeptide, or only the polymerase is provided as the IDR-polypeptide, or only an exonuclease is provided as the IDR-polypeptide.

17. 10. The method of any of the preceding claims, wherein one or more functional IDRs of an IDR-polypeptide are tagged to the IDR-polypeptide as an amino acid sequence comprising or consisting of said one or more IDRs, such that said IDR-polypeptide is a genetically engineered fusion protein, and wherein said one or more functional IDRs are located at the C-terminus of said IDR-polypeptide, at the N-terminus of said IDR-polypeptide, or at both the C-terminus of said IDR-polypeptide and the N-terminus of said IDR-polypeptide, or at any amino acid position along the length of said polypeptide.

18. 10. The method according to any of the preceding claims, wherein one or more functional IDRs of an IDR-macromolecule or an IDR-polypeptide are characterized as sequences of amino acids that exhibit a score of more than 0.5 when analyzed by the algorithm MetaDisorder.

19. 10. The method according to any of the preceding claims, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of an amino acid sequence comprising one or more repeats of the tripeptide sequence RGG.

20. 20. The method of claim 19, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of an amino acid sequence further comprising one or more repeats of the dipeptide sequence FG.

21. 21. The method of claim 19 or claim 20, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of an amino acid sequence further comprising at least one aromatic amino acid residue consisting of tyrosine or phenylalanine.

22. One or more functional IDRs of an IDR-macromolecule or an IDR-polypeptide are i. (YNPQGGYQQ) ｎ , wherein n is a positive integer from 1 to 10, and optionally n=1, 2, or 3; or ii. (YSPTSPS) ｎ , wherein n is a positive integer from 1 to 10, and optionally n=1, 2, or 3; or iii. (FSPTSPT) ｎ , wherein n is a positive integer from 1 to 10, and optionally n=1, 2, or 3; or iv. (YSPTSP-A / N / G) ｎ where n is a positive integer from 1 to 10, and optionally n=1, 2, or 3), or v. (YSPGSPA) ｎ , wherein n is a positive integer from 1 to 10, and optionally n=1, 2, or 3.

10. The method of any of the preceding claims, comprising or consisting of the amino acid sequence of

23. 10. The method of any of the preceding claims, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of a glutamine-rich amino acid sequence, optionally said amino acid sequence comprising at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive glutamine residues.

24. 24. The method of claim 23, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of an amino acid sequence comprising one or more repeats of the tripeptide sequence QQQ.

25. One or more functional IDRs of the IDR-macromolecule or IDR-polypeptide are (QQQPQY) ｎ 25. The method of claim 23 or claim 24, comprising or consisting of an amino acid sequence of:

26. 10. The method of any of the preceding claims, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of a sequence of at least 5 consecutive amino acids of SEQ ID NO:

1.

27. 10. The method of any of the preceding claims, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise or consist of an amino acid sequence of at least 5 consecutive amino acids of SEQ ID NO:

9.

28. 10. The method according to any of the preceding claims, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise an amino acid sequence containing one or more aromatic tyrosine residues and one or more phenylalanine residues capable of participating in aromatic cation-π interactions with polyvalent metal ions, preferably divalent metal ions.

29. 10. The method according to any of the preceding claims, wherein one or more functional IDRs of the IDR-macromolecule or IDR-polypeptide comprise an amino acid sequence comprising one or more arginine residues capable of participating in guanidine-metal interactions with polyvalent metal ions, preferably divalent metal ions.

30. 10. A method according to any of the preceding claims, wherein the IDR-polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 43, or comprises or consists of a functional variant amino acid sequence of SEQ ID NOs: 1 to 43, for example a polypeptide tagged with an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 1 to 43.

31. The method according to any one of claims 9 to 30, wherein the IDR-polypeptide is Gp32, a single-chain stabilizer having the amino acid sequence of any one of SEQ ID NOs: 65 to 88, or the IDR-polypeptide is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity with any one of SEQ ID NOs: 65 to 88.

32. The method according to any one of claims 9 to 30, wherein the IDR-polypeptide is UvsX, a recombinase agent having the amino acid sequence of any one of SEQ ID NOs: 44 to 59, or the IDR-polypeptide is a functional variant thereof, for example an IDR-polypeptide having an amino acid sequence having 80% or more identity with any one of SEQ ID NOs: 44 to 59.

33. The method according to any one of claims 9 to 30, wherein the IDR polypeptide is UvsY, a recombinase loading protein having the amino acid sequence of any one of SEQ ID NOs: 60 to 64, or the IDR polypeptide is a functional variant thereof, for example an IDR polypeptide having an amino acid sequence having 80% or more identity with any one of SEQ ID NOs: 60 to 64.

