WO2024086949A1

WO2024086949A1 - Isolation and purification of double stranded rna

Info

Publication number: WO2024086949A1
Application number: PCT/CA2023/051438
Authority: WO
Inventors: Mamadou Lamine FALL; Dong Xu
Original assignee: His Majesty The King In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food
Priority date: 2022-10-27
Filing date: 2023-10-27
Publication date: 2024-05-02

Abstract

A method of isolating and purifying double stranded RNA (dsRNA) from a sample includes treating a crude RNA extract containing dsRNA obtained from a sample with a B2 dsRNA-binding protein to form a B2-dsRNA mixture containing a B2-dsRNA complex; separating the B2-dsRNA complex from the B2-dsRNA mixture, wherein the B2-dsRNA complex is not immobilized on a substrate; dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA; and separating the B2 dsRNA-binding protein from the dsRNA. Also provided is a kit for carrying out the method.

Description

ISOLATION AND PURIFICATION OF DOUBLE STRANDED RNA

Field

[0001] The present application relates to an improved method for isolating and purifying double stranded RNA (dsRNA). More specifically, the present application relates to an improved method for isolating and purifying dsRNA from a sample which can be used to detect and/or identify viruses and viral genomes in a sample.

Background

[0002] The presence of high molecular weight or long double stranded RNA (dsRNA) can be indicative of the presence of a virus, which may use dsRNA as its genomic material or may generate dsRNA as an intermediate during replication in a host cell. Because viral hosts generally do not produce significant quantities of high molecular weight dsRNA in the absence of viruses, isolation and sequencing of dsRNA from a sample can provide useful information regarding the identity of any viruses present in the sample. Such methods are especially useful for identifying viruses and viroids infecting plant tissue, as the majority of plant viruses contain RNA genomes and/or produce dsRNA as an intermediate during viral replication.

[0003] Methods of isolating and characterizing dsRNA are known. For example, Kesanakurti et al (Journal of Virological Methods (2016), 236: 35-40) describe a method of extracting and purifying dsRNA from homogenized virally-infected plant tissue which involves adsorbing the dsRNA on cellulose, washing the cellulose and subsequently eluting the purified dsRNA. Okada et al (Journal of General Plant Pathology (2015), 81 : 103-107) describe a similar method for isolating viral dsRNA from infected plant and fungal sources. In addition, kits for isolating dsRNA from plant tissue are commercially available, including the Double RNA Viral dsRNA Extraction Mini Kit (for Plant Tissue) (iNtRON Biotechnology, Inc.) and the Plant Viral dsRNA Enrichment Kit (MBL). Use of the Double RNA Viral dsRNA Extraction Mini Kit (for Plant Tissue) involves binding dsRNA to a spin column, washing the column to purify the dsRNA and subsequently eluting the isolated dsRNA from the column. Use of the Plant Viral dsRNA Enrichment Kit involves binding the dsRNA to a dsRNA-binding protein derived from Arabidopsis thaliana, immobilizing the complex on glutathione resin, washing the resin and subsequently eluting the isolated dsRNA from the resin.

[0004] It was reported by Chao et al (Nature Structural & Molecular Biology (2005), 12(11): 952-957) that the Flock House virus (FHV) B2 protein can bind to dsRNA. Monsion et al (Front. Plant Sci. (2018): 9: 70) describe methods of detecting viral dsRNA in vitro and in vivo using the FHV B2 protein. Incarbone et al (Methods in Molecular Biology (2020), 2166: 307-327) describe a method of isolating viral dsRNA from virally infected Arabidopsis thaliana plants which have been genetically modified to express a fusion protein of the FHV B2 protein with green fluorescent protein (B2-GFP). The method involves isolating a complex of the viral dsRNA and the B2-GFP protein from homogenized plant tissue by immobilizing the complex on magnetic beads bearing an anti-GFP antibody. Similar procedures are described in Incarbone et al, The Plant Cell (2021), 33: 3402-3420, and Incarbone et al, Viruses (2020), 12: 1121.

[0005] However, these methods have the disadvantages of being time-consuming, labour-intensive and/or expensive. Therefore, there is a need for an alternative method of isolating and purifying dsRNA.

Summary

[0006] In one aspect, the present application provides a method of isolating and purifying double stranded RNA (dsRNA) from a sample. The method includes:

• treating a crude RNA extract obtained from the sample with a B2 dsRNA-binding protein to form a B2-dsRNA mixture comprising a B2-dsRNA complex;

• separating the B2-dsRNA complex from the B2-dsRNA mixture, wherein the B2-dsRNA complex is not immobilized on a substrate;

• dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA; and

• separating the B2 dsRNA-binding protein from the dsRNA.

[0007] In at least one embodiment, the sample is a sample of plant tissue.

