METHOD FOR RNA SELECTION AND/OR ENRICHMENT, RNA MOLECULE AND USE THEREOF

Abstract

A method for RNA selection and/or enrichment, especially with mRNA molecules, from a pool of RNA molecules, the method comprising a step of incubating a sample containing a pool of RNA molecules with an RNA binding protein to form RNA-RNA binding protein complexes, wherein a protein of IFIT family of proteins or its functional variants, homologues or mutants are used as the RNA binding protein. The RNA molecule selected and/or enriched by the method and is used for detection in RNA pathogen-based diagnostic tests and for preparation of libraries for RNA sequencing.

Claims

1-31. (canceled)

32. A method for RNA selection and/or enrichment, especially in mRNA molecules, from a pool of RNA molecules, the method comprising the following steps: (a) preparing an RNA binding protein (b) binding a bed to RNA molecules by means of RNA binding protein (c) washing away the unbound RNA molecules (d) detecting selected RNA molecules whereby a protein of the IFIT protein family or its functional variants, homologues or mutants are is used as the RNA binding protein.

33. The method according to claim 32, wherein the RNA binding protein for use in step (a) is used as a result of conducting the following steps: (a1) producing the RNA binding protein in bacterial protein overexpression systems (a2) purifying the RNA binding protein.

34. The method according to claim 32, wherein the RNA binding protein for use in step (a) is in a form bound to a reporter molecule.

35. The method of claim 34, wherein a released reporter molecule is also washed away in step (c).

36. The method according to claim 32, wherein the binding the bed to RNA by means of RNA binding protein in step (b) is achieved in the following steps: (b1) immobilizing the RNA binding protein on the bed (b2) incubating a sample comprising a pool of RNA molecules with the RNA binding protein to form RNA-RNA binding protein complexes.

37. The method of claim 32, wherein the binding the bed to RNA by means of RNA binding protein in step (b) is achieved in the following steps: (b1′) incubating a sample comprising a pool of RNA molecules with the RNA binding protein to form RNA-RNA binding protein complexes (b2′) immobilizing the RNA binding protein in form of RNA-RNA binding protein complexes on the bed.

38. The method according to claim 32, wherein after the step (c) and before the step (d) the RNA molecules are released from RNA-RNA binding protein complexes.

39. The method according to claim 32, wherein detecting of the RNA molecules in step (d) is performed based on the presence of the released reporter molecule

40. The method according to claim 32, wherein detecting the selected and detected RNA molecules are RNA molecules, especially mRNA, with a Cap 0 structure or a triphosphate group at the 5′ end

41. The method according to claim 40, wherein the Cap 0 structure comprises m7Gppp and/or its forms differing in methylation, such as Gppp.

42. The method according to claim 40, wherein the selected and detected RNA molecules are RNA molecules, especially mRNA, comprising a region of at least 4 unpaired nucleotides at the 5′ end.

43. A method according to claim 32, wherein the selected and detected RNA molecules are selected from: mRNA molecules of viruses, bacteria, fungi, protozoa, invertebrates and/or plants; mtRNA molecules of vertebrates; or RNA molecules being an intermediate in the RNA maturation process.

44. The method according to claim 32, wherein the protein of the IFIT protein family is a protein containing a tetratricopeptide repeat (TPR) region and a structural motif with an amino acid sequence CHFxW present in a helical twist in a loop between alpha-helices, where x means T or N or another amino acid.

45. The method according to claim 32, wherein the protein of the IFIT protein family is a protein comprising an amino acid sequence which selected from (i) a sequence at least 20% identical to the amino acid sequence of the human IFIT1 protein shown as SEQ ID NO: 1, or a functional fragment thereof, and (ii) a sequence at least 20% identical to the amino acid sequence of the human IFIT5 protein shown as SEQ ID NO: 2, or a functional fragment thereof.

46. The method according to claim 32, wherein protein of the IFIT protein family is selected from: human IFIT1 protein having the amino acid sequence shown as SEQ ID NO: 1; human IFIT5 protein having the amino acid sequence shown as SEQ ID NO: 2; a functional variant of IFIT1 and/or IFIT5 protein from a non-human organism; or mutants thereof.

47. The method according to claim 32, wherein the IFIT1 and/or IFIT5 protein is a recombinant protein.

48. The method according to claim 47, wherein the recombinant IFIT1 and/or IFIT5 protein additionally contains an oligohistidine tag, a Strep-tag, a One-Strep or other elements for purifying or immobilizing the protein, and optionally a protease cleavage site between the tag and the tag removal protein and optionally the SUMO tag or other domains improving the protein solubility or stability.