34. 10. The method of any preceding claim, further comprising providing a polyvalent metal ion to an IDR-macromolecule or IDR-polypeptide in an in vitro reaction system, thereby stimulating or enhancing said liquid-liquid demixing in said in vitro reaction system, thereby increasing the efficiency of a biochemical reaction, preferably wherein said liquid-liquid demixing promotes the formation of detectable phase-separated particles in said in vitro reaction system, thereby increasing said efficiency of said biochemical reaction, optionally wherein said polyvalent metal ion is provided at a concentration of about 22 mM or greater, preferably wherein said polyvalent metal ion is provided at a concentration of about 22 mM to 50 mM.

35. The polyvalent metal ion is a divalent metal ion, optionally Mg ２＋ , Mn ２＋ , Ca ２＋ , Co ２＋ or Ni ２＋ , preferably Mg ２＋ , Mn ２＋ or Ca ２＋ , more preferably Mg ２＋ 35. The method of claim 34, wherein:

36. 10. The method of any of the preceding claims, wherein the biochemical reaction is carried out in a solid phase reaction system comprising a surface.

37. The method of claim 36, wherein the biochemical reaction is a method for amplifying a single-stranded or double-stranded target nucleic acid molecule in an in vitro reaction system according to claim 3 or claim 4, and wherein at least one nucleic acid primer and / or IDR-macromolecule and / or the one or more polypeptide cofactors are bound to the surface.

38. 35. The method of any one of claims 9 to 34, wherein the biochemical reaction is a recombinase polymerase amplification method for amplifying a double-stranded target nucleic acid molecule in an in vitro reaction system, the reaction being carried out in a solid-phase reaction system comprising a surface, and the recombinase agent and / or the recombinase loading protein and / or the single-strand stabilizing agent and / or the polymerase and / or the exonuclease and / or the first nucleic acid primer and / or the second nucleic acid primer being bound to the surface, preferably, (i) the first nucleic acid primer or the second nucleic acid primer is bound to the surface, or (ii) both the first and second nucleic acid primers are bound to the surface.

39. 39. The method of any of claims 36 to 38, wherein the surface is a plane or a microbead, preferably the surface comprises silicon, glass, a gel-based material and / or a polymeric material such as polystyrene, more preferably the surface is a microbead comprising a polymeric material such as polystyrene.

40. 40. The method of claim 39, wherein the surface is attached to a substrate, preferably said surface is planar and / or said substrate comprises glass, optionally said surface and / or said substrate being provided as a flow cell.

41. 1. A non-naturally occurring IDR-macromolecule comprising a macromolecule and a tag amino acid sequence, wherein the tag sequence comprises or consists of one or more functional intrinsically disordered regions (IDRs), and wherein the IDR-macromolecule is capable of promoting liquid-liquid demixing in solution.

42. 42. The IDR-macromolecule of claim 41, wherein liquid-liquid demixing promotes the formation of detectable phase-separated particles in an in vitro reaction system.

43. 43. The IDR-macromolecule of claim 41 or claim 42, wherein liquid-liquid demixing and the formation of phase-separated aqueous compartments thereby enhances the efficiency of a biochemical reaction in the system, or wherein the formation of detectable phase-separated aqueous particles thereby enhances the efficiency of a biochemical reaction in the system.

44. 44. The IDR-macromolecule according to any one of claims 41 to 43, which is a non-naturally occurring, artificial or genetically engineered IDR-polypeptide comprising a polypeptide and a tag amino acid sequence.

45. 45. The IDR-polypeptide of claim 44, wherein the tag amino acid sequence is located at the C-terminus of the polypeptide, the N-terminus of the polypeptide, or at both the C-terminus of the polypeptide and the N-terminus of the polypeptide, or at any amino acid position along the length of the polypeptide.

46. 46. ​​The IDR-polypeptide of claim 45, wherein one or more functional IDRs of the tag amino acid sequence are functional IDRs according to any one of claims 14 to 28.

47. The tag sequence is selected from the group consisting of a polyvalent metal cation, preferably a divalent metal cation, more preferably Mg ２＋ , Mn ２＋ , Ca ２＋ , Co ２＋ or Ni ２＋ ions, even more preferably Mg ２＋ , Mn ２＋ or Ca ２＋ , and even more preferably Mg ２＋ 47. The IDR-macromolecule or polypeptide of any one of claims 41 to 46, comprising amino acid residues capable of participating in aromatic cation-π interactions with

48. 48. The IDR-polypeptide according to any one of claims 44 to 47, wherein the polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs is an enzyme, such as a helicase, a gyrase, a recombinase, such as an RPA recombinase agent, a nuclease, such as an exonuclease and an endonuclease, a ligase, a glycolyase, a methylase, a methyltransferase, a glucosyltransferase, a polymerase, a kinase, a phosphatase, a gene editing enzyme such as a CRISPR enzyme, such as the Cas9 enzyme; or a cofactor, such as an RPA recombinase loading protein and an RPA single-strand stabilizer.