[0008] In at least one embodiment, the step of treating the crude RNA extract with a B2 dsRNA-binding protein to form a B2-dsRNA complex comprises mixing the crude RNA extract with a reaction solvent to form a crude dsRNA mixture; and mixing the crude dsRNA mixture with the B2 dsRNA-binding protein to form the B2-dsRNA mixture, such that a precipitate comprising the B2-dsRNA complex is formed. In at least one embodiment, the precipitate comprising the B2-dsRNA complex forms in the B2-dsRNA mixture. In at least one embodiment, the precipitate comprising the B2-dsRNA complex forms upon addition of a second reaction solvent to the B2-dsRNA mixture. In at least one embodiment, components of the crude dsRNA mixture which are insoluble in the reaction solvent are removed from the crude dsRNA mixture before mixing the crude dsRNA mixture with the B2 dsRNA-binding protein to form the B2-dsRNA mixture.

[0009] In at least one embodiment, the reaction solvent is a buffer having a pH of about 8 to about 9 and containing a chelating agent and a salt. In at least one embodiment, the buffer further contains a blocking agent. [0010] In at least one embodiment, the step of separating the B2-dsRNA complex from at least one other component of the crude RNA extract includes separating the precipitate comprising the B2-dsRNA complex from the B2-dsRNA mixture.

[0011] In at least one embodiment, the step of dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA comprises mixing the precipitate comprising the B2-dsRNA complex with a dissociation solvent to form a dissociation mixture. In at least one such embodiment, the step of separating the B2 dsRNA-binding protein from the dsRNA comprises lowering the temperature of the dissociation mixture to below 0°C, such that a precipitate comprising the dsRNA forms, and separating the precipitate comprising the dsRNA from the dissociation mixture.

[0012] A further aspect of the present application provides a kit for use in isolating and purifying dsRNA from a sample using a method as described herein. In at least one embodiment, the kit comprises one or more reagents used to carry out a method as described herein. In at least one embodiment, the one or more reagents include one or more reagents selected from the group consisting of a reaction solvent and a dissociation solvent. In at least one embodiment, the one or more reagents further include a second reaction solvent. In at least one embodiment, the kit further includes instructions for carrying out the method, including but not limited to instructions for preparation and/or use of the one or more reagents in a method as described herein.

Detailed Description

[0013] One aspect of the present application provides a method of isolating and purifying double stranded RNA (dsRNA) from a sample. As used herein, the terms “double stranded RNA” or “dsRNA” are intended to mean polyribonucleotides in which two complementary RNA strands are chemically base-paired with each other. In at least one embodiment the dsRNA is long dsRNA having at least 20 base pairs. In at least one embodiment, the dsRNA is indicative of the presence of one or more viruses or viroids in the sample. In at least one embodiment, the dsRNA can be sequenced or otherwise used to identify or characterize the one or more viruses or viroids present in the sample. The person of skill in the art would be readily able to identify and use methods for identifying or characterizing viruses and/or viroids on the basis of isolated and/or purified dsRNA obtained by the method described herein, including but not limited to sequencing the dsRNA and comparing the sequence or other properties of the dsRNA to the corresponding sequence or other properties of dsRNA from a known origin or with a known sequence or identity.

[0014] The method includes a step of treating a crude RNA extract obtained from a sample with a B2 dsRNA-binding protein to form a B2-dsRNA mixture comprising a B2-dsRNA complex. Suitable samples include but are not limited to plant tissue, insect tissue, animal tissue, biological fluids, fungal tissue and other samples which may contain dsRNA, as would be understood in the art. In at least one embodiment, the sample is a sample of plant tissue.

[0015] As used herein, the term “crude RNA extract” is intended to mean an extract prepared from a sample which contains RNA, including but not limited to dsRNA. For example, in at least one embodiment, the crude RNA extract can be a total RNA extract, including any double stranded RNA (dsRNA) present in the sample, in addition to any one or more of single stranded RNA (ssRNA), small interfering RNA (siRNA), circular RNA (cirRNA), long non-coding RNA (IncRNA), micro RNA (miRNA), and other forms of RNA known in the art which are present in the sample. In at least one embodiment, the crude RNA extract can be a liquid mixture or solution containing RNA, including but not limited to dsRNA. In at least one embodiment, the crude RNA extract can be a solid mixture containing RNA, including but not limited to dsRNA. In at least one embodiment, the crude RNA extract can be a precipitate containing RNA, including but not limited to dsRNA. As used herein, the term “precipitate” is intended to mean a solid portion of a mixture which can be separated from a liquid portion of the mixture by filtration or by centrifugation as a pellet, for example, as is well understood in the art.

[0016] Methods of obtaining a crude RNA extract, including but not limited to a total RNA extract, from a sample are well known in the art. For example, a crude RNA extract can be obtained from plant tissue by using methods described herein or in Kesanakurti et al (Journal of Virological Methods (2016), 236: 35-40) or in Okada et al (Journal of General Plant Pathology (2015), 81 : 103-107). In addition, total RNA can be extracted from plant tissue using a commercially available kit, such as the Spectrum™ Plant Total RNA Kit (Millipore Sigma). It will be clear to the person of skill in the art that other methods known in the art can be used for obtaining crude RNA extracts from samples obtained from other sources, and the skilled person would be readily able to identify and use appropriate methods for obtaining such crude RNA extracts in view of the teaching herein.