49. An RNA molecule selected and detected by the method defined in claim 32.

50. Use of the RNA molecule selected and detected by the method of claim 32 in RNA pathogen-based diagnostic tests.

51. Use of the RNA molecule selected and detected by the method of claim 32 for preparing cDNA libraries for RNA sequencing or for preparing samples for direct RNA sequencing.

Description

[0048] The invention is illustrated in a drawing, in which:

[0049] FIG. 1 is a scheme of the method of the invention illustrating the selection and enrichment of RNA molecules having appropriate modification of the 5′ end using the rhIFIT1 (A) and rhIFIT5 (B) proteins. Proteins are immobilized on a nickel support, and placed in a mixture of RNA molecules with different 5′ ends. After washing away the unbound RNA molecules, the rhIFIT1 complexes with 5′Cap 0-RNA and rhIFIT5 with 5′ppp-RNA are broken down and the RNA molecules released from the complexes are purified and analyzed.

[0050] FIG. 2 shows images after RNA separation in a polyacrylamide gel of a mixture of synthetic RNA molecules with different 5′ ends and RNA molecules released from the complexes with IFIT1 (A) or IFIT5 (B) proteins following a pull-down experiment (test sample “+IFIT+RNA” and negative control “−IFIT+RNA”). M—size marker, PAA—polyacrylamide gel with a given polyacrylamide content.

[0051] FIG. 3 shows plots of enrichment of selected mRNA from Saccharomyces cerevisiae yeast following a pull-down reaction with rhIFIT1 with respect to 18S rRNA. RT-qPCR results were analyzed by relative quantification, Act1—actin 1, Tdh3—3-phosphoglycerin aldehyde dehydrogenase, RPS26A—component of the small ribosomal 40S unit, GCN4—general control protein 4.

[0052] FIG. 4 shows plots of enrichment of selected RNA from Listeria monocytogenes bacteria following a pull-down reaction with IFIT5: A) with respect to 16S rRNA; B) with respect to 23S rRNA; C) an increase in Cq value means lower amounts of rRNA in samples subjected to pulldown with IFIT5 (e.g. a Cq difference of 1 means a two-fold decrease in RNA amount).

[0053] FIG. 5 shows the image after RNA separation in a polyacrylamide gel of a mixture of synthetic RNA molecules with different 5′ ends and RNA molecules released from rrIFIT1 protein complexes following a pull-down experiment (test sample+IFIT+RNA), M—size marker.

[0054] FIG. 6 shows a comparison and determination of the % of amino acid sequence identity of IFIT5 protein homologs in various animal species. Results of the search in the NCBI protein sequence database using the BLAST-P algorithm by means of the hIFIT5 sequence (SEQ ID NO: 2) have been collated. Parameters are given for selected sequences of human (H), rabbit (R), turtle (T), fish (F) and invertebrate (N) homologues. The proteins marked (*) in the last column are most likely TPR repeat proteins that are not structural and functional IFIT homologs because they do not contain the CHFxW motif (FIG. 7).

[0055] FIG. 7 shows the alignment of selected IFIT protein homologs in the region of the conserved CHFTW structural motif. H—human protein sequences, F—fish protein sequences, N*—invertebrate protein sequences most likely not being structural and functional counterparts of IFIT proteins.

[0056] FIG. 8 shows images of the separation during capillary electrophoresis in a Bioanalyzer, Agilent of RNA molecules obtained following the pull-down experiments described in Example 2 (A) and Example 3 (B).

[0057] The invention has been illustrated in detail in the following examples. The exemplary embodiments do not limit the scope of the invention.

WORKING EXAMPLES

Example 1

[0058] A Method for RNA Selection and/or Enrichment with RNA Molecules Having a Cap 0 Structure at the 5′ End and with RNA Molecules Having a Triphosphate Group at the 5′ End, from a Pool of Synthetic RNA Molecules with Different 5′ Ends

[0059] In the RNA selection and enrichment method, recombinant human protein IFIT1 (rhIFIT1) with SEQ ID NO:1 and recombinant human protein IFIT5 (rhIFIT5) with SEQ ID NO:2 sequence, both in the fusion form with a histidine tag (His-tag) were used.