49. 49. The IDR polypeptide of claim 48, wherein the polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs is an RPA single-stranded stabilizer, preferably Gp32, and optionally the IDR polypeptide has the amino acid sequence of any one of SEQ ID NOs: 65 to 88, or the IDR polypeptide is a functional variant thereof, for example an IDR polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 65 to 88.

50. 49. The IDR polypeptide of claim 48, wherein the polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs is an RPA recombinase agent, preferably UvsX, and optionally the IDR polypeptide has the amino acid sequence of any one of SEQ ID NOs: 44 to 59, or is a functional variant thereof, for example an IDR polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 44 to 59.

51. 49. The IDR polypeptide according to claim 48, wherein the polypeptide tagged with a sequence comprising or consisting of one or more functional IDRs is an RPA recombinase loading protein, preferably UvsY, and optionally wherein the IDR polypeptide has the amino acid sequence of any one of SEQ ID NOs: 60 to 64, or wherein the IDR polypeptide is a functional variant thereof, for example an IDR polypeptide having an amino acid sequence having 80% or more identity to any one of SEQ ID NOs: 60 to 64.

52. 52. An isolated nucleic acid molecule comprising a first nucleic acid sequence encoding an IDR-polypeptide according to any one of claims 41 to 51, and optionally a second nucleic acid sequence encoding a promoter, said first nucleic acid sequence being operably linked to said second nucleic acid sequence.

53. 53. A recombinant polynucleotide expression vector comprising the nucleic acid molecule of claim 52.

54. 54. A host cell comprising the nucleic acid molecule of claim 52 or the recombinant polynucleotide expression vector of claim 53.

55. 55. A cell culture comprising a growth medium and a population of host cells, said population comprising the host cell of claim 54.

56. A kit comprising a non-naturally occurring IDR macromolecule or IDR polypeptide according to any one of claims 41 to 51.

57. 57. The kit of claim 56, further comprising additional RPA components including an RPA recombinase agent, and / or an RPA recombinase loading protein, and / or a polymerase, and / or first and second nucleic acid primers, and / or an exonuclease, and / or a buffer, and / or a source of polyvalent metal ions, preferably divalent metal cations.

58. 58. The kit of claim 56 or claim 57, wherein all components are provided in lyophilized form.

59. 52. A method for stimulating or enhancing liquid-liquid demixing in a solution, said method comprising providing a solution comprising an IDR-macromolecule or IDR-polypeptide according to any one of claims 41 to 51, and contacting said IDR-macromolecule or IDR-polypeptide in said solution with a polyvalent metal ion, wherein liquid-liquid demixing in said solution is stimulated or enhanced.

60. 60. The method of claim 59, wherein the liquid-liquid demixing results in the formation of detectable phase-separated particles in the solution.

61. The polyvalent metal ion is a divalent metal ion, optionally Mg ２＋ , Mn ２＋ , Ca ２＋ , Co ２＋ or Ni ２＋ , preferably Mg ２＋ , Mn ２＋ or Ca ２＋ , more preferably Mg ２＋ 61. The method of claim 59 or claim 60, wherein:

62. 52. Use of a polyvalent metal ion in stimulating or enhancing liquid-liquid demixing in a solution, said demixing being mediated by an IDR-macromolecule or IDR-polypeptide according to any one of claims 41 to 51.

63. 63. The use of claim 62, wherein the liquid-liquid demixing results in the formation of detectable phase-separated particles in the solution.

64. The polyvalent metal ion is a divalent metal ion, optionally Mg ２＋ , Mn ２＋ , Ca ２＋ , Co ２＋ or Ni ２＋ , preferably Mg ２＋ , Mn ２＋ or Ca ２＋ , more preferably Mg ２＋ 64. The use according to claim 62 or claim 63, wherein

65. 65. The use of any one of claims 62 to 64, wherein the polyvalent metal ion participates in aromatic cation-π interactions with amino acid residues in one or more functional IDR amino acid sequences, thereby promoting liquid-liquid demixing.

66. A non-naturally occurring IDR-macromolecule or IDR-polypeptide according to any one of claims 41 to 48 for use in therapy or as a diagnostic agent.

67. 1. A method for determining the nucleotide sequence of one or more target polynucleotide molecules, comprising: (i) performing a method according to any one of claims 1 to 40 to amplify one or more target polynucleotide molecules, thereby obtaining a population comprising multiple copies of said one or more target polynucleotide molecules; (ii) performing one or more nucleic acid sequencing reactions on the population comprising the multiple copies of the target polynucleotide molecule; Preferably, the method is carried out in a solid phase reaction system comprising a surface.

68. Use of an IDR-macromolecule or an IDR-polypeptide according to any of claims 41 to 51 in a method for determining the nucleotide sequence of one or more target polynucleotide molecules, preferably DNA molecules, wherein the method is preferably a method according to claim 67.

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