[0017] For example, the crude RNA extract can be obtained from plant tissue by contacting the sample of plant tissue with a buffer, lysing or homogenizing the mixture, and centrifuging the mixture to remove tissue debris and other insoluble impurities. The buffer can have a pH value of at least about 8, preferably between about 8 and about 9, and can further include components such as detergents, chelating agents, |3-mercaptoethanol and materials to remove polyphenol contaminants, such as polyvinylpyrrolidone (PVP). A precipitate containing crude RNA, including but not limited to dsRNA, can be obtained by adding an alcohol, such as ethanol or isopropanol, to the supernatant solution.

[0018] The present method includes treating the crude RNA extract with a B2 dsRNA- binding protein to form a B2-dsRNA mixture comprising a B2-dsRNA complex. In at least one embodiment, including but not limited to when the crude RNA extract is a precipitate containing dsRNA as described herein, the crude RNA extract can be mixed with a reaction solvent to form a crude dsRNA mixture in preparation for treatment with the B2 dsRNA-binding protein. In at least one embodiment, the reaction solvent comprises a mixture of components. In at least one embodiment, the mixture of components is an aqueous mixture. In at least one embodiment, the aqueous mixture is a buffer solution. [0019] In at least one embodiment, the reaction solvent is a buffer solution having a pH of about 8 to about 9. In at least one embodiment, the buffer solution has a pH of about 8. In at least one embodiment, the buffer solution has a pH of about 8.0. In at least one embodiment, the buffer solution further comprises a chelating agent. In at least one embodiment the chelating agent is EDTA. In at least one embodiment, the buffer solution further contains a salt. In at least one embodiment, the salt is NaCI. In at least one embodiment, the buffer solution further comprises a blocking agent. In at least one embodiment, the blocking agent is present in the buffer in a concentration of about 0.1% (w/v) to about 50% (w/v). In at least one embodiment, the blocking agent is bovine serum albumin (BSA). In at least one embodiment, the buffer solution is prepared by mixing 20 ml of 1 M Tris buffer (pH 8.3), 20 ml of 0.5 M EDTA solution, 80 ml of 1 M NaCI solution and 880 ml of ultrapure water. In at least one embodiment, the buffer contains Tris (20 mM, pH 8.0), EDTA (10 mM, pH 8.0), NaCI (180 mM) and BSA (1% w/v).

[0020] In at least one embodiment, the crude dsRNA mixture contains impurities and other components which are insoluble or have a low solubility in the reaction solvent. In at least one embodiment, components of the crude dsRNA mixture which are insoluble in the reaction solvent are removed from the crude dsRNA mixture before treating the crude dsRNA mixture with the B2 dsRNA-binding protein. In such embodiments, the dsRNA dissolved in the reaction solvent can be at least partially separated from components of the precipitate having a lower solubility in the reaction solvent by well- known methods including but not limited to centrifugation and filtration.

[0021] As used herein, the term “B2 dsRNA-binding protein” is intended to mean the Flock House virus (FHV) B2 protein, or a fragment thereof which is capable of binding to polynucleotides, including but not limited to dsRNA, so as to form a B2-dsRNA complex as described herein. In at least one embodiment, the B2 dsRNA-binding protein has an amino acid sequence defined as SEQ ID NO:1. In at least one embodiment, the B2 dsRNA-binding protein has an amino acid sequence defined as SEQ ID NO:2. In at least one embodiment, the preparation of the B2 dsRNA-binding protein is prepared by cloning a polydeoxyribonucleotide having a sequence defined as SEQ ID NO:3 into a vector, expressing the polydeoxyribonucleotide in a host cell and isolating the preparation of the B2 dsRNA-binding protein. In at least one embodiment, the preparation of the B2 dsRNA-binding protein is a crude preparation. In at least one embodiment, the preparation of the B2 dsRNA-binding protein is a purified preparation. [0022] In at least one embodiment, the crude dsRNA mixture and the B2 dsRNA-binding protein are mixed to form the B2-dsRNA mixture under conditions in which the B2-dsRNA complex has a lower solubility in the B2-dsRNA mixture than the solubility of other components present in the B2-dsRNA mixture. In at least one such embodiment, the B2-dsRNA complex forms as a precipitate upon mixing the crude dsRNA mixture with the B2 dsRNA-binding protein to form the B2-dsRNA mixture.

[0023] In at least one alternative embodiment, the dsRNA and the B2 dsRNA-binding protein are mixed under conditions in which the B2-dsRNA complex remains soluble in the reaction solvent. In such alternative embodiments, the conditions may be modified during the reaction such that the B2-dsRNA complex has a lower solubility in the B2-dsRNA mixture than the solubility of other components present in the B2-dsRNA mixture under the modified conditions, and forms as a precipitate. Modification of the conditions includes but is not limited to addition of a second reaction solvent in which the solubility of the B2-dsRNA complex is reduced, and adjusting the temperature of the B2-dsRNA mixture such that the solubility of the B2-dsRNA complex is reduced. In at least one embodiment the second reaction solvent contains a higher concentration of salt than the reaction solvent. In at least one embodiment the second reaction solvent is a buffer containing Tris (20 mM, pH 8.0), EDTA (10 mM, pH 8.0), NaCI (240 mM) and BSA (1% w/v). In at least one embodiment, the temperature of the B2-dsRNA mixture is from about 4°C to about 10°C.