[0060] The rhIFIT1 and rhIFIT5 proteins were obtained in a bacterial protein overexpression system using standard techniques, utilizing Escherichia coli BL21-CodonPlus (DE3)-RIL strain. Plasmid with rhIFIT1 or rhIFIT5 protein encoding sequence was introduced into bacterial cells by a heat shock method, and the bacterial clones that collected the introduced genetic material were selected by culturing on LB selection supports with antibiotics. The production of proteins was induced after the bacterial culture reached OD.sub.600 0.6 by adding isopropyl-β-D-galactopyranoside (IPTG) to the final concentration of 0.5 mM. After inducing the protein production, bacterial culture was continued overnight (about 16 hours) at 25° C., with shaking at 1200-1600 rpm. Once the culturing was ended, the bacteria were centrifuged (4000 rpm, 20 min. 4° C.), the supernatant was removed and the remaining sediment was suspended in a lysis buffer (50 mM Tris pH 7.5, 0.5 M NaCl, 20 mM imidazole, 10% glycerol, 0.5 mM TCEP), to which protease inhibitor and lysozyme (Sigma) were added. The bacterial suspension was sonicated (breaking down by ultrasounds), then centrifuged (20000 rpm, 40 min. 4° C.) and a clear bacterial lysate was collected containing, among others, IFIT soluble proteins produced in bacterial cells.

[0061] The IFIT proteins were purified by affinity chromatography and gel filtration. For this purpose, the bacterial lysate was passed through a HisTrap HP crude column (GE Healthcare Life Sciences). The rhIFIT1 or rhIFIT5 protein was eluted from the column in a buffer gradient with a high content of imidazole (50 mM Tris pH 7.5, 0.5 M NaCl, 500 mM imidazole, 10% glycerol, 0.5 mM TCEP). The collected fractions containing the purified protein were combined. The sample was diluted 5× in a buffer containing 50 mM Tris pH 7.5, 10% glycerol, 0.5 mM TCEP and passed through a HiTrap Heparin HP column (GE Healthcare Life Sciences) equilibrated with a heparinA buffer (50 mM Tris pH 7.5, 100 mM NaCl, 10% glycerol, 0.5 mM TCEP). The rhIFIT1 or rhIFIT5 protein was eluted from the heparin column in a heparinB buffer gradient with a high salt content (50 mM Tris pH 7.5, 1 M NaCl, 10% glycerol, 0.5 mM TCEP). The collected fractions containing the purified protein were combined. The sample was concentrated to a volume of 1 ml using membrane filters (Vivaspin 20, Sartorius) and passed through a Superdex 200 Increase column (GE Healthcare Life Sciences) in a SEC buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP). The rhIFIT proteins purified in this manner were stored at 4° C. for a few days or frozen in a stream of liquid nitrogen and stored at −80° C.

[0062] The rhIFIT1 and rhIFIT5 proteins were then used for pull-down experiments, which were started by immobilizing the protein to a Ni Sepharose 6 Fast Flow nickel resin support (GE Healthcare). For this purpose, 10 μl of the support was transferred to an Eppendorf tube, washed with 1 ml of RNase-free water, followed by 1 ml of Binding Buffer (BB) of the following composition: 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, 5 mM imidazole, 3 mM MgCl.sub.2, 0.01% Tween 20, centrifuging each time at 100 rpm, 4° C., for 1 min. and removing supernatant. 1 ml of the BB buffer and 2 μg (35 pmol) of rhIFIT1 or rhIFIT5 protein were added to the prepared support. The sample was incubated for 30 min. at 4° C. with gentle mixing. Once the protein was bound to the support, the sample was centrifuged as before, the support was washed with 1 ml of the BB buffer and centrifuged again.

[0063] 1 ml of a mixture containing the BB buffer, about 10 μg of RNA and poly (dIdC) (Poly (deoxyinosinic-deoxycytidylic) acid sodium salt, Sigma) was added to the support at a final concentration of 2 μg/ml. Protein binding to RNA was carried out for 60 min. at 4° C. with gentle mixing. Alternatively, protein binding to RNA was performed prior to immobilizing the protein on the support, starting by mixing 2 μg rhIFIT1 or rhIFIT5 protein with about 10 μg of RNA in 1 ml of the BB buffer. Protein binding to RNA was carried out for 60 min. at 4° C. with gentle mixing. After that the formed protein complexes with RNA were immobilized on a nickel support by incubating the sample with an addition of poly(dIdC) at a final concentration of 2 μg/ml with the support (washed with water and the BB buffer) for 30 min. at 4° C. with gentle mixing. In both cases, after incubation and immobilization, RNA molecules that did not bind to the protein were removed by washing them away. For this purpose, RNA-protein complexes immobilized on the nickel support were washed three times with 1 ml of a Wash Buffer (WB) of the composition: 50 mM Tris pH 7.5, 250 mM NaCl, 1 mM DTT, 5 mM imidazole, 3 mM MgCl.sub.2, 0.01% Tween 20, each time being centrifuged at 100 rpm, 4° C., for 1 min., followed by supernatant removal.