[0024] The present method further includes separating the B2-dsRNA complex from the B2-dsRNA mixture. In at least one embodiment, separating the B2-dsRNA complex from the B2-dsRNA mixture comprises separating the precipitate containing the B2-dsRNA complex from other components which remain in solution in the B2-dsRNA mixture. In at least one embodiment, the precipitate containing the B2-dsRNA complex can be separated from other components remaining in solution in the B2-dsRNA mixture by methods well known in the art including but not limited to centrifugation and filtration. It is notable that separation of the B2-dsRNA complex from the B2-dsRNA mixture by the present method does not involve immobilizing or adsorbing the B2-dsRNA complex on a solid substrate, such as a magnetic bead or a resin. [0025] The present method further includes dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA. In at least one embodiment, dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA comprises mixing a precipitate containing the B2-dsRNA complex with a dissociation solvent to form a dissociation mixture. In at least one embodiment, the dissociation solvent comprises a mixture of components. In at least one embodiment, the mixture of components is an aqueous mixture. In at least one embodiment, the aqueous mixture is a buffer solution.

[0026] In at least one embodiment, the buffer solution comprises a detergent. In at least one embodiment, the detergent is an ionic detergent. In at least one embodiment, the detergent is sodium dodecyl sulfate or lithium dodecyl sulfate. In at least one embodiment, the ionic detergent is preferably soluble in the buffer solution at temperatures in the range of 0°C to 4°C. In at least one such embodiment, the detergent is lithium dodecyl sulphate.

[0027] In at least one embodiment, the buffer solution further contains a salt. In at least one embodiment, the salt is a salt of a weak acid. In at least one embodiment, the salt is sodium acetate. In at least one embodiment, the buffer solution is prepared by adding 10 ml of 3 M sodium acetate solution (pH 5.2) and 10 ml of 10% lithium dodecyl sulphate solution to 80 ml of a buffer solution prepared by mixing 200 ml of 1 M Tris buffer (pH 8.3), 20 ml of 0.5 M EDTA solution and 700 ml of ultrapure water.

[0028] In at least one embodiment, dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA further comprises mixing a non-aqueous solvent comprising chloroform with the dissociation mixture, to provide an aqueous phase containing dsRNA and a non-aqueous phase. In at least one embodiment, the nonaqueous solvent is chloroform. In at least one embodiment, the non-aqueous solvent is a mixture of chloroform and isoamyl alcohol in a ratio of 24:1 (v/v). In at least one embodiment, the non-aqueous solvent is a mixture of phenol, chloroform and isoamyl alcohol.

[0029] Without being bound by theory, it is contemplated that mixing the precipitate containing the B2-dsRNA complex with the dissociation solvent results in dissociation of the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA. Addition of a non-aqueous solvent containing chloroform to this mixture is believed to result in denaturation of the B2 dsRNA-binding protein, which then forms a precipitate at the interface between the more dense non-aqueous phase and the less dense aqueous phase. The dsRNA is believed to remain in the less dense aqueous phase. Thus, in at least one embodiment, separating the B2 dsRNA-binding protein from the dsRNA includes separating the aqueous phase from the non-aqueous phase, and from any precipitate located at the interface between the aqueous and non-aqueous phases. In at least one embodiment, the aqueous phase is separated from the non-aqueous phase by using a pipette to carefully remove the aqueous phase. The skilled person would be well aware of other means by which the aqueous phase can be separated from the non-aqueous phase.

[0030] In at least one embodiment, the present method further comprises precipitation of dsRNA from the aqueous phase containing the dsRNA. In at least one embodiment, precipitation of dsRNA from the aqueous phase containing dsRNA can be effected by adding an alcohol to the aqueous phase containing dsRNA and cooling the mixture such that dsRNA precipitates from the mixture. In at least one embodiment, the alcohol is ethanol or isopropanol. In at least one embodiment, the alcohol is ethanol. In at least one embodiment, the precipitated dsRNA can be separated from the mixture by methods well known in the art including but not limited to centrifugation and filtration. In at least one embodiment, the precipitated dsRNA is sufficiently pure that it can be sequenced or used to identify viruses or viroids present in the initial sample.

[0031] In at least one alternative embodiment, the dissociation solvent comprises an aqueous solution containing a high salt concentration. Any suitable mineral or inorganic salt can be used, as will be recognized by the skilled person. In at least one such embodiment, the salt can contain an alkali metal cation and an anion. In at least one such embodiment, the salt is lithium chloride (LiCI). In at least one such embodiment, the concentration of the salt is from about 0.5 M to about 5 M. In at least one such embodiment, the concentration of the salt is from about 2 M to about 3 M. In at least one such embodiment, the concentration of LiCI is about 2.5 M.

[0032] In at least one such embodiment, the dissociation solvent further comprises an alcohol. In at least one such embodiment, the alcohol is ethanol. In at least one such embodiment, the ethanol is anhydrous. In at least one such embodiment, the alcohol is present in a concentration of between 10% and 70% (v/v). In at least one such embodiment, the alcohol is present in a concentration of about 60% to about 70% (v/v). [0033] In at least one such embodiment, the dissociation solvent further comprises glycogen. In at least one such embodiment, the temperature of the dissociation mixture is lowered to below 0°C. In at least one such embodiment, the temperature of the dissociation mixture is lowered to about -20°C.