[0064] In the last step, RNA was released from the complexes with IFIT1 and IFIT5, by removing proteins. For this purpose, 400 μL of the WB buffer with proteinase K (Sigma) was added to the support with immobilized complexes at a final concentration of 50 μg/ml. The sample was incubated for 60 min. at 37° C., with shaking at 800 rpm, then centrifuged at 100 rpm, 4° C., for 1 min. The supernatant was collected, 5 μl of linear acrylamide (Sigma) and 2.5 volumes of 100% ethanol were added to it. Alternatively, 400 μL of the WB buffer was added to the support with immobilized complexes and then RNA was purified by phenol and chloroform extraction. RNA was precipitated by adding 1/10 volume of 3M sodium acetate pH 4.8, 5 μl of linear acrylamide (Sigma) and 2.5 volumes of 100% ethanol to the purified sample. The samples were incubated overnight at −20° C., then centrifuged for at least 30 min. at 4° C. at maximum speed. The supernatant was removed and the pellet was washed with 1 ml 80% ethanol and centrifuged for 15 min as before. The RNA precipitate was dried (5-10 minutes) at room temperature, then dissolved in 10-20 μL RNase-free water.

[0065] A scheme illustrating the described pull-down experiment aimed for the selection and enrichment of RNA molecules by means of IFIT proteins is shown in FIG. 1.

[0066] A pull-down using rhIFIT1 or rhIFIT5 protein and a pool of synthetic RNA molecules with different 5′ end was carried out to check whether it is possible to select and/or enrich the RNA sample with RNA molecules having a Cap 0 structure at the 5′ end and with RNA molecules having a triphosphate group at the 5′ end by means of rhIFIT proteins. For this purpose, a mixture was prepared of four types of synthetic RNA molecules, obtained by in vitro transcription (using the HiScribe™ T7 In Vitro Transcription Kit, New England BioLabs, according to the manufacturer's protocol) and enzymatic modification of 5′ ends. The 5′OH modification was obtained by incubating RNA with CIP dephosphorylase (Alkaline Phosphatase, Calf Intestinal, New England BioLabs, according to the manufacturer's protocol). Modification of 5′p was obtained by RNA digestion with 5′ RNA pyrophosphohydrolase (RppH, New England BioLabs, according to the manufacturer's protocol). The 5′ppp modification is a natural effect of in vitro transcription. The 5′ Cap 0 modification was achieved using the capping enzyme of Vaccinia virus (using the Vaccinia Capping System kit, New England BioLabs, according to the manufacturer's protocol). The mixture was prepared by mixing 70 picomoles of each type of molecules:

[0067] RNA 80mer (5′OH) 2 μg (70 pmol)

[0068] RNA 100mer (5′p) 2.4 μg (70 pmol)

[0069] RNA 135mer (5′ppp) 3.2 μg (70 pmol)

[0070] RNA 160mer (5′Cap 0) 3.8 μg (70 pmol)

[0071] The RNA sample prepared in this way was added to the rhIFIT1 or rhIFIT5 protein immobilized on the nickel support and the protein was bound to RNA followed by recovery of the selected RNA molecules from the complexes following the pull-down protocol described above. The obtained RNA was separated by electrophoretic separation in a 12% polyacrylamide gel containing 7M urea, and finally visualized by staining the gel with SYBR™ Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific) and documented using a ChemiDoc MP Imaging System Bio-Rad (FIG. 2).

[0072] The results presented in FIG. 2 show that significant enrichment of the sample with RNA molecules having a Cap 0 structure compared to the remaining RNAs was obtained when using the rhIFIT1 protein and in RNA molecules with a triphosphate group when using the rhIFIT5 protein. RNA samples recovered from the complexes with rhIFIT1 and rhIFIT5 proteins are of good quality (no signs of RNA degradation), which allows to use them in further procedures: transcription into DNA, amplification and sequencing or detection with a tagged probe. Therefore, an efficient, fast and simple method of selecting and enriching the tested RNA sample with RNA molecules having a Cap 0 structure and/or a triphosphate group at the 5′ end was developed.