[0034] Without being bound by theory, it is contemplated that in such alternative embodiments, the high salt concentration in addition to the presence of an alcohol and glycogen contribute to precipitation of the dsRNA, leaving the B2 dsRNA binding protein and other impurities in the remaining liquid phase. Thus, in at least one such embodiment, separating the B2 dsRNA-binding protein from the dsRNA includes separating the liquid phase containing the B2 dsRNA-binding protein from the precipitate containing the dsRNA. The precipitate can be removed from the liquid phase by methods well known in the art including but not limited to centrifugation and filtration. As will be evident to the skilled person, the precipitate containing the dsRNA can be further purified by rinsing with a solvent, such as 70% aqueous ethanol, in which the dsRNA is not soluble, but any impurities present in the precipitate are soluble. Again, in at least one such embodiment, the precipitated dsRNA is sufficiently pure, including after rinsing, that it can be sequenced or used to identify viruses or viroids present in the initial sample.

[0035] Embodiments of the current method can show advantages over previously known methods of isolating and purifying dsRNA from samples. In at least one embodiment, the present method can take less time to perform than one or more previously known methods. In at least one embodiment, the present method can be less expensive to carry out than one or more previously known methods. In at least one embodiment, the present method can provide a better yield of dsRNA than one or more previously known methods. In at least one embodiment, the present method can provide dsRNA of higher purity than one or more previously known methods.

[0036] A further aspect of the present application provides a kit for use in isolating and purifying dsRNA from a sample. In at least one embodiment, the kit comprises reagents used to carry out the steps of

• separating the B2 dsRNA-binding protein from the dsRNA.

[0037] In at least one embodiment, the one or more reagents include one or more reagents selected from the group consisting of a reaction solvent and a dissociation solvent, as described herein. In at least one embodiment, the one or more reagents further include a second reaction solvent as described herein. In at least one embodiment, the kit can further include reagents for use in preparing the crude RNA extract from a sample. In at least one embodiment, the reagents include ingredients which can be combined with water and/or other readily available ingredients. In at least one embodiment, the kit further includes instructions for use in carrying out the method, including but not limited to instructions for preparation and/or use of the one or more reagents in a method as described herein. [0038] As used herein, the terms “a” and “an” are intended to mean “at least one”, and include both singular and plural, unless otherwise indicated.

[0039] As used herein, the terms “about” or “approximately” as applied to a numerical value or range of values are intended to mean that the recited values can vary within an acceptable degree of error for the quantity measured given the nature or precision of the measurements, such that the variation is considered in the art as equivalent to the recited values and provides the same function or result. For example, the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of ±1 in the most precise significant figure reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" can mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value.

Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0040] As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” aligned would mean that the object is either completely aligned or nearly completely aligned. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.

[0041] The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of’ particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is “substantially free of’ an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.

[0042] As used herein, terms indicating relative direction or orientation, including but not limited to “upper”, “lower”, “top”, “bottom”, “vertical”, “horizontal”, “outer”, “inner”, “front”, “back”, and the like, are intended to facilitate description of the present invention by indicating relative orientation or direction in usual use or as illustrated, and are not intended to limit the scope of the present invention in any way to such orientations or directions. EXAMPLES

[0043] Other features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.

Example 1 : FHV B2 gene cloning

[0044] A polydeoxyribonucleotide encoding the dsRNA-binding N-terminal fragment of the Flock House virus (FHV) B2 protein sequence (GenBank Accession No. X77156), was synthesized for cloning. The sequence of the B2 protein is MPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSL LEKPSWAYLEGKAPEEAKPTLEERLRKLELSHSLPTTGSDPPPAKL (SEQ ID NO:1) [0045] The sequence of the 75-amino acid dsRNA-binding N-terminal fragment is MPSKLALIQELPDRIQTAVEAAMGMSYQDAPNNVRRDLDNLHACLNKAKLTVSRMVTSL LEKPSWAYLEGKAPE (SEQ ID NO:2)

[0046] The 225-nucleotide DNA sequence encoding the 75-amino acid N-terminal fragment is

ATGCCAAGCAAACTCGCGCTAATCCAGGAACTTCCCGACCGCATTCAAACGGCGGT GGAAGCAGCCATGGGAATGAGCTACCAAGACGCACCGAACAACGTGCGCAGGGAC CTCGACAACCTGCACGCTTGCCTAAACAAGGCAAAACTAACGGTAAGTCGGATGGT AACATCACTGCTGGAGAAACCCAGCGTGGTGGCATACCTAGAGGGAAAGGCCCCC GAG (SEQ ID NO:3)

[0047] The B2 fragment-encoding polydeoxyribonucleotide was prepared by the polymerase chain reaction (PCR) using the overlapping oligomers shown in Table 1 and the primers shown in Table 2, which include 5’ tags optimized for cloning into the plasmid pLATE31. Oligomers and primers were synthesized by a commercial provider (Integrated DNA Technology).

Table 1 : B2 oligomer fragments

Table 2: B2 amplification primers

[0048] The PCR reaction mixture (100 pL) contained 50 pL of 2X TopTaq™ Master Mix, a mixture containing a 1 pM concentration of each of the five oligomers shown in Table 1 (1 pL), the forward and reverse primers shown in Table 2 (20 pM, 2 pL of each solution) and water (45 pL). Cycling conditions consisted of an initial denaturation at 94°C for 2 min, followed by 25 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 60 s, and a final step at 72°C for 2 min. The identity of the B2 DNA fragment in the PCR product was verified by 1 % agarose gel electrophoresis. The B2 DNA fragment was purified from the PCR product using a PCR clean-up and Gel extraction kit (Machery-Nagel) and quantitated using a Qubit™ 3.0 fluorometer.

[0049] Ligation-independent cloning (LIC) of the B2 DNA fragment into the pLATE31 vector was carried out using the aLICator™ LIC Cloning and Expression Kit 3 (C-terminal His-tag), #K1261 (Thermo Fisher Scientific) and following the instructions provided by the manufacturer. The resulting annealed mixture is used directly for bacterial cell transformation.

Example 2: B2 protein production in Escherichia coli BL21 (DE3) cells

Transformation of Escherichia coli BL21 (DE3) cells

[0050] The annealed mixture from Example 1 above (2 pl) is added to a vial of Escherichia coli BL21 (DE3) competent cells (separated into two tubes, 50 pl each) and gently mixed by stirring with a pipette tip. The mixture is incubated on ice for 5 to 30 minutes, then the cells are heat-shocked by incubating the vial at 42°C for 30 seconds without shaking. The vial is immediately placed on ice and 250 pl of SOC medium (2% Tryptone, 0.5% Yeast Extract, 10 mM NaCI, 2.5 mM KCI, 10 mM MgCI₂, 10 mM MgSO4, 20 mM glucose; Thermo Fisher Scientific) at room temperature is added. The tube is capped tightly and shaken at 37°C for 1 h, then 10-50 pl of the mixture is spread on a prewarmed selective plate (nutrient agar with ampicillin, 50 pg/ml) and incubated at 37°C for two days. Five clones are selected for multiplication in nutrient broth (NB). Clone cells are conserved in 30% glycerol at -80°C.

Confirmation of expression of the FHV B2 fragment

[0051] The cloned transformed E. coli BL21 (DE3) cells are refreshed by streaking on a nutrient agar plate. A single colony is inoculated in 5 ml of nutrient broth containing 100 pg/ml ampicillin and incubated overnight with shaking at 220-250 rpm to provide a fresh culture.

[0052] The fresh culture (0.6 ml) is transferred to 30 ml of nutrient broth with ampicillin in a 125 ml Erlenmeyer flask and incubated at 37°C with shaking at 220-250 rpm for about

2 h (ODeoo of 0.5-0.6). A control portion (1.0 ml) of the culture is removed, centrifuged at 5000 x g at 4°C for 15 minutes, and the pellet is weighed and conserved at -20°C. Isopropyl p-D-1 -thiogalactopyranoside (IPTG) is added to the remaining culture to a final concentration of 1 mM and the culture is incubated at 37°C with shaking at 250 rpm for

3 h.

[0053] Samples are removed after culturing for 1 h, 2 h and 3 h and centrifuged at 5000 x g at 4°C for 10 minutes. The pellets are weighed, suspended in 5 ml of lysis buffer (20 mM sodium phosphate, 300 mM NaCI, pH 7.4) with lysozyme at 0.4 mg/ml per gram of cells, and incubated at 37°C for 30 minutes. The culture undergoes three freeze/thaw cycles (incubation at -80°C for 15 minutes followed by 30°C for 15 minutes) to break cell walls and the mixture is agitated by pipetting or vortexing until the cell suspension is homogeneous. Alternatively, the culture can be homogenized using a homogenizer such as PowerLyzer™ or FastPrep-24™. The lysate is centrifuged at 20,000 x g at 4°C for 30 minutes and the clear supernatant is analyzed by polyacrylamide gel electrophoresis (PAGE) to verify the presence of the B2 fragment protein.

Preparation of a crude extract of the FHV B2 fragment

[0054] A fresh culture of a transformed E. coli BL21 (DE3) clone is added to nutrient broth with ampicillin at a ratio of 1 :50, and incubated at 37°C with shaking at 220-250 rpm until OD₆oo of 0.5-0.6 is attained. IPTG is added to a final concentration of 1 mM and the culture is incubated at 37°C with shaking at 250 rpm for 3 h.