[0073] On this basis, it was assumed that the RNA selection and enrichment method using the rhIFIT1 and rhIFIT5 proteins of the invention is universal and allows to selectively capture RNA molecules with Cap 0 and a triphosphate group at the 5′ end also in complex tests of total RNA isolated from cells or tissues of various organisms whose RNA has specific Cap 0 and ppp structures at the 5′ end.

[0074] To verify the above, pull-down experiments analogous to those described above were performed using rhIFIT1 protein and total RNA isolated from Saccharomyces cerevisiae yeast, strain BY4741 (Example 2), as well as for rhIFIT5 protein with total RNA from Listeria monocytogenes bacteria, strain EGDe (Example 3) cultured under stress conditions stimulating transcription of active genes during infection. On this basis, the versatility of the method in terms of the origin of the RNA sample was concluded, since the method allows to enrich RNA molecules from organisms belonging even to distant kingdoms such as fungi or bacteria.

[0075] In addition, in order to confirm that in the method of the invention not only human-derived rhIFIT1 and rhIFIT5 proteins can be used, but also non-human homologues of the rhIFIT1 and rhIFIT5 proteins, the amino acid sequence of these proteins was compared (Example 5) and in further experiments a rabbit homolog of rhIFIT1 protein (Example 4) and fish homolog of rhIFIT5 protein (Example 4) were used.

Example 2

[0076] Method for Selection and/or Enrichment of RNA Isolated from Yeast with mRNA Molecules Having a Cap 0 Structure at the 5′ End Using the rhIFIT1 Protein

[0077] The selection and enrichment method, including pull-down experiments, was carried out analogous to that described in the Example 1, with the difference that the RNA molecule pool was a total RNA sample isolated from Saccharomyces cerevisiae yeast, strain BY4741, by extraction using phenol and chloroform. The protein used was rhIFIT1 with the sequence SEQ ID NO: 1, fused to the His-tag.

[0078] After pull-down type experiments, the RNA released from the complexes with the rhIFIT1 protein was subjected to a reverse transcription reaction (using a commercial kit, e.g. First Strand cDNA Synthesis Kit, Roche, according to the manufacturer's protocol). The resulting cDNA was used for real-time quantitative PCR (RT-qPCR). The results were analyzed by relative quantification, comparing the level of selected transcripts: Act1 (actin 1), Tdh3 (3-phosphoglycerylaldehyde dehydrogenase), RPS26A—(component of the small ribosomal 40S unit), GCN4 (general control protein 4, transcription factor) to the level of 18S rRNA. The enrichment of the material with selected RNA molecules was then determined by comparing the relative amount before and after the pull-down procedure.

[0079] The results presented in FIG. 3 show that the use of the rhIFIT1 protein enriches the yeast-derived RNA sample with mRNA molecules from a few to several dozen times relative to 18S rRNA.

Example 3

[0080] A Method for Selection and/or Enrichment of RNA Isolated from Bacteria with RNA Molecules Having a Triphosphate Group at the 5′ End Using the rhIFIT5 Protein

[0081] The selection and enrichment method, including pull-down experiments, was carried out analogous to that described above in the Example 1, with the difference that the RNA molecule pool was a total RNA sample isolated from Listeria monocytogenes bacteria cultured under stress conditions stimulating transcription of virulence-associated genes and the protein used was rhIFIT5 with the sequence SEQ ID NO: 2, fused to a His-tag.

[0082] RNA released from the complexes with the rhIFIT5 protein after pull-down experiments was subjected to reverse transcription (using a commercial kit, e.g., the First Strand cDNA Synthesis Kit from Roche). The resulting cDNA was used for real-time quantitative PCR (RT-qPCR). The results were analyzed by relative quantification, comparing the level of selected transcripts to the level of ribosomal RNA. The enrichment of the material with selected RNA molecules was then determined by comparing the relative amount before and after the pull-down procedure.

[0083] The results are shown in FIG. 4. Following the procedure using the rhIFIT5 protein, approximately 10 fold enrichment relative to 16S rRNA (FIG. 4A) and over 20 fold enrichment relative to 23S rRNA (FIG. 4B) was noted. Enrichment with molecules belonging to the mRNA fraction (Lmo2210) as well as with small non-coding sRNA (LhrC) took place.