[0055] The culture is harvested by centrifugation at 5000 x g at 4°C for 10 minutes. The pellet is weighed, suspended in 5 ml of lysis buffer (20 mM sodium phosphate, 300 mM NaCI, pH 7.4) with lysozyme at 0.4 mg/ml per gram of cells, and incubated at 37°C for 30 minutes. Cells are lysed by vibration in a FastPrep-24™ homogenizer for 45 seconds at a speed of 5.5 m/s, and the lysate is centrifuged at 20,000 x g at 4°C for 30 minutes. The clear supernatant is collected and digested with DNase I (1 unit/10 pl of sample) and RNase T1 (1 unit/1 pl of sample) at 37°C for 30 minutes. The reaction is stopped by addition of ethylenediaminetetraacetic acid (EDTA) and RNAsecure™ RNase inactivation reagent (Invitrogen), and heating at 65°C for 10 minutes. The resulting crude B2 fragment preparation is used as is, or after further purification by column chromatography using a 1 ml HisTrap™ HP column to a purity of about 95%. Example 3: Isolation of crude dsRNA extract from plant tissue

Preparation of buffer solutions

[0056] Buffer I is prepared by mixing 200 ml of 1 M Tris buffer (pH 8.3), 20 ml of 0.5 M ethylenediaminetetraacetic acid (EDTA) solution, 12.7 g of lithium chloride, 15 g of lithium dodecyl sulphate, 10 g of deoxycholic acid, 20 g of polyvinylpyrrolidone

(PVP 40,000) and 10 ml of Nonidet™ P-40, and adding ultrapure water to a total volume of 1 L. p-Mercaptoethanol is added to a final concentration of 1% immediately before use.

[0057] Buffer II is prepared by mixing 104 ml of glacial acetic acid and 384 g of potassium acetate in 500 ml of ultrapure water and adding ultrapure water to a total volume of 1 L.

Isolation of crude dsRNA extract

[0058] At room temperature, 500 pl of Buffer I and about 100 mg of fresh or frozen plant tissue are added to a 2-ml tube with a screw cap containing four ceramic beads. The tube is mounted in PowerLyzer™ homogenizer and the sample is homogenized at 4200 rpm for 2 cycles of 45 s - 30 s each, depending on the toughness of the plant tissue to be homogenized. Buffer II (500 pl) is added and the mixture is mixed thoroughly by vortex and centrifuged at 16,500 x g for 10 min at 10°C. Isopropanol (0.67 ml) is added to the supernatant, and the mixture is mixed well and centrifuged at 15,000 x g for 15 min at 4°C. The supernatant is discarded and the pellet containing crude dsRNA is rinsed twice with cold 70% ethanol.

Example 4: Purification of dsRNA

Preparation of buffer solutions

[0059] Buffer III is prepared by mixing 20 ml of 1 M Tris buffer (pH 8.3), 20 ml of 0.5 M EDTA solution, 80 ml of 1 M NaCI solution and 880 ml of ultrapure water.

[0060] Buffer IV is prepared by adding 10 ml of 3 M sodium acetate solution (pH 5.2) and 10 ml of 10% (w/v) lithium dodecyl sulphate solution to 80 ml of Buffer III above.

Purification method

[0061] The crude dsRNA pellet prepared as described in Example 3 is suspended in 400 pl Buffer III and the mixture is centrifuged at 16500 x g for 5 minutes at 4°C. The supernatant is transferred to a clean tube and stabilized at 37°C and 5 pl of the crude B2 fragment preparation of Example 2 (40-50 pg) is added. The mixture is briefly mixed by vortex, incubated at room temperature RT for 5 minutes and cooled on ice for about 5 minutes, then centrifuged at 16500 x g for 10 minutes at 4°C. The supernatant is discarded and the pellet is rinsed with 500 pl of Buffer III and centrifuged at 16500 x g for 10 minutes at 4°C. Again, the supernatant is discarded and the pellet is re-suspended in 50 pl of Buffer IV and mixed well by vortex. Chloroform, or a mixture of chloroform and isoamyl alcohol (24:1 v/v), or a mixture of phenol, chloroform, and isoamyl alcohol (50 pl) is added and the mixture is vortexed and centrifuged at 15000 x g for 15 minutes at 4°C. The clear upper aqueous phase is carefully removed by pipet and transferred to a clean tube. Two volumes of anhydrous ethanol are added and the mixture is mixed and allowed to remain at -20°C for 1 h, then centrifuged at 15000 x g for 15 minutes at 4°C. The supernatant is discarded and the pellet is rinsed twice with cold 70% ethanol, then allowed to air dry. The purified dsRNA pellet is suspended in 25 pl H₂O and may be treated with a deoxyribonuclease and/or a ribonuclease before further characterization or use, including but not limited to sequencing.

Example 5: Alternative purification of dsRNA

Buffer solutions

[0062] Binding buffer (Tris (20 mM, pH 8.0), EDTA (10 mM, pH 8.0), NaCI (180 mM) and bovine serum albumin (BSA, 1% w/v)). A stock buffer can be prepared including all components except BSA, and the BSA can be added immediately prior to use.

[0063] Treatment buffer (Tris (20 mM, pH 8.0), EDTA (10 mM, pH 8.0), NaCI (240 mM) and BSA (1% w/v)). The buffer is chilled on ice before use. A stock buffer can be prepared including all components except BSA, and the BSA can be added immediately prior to use.

[0064] Wash buffer (Tris (20 mM, pH 8.0), EDTA (10 mM, pH 8.0) and NaCI (80 mM)).

Preparation of Eppendorf tubes

[0065] Aqueous BSA solution (10% w/v) is added to clean empty Eppendorf tubes (1 mL of solution for a 5-mL tube; 0.25 mL of solution for a 0.5 mL tube) and the tubes are capped and vortexed to ensure that the BSA solution covers the complete inner surface of the tube and cap. The tubes are centrifuged and the BSA solution is removed and recovered. Treated tubes can be used immediately or dried at a temperature no higher than 45°C and stored at room temperature, if completely dry, or at 4°C or -20°C for future use.

Purification method

[0066] A mixture of a crude RNA extract containing about 40 pg of total RNA and binding buffer (200 pL) is centrifuged at 18,000 x g for 5-10 minutes to remove material which is not soluble in the binding buffer and the supernatant is added to a 0.5 mL Eppendorf tube treated with BSA as described above. The tube is mixed by vortex during addition of the purified B2 fragment preparation of Example 2 (2-2.5 pg of pure B2 dsRNA-binding protein per pg of total RNA; 80-100 pg for a sample containing 40 pg total RNA) and the mixture is mixed well. The tube is capped, secured in a horizontal position on a shaker plate and shaken at 350 rpm for 10 minutes at 37°C.

[0067] The tube is briefly centrifuged to a maximum speed of no higher than 10,000 x g to collect all the liquid, and the collected mixture is transferred to a 5 mL Eppendorf tube treated with BSA as described above containing 3.8 mL of treatment buffer pre-chilled on ice. The tube is capped, secured in a horizontal position on a shaker plate and shaken at 10°C at 200 rpm for 30 minutes, then centrifuged at 18,000 x g at 4°C for 15 minutes. The supernatant is removed carefully by pipette and discarded. Wash buffer (1 mL) is added to the tube containing the pellet, the tube is centrifuged at 18,000 x g at 4°C for 10 minutes and the supernatant is carefully removed and discarded.

[0068] Aqueous LiCI solution (2.5 M, 72 pL) is added to the tube containing the pellet, and the mixture is mixed well by vortex and spun down briefly. The liquid is transferred to a 1.5 mL Eppendorf tube which has not been pre-treated with BSA, and anhydrous ethanol (150 pL) and aqueous glycogen solution (20 pg/pL, 3 pL) are added. The mixture is mixed and allowed to stand at -20°C for 20-30 minutes, then centrifuged at 18,000 x g at 4°C for 10 minutes. The supernatant is removed by pipette and the pellet is carefully rinsed with cold 70% ethanol (v/v). The mixture is centrifuged and the supernatant is discarded. The pellet containing the purified dsRNA is air dried and suspended in 15-20 pL water or TE (T ris-EDTA) buffer for sequencing or other further use.

[0069] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to the person of skill in the art are intended to be included. The appended claims should not be limited by the specific embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS A method of isolating and purifying double stranded RNA (dsRNA) from a sample, the method comprising: treating a crude RNA extract obtained from the sample with a B2 dsRNA-binding protein to form a B2-dsRNA mixture comprising a B2-dsRNA complex; separating the B2-dsRNA complex from the B2-dsRNA mixture, wherein the B2-dsRNA complex is not immobilized on a substrate; dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA; and separating the B2 dsRNA-binding protein from the dsRNA. The method according to claim 1 , wherein the sample is a sample of plant tissue. The method according to claim 1 or 2 wherein the step of treating the crude RNA extract with a B2 dsRNA-binding protein to form a B2-dsRNA complex comprises: mixing the crude RNA extract with a reaction solvent to form a crude dsRNA mixture; and mixing the crude dsRNA mixture with the B2 dsRNA-binding protein to form the B2-dsRNA mixture; wherein a precipitate comprising the B2-dsRNA complex is formed. The method according to claim 3, wherein the precipitate comprising the B2-dsRNA complex forms in the B2-dsRNA mixture. The method according to claim 3, wherein the precipitate comprising the B2-dsRNA complex forms upon addition of a second reaction solvent to the B2-dsRNA mixture. The method according to any one of claims 3 to 5 wherein components of the crude dsRNA mixture which are insoluble in the reaction solvent are removed from the crude dsRNA mixture before mixing the crude dsRNA mixture with the B2 dsRNA- binding protein to form the B2-dsRNA mixture. The method according to any one of claims 3 to 6, wherein the reaction solvent is a buffer having a pH of about 8 to about 9 and containing a chelating agent and a salt. The method according to claim 7, wherein the buffer further contains a blocking agent. The method according to any one of claims 3 to 8, wherein the step of separating the B2-dsRNA complex from the B2-dsRNA mixture comprises separating the precipitate comprising the B2-dsRNA complex from the B2-dsRNA mixture. The method according to claim 9, wherein the step of dissociating the B2-dsRNA complex to form the B2 dsRNA-binding protein and the dsRNA comprises mixing the precipitate comprising the B2-dsRNA complex with a dissociation solvent to form a dissociation mixture. The method according to claim 10, wherein the step of separating the B2 dsRNA- binding protein from the dsRNA comprises: lowering the temperature of the dissociation mixture to below 0°C, such that a precipitate comprising the dsRNA forms, and separating the precipitate comprising the dsRNA from the dissociation mixture. A kit for use in the method according to any one of claims 1 to 11 , the kit comprising one or more reagents used to carry out the method and instructions for carrying out the method. The kit according to claim 12 wherein the one or more reagents comprise one or more reagents selected from the group consisting of a first extraction solvent, a second extraction solvent, a reaction solvent and a dissociation solvent.