Example 4

[0084] A Method for Selection and/or Enrichment of RNA with RNA Molecules Having a Cap 0 Structure at the 5′ End Using an IFIT Protein Homologue from a Non-Human Organism—Example of rrIFIT1 (Rabbit Homologue of rhIFIT1 Protein) or rfIFIT12B (Fish Homolog of IFIT1 Protein)

[0085] The selection and enrichment method, including pull-down experiments, was carried out analogous to that described above in the Example 1, with the difference that the protein was a rabbit protein rrIFIT1 with the sequence SEQ ID NO: 3 or a fish protein rfIFIT12B with the sequence SEQ ID NO: 4, fused with SUMO-tag and His-tag. A mixture of four types of synthetic RNA molecules with different 5′ ends obtained by in vitro transcription was prepared as described in Example 1.

[0086] The results presented in FIG. 5 show that, as in the case of rhIFIT1, a significant enrichment of the sample with RNA molecules having a Cap 0 structure was obtained with the rabbit homolog. RNA samples recovered from the protein complexes are of good quality (no signs of RNA degradation), they can be used in further procedures: transcription into DNA, amplification and sequencing or detection with a tagged probe.

[0087] On this basis, it has been concluded that the RNA selection and enrichment method utilizing the IFIT1 protein homologue from a non-human organism allows selective capture of RNA molecules with Cap 0.

Example 5

Comparison and Determination of Amino Acid Sequence Conservation of IFIT1 and IFIT5 Protein Homologs in Various Animal Species

[0088] The amino acid sequence comparison was performed using the BLASTp algorithm, searching the NCBI database, available at https://blast.ncbi.nlm.nih.gov, using the human IFIT1 (SEQ ID: 1) or IFIT5 (SEQ ID: 2) protein sequences. The results obtained for the selected sequences found are shown in FIG. 6, where the scores and % of sequence identity are given for a given human IFIT5 sequence coverage in individual alignments. Examples of homologous IFIT proteins from various organisms were selected—human, rabbit, turtle and fish, with some fish IFIT homologs having a sequence that was similar in approximately 20% to the human IFIT5 protein sequence. IFIT1 and IFIT5 protein homologs were found to be widespread among vertebrates and the lower limit of sequence identity for probable IFIT homologs was set to 20% for the coverage of most sequences of a human IFIT protein.

[0089] In FIG. 6 also several examples of invertebrate proteins were provided that were designated in the NCBI database as IFIT protein homologs, as well as the UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase enzyme, which was a frequent search result. Some of these proteins have TPR motifs and % of sequence identity exceeding 20% with respect to human IFIT proteins, although they are unlikely structural and functional equivalents of the IFIT protein. Therefore, an auxiliary criterion for identifying IFIT protein homologues was introduced, which is the presence of a structurally important motif having the sequence CHFxW (where x is an amino acid that is usually T). The CHFxW motif adopts a helical twist conformation between alpha-helices in the subdomain I of IFIT proteins and is most likely important for the correct folding or stability of the IFIT protein. Structural homologues of the IFIT proteins, for which the full sequence is available in the NCBI database, are characterized by the presence of the CHFxW motif, as shown by the alignment of IFIT proteins in this region presented in FIG. 7 (achieved using the Clustal Omega algorithm available in NCBI tools).

Example 6

[0090] Evaluation of the Quality and Composition of the RNA Sample Obtained by the Method of the Invention after Pull-Down Experiments

[0091] RNA molecules selected by the method of the invention described in the Example 1 using rhIFIT1 and total RNA from yeast as well as rhIFIT5 and total RNA from bacteria were analyzed by capillary electrophoresis in a Bioanalyzer (with Agilent RNA 6000 Nano Kit according to the manufacturer's protocol).

[0092] It has been shown that the rRNA fraction in RNA samples obtained by pull-down experiments is significantly reduced (FIG. 8), suggesting that the method of the invention can be used for ribodepletion (removal of rRNA) and enrichment with mRNA molecules and some non-coding regulatory RNAs to prepare for a sample for RNA sequencing.

Example 7

Enrichment of RNA Material by the Method of the Invention for the Preparation of an RNA Sample for High-Throughput Nanopore-Based Sequencing

[0093] RNA molecules selected by the method of the invention described in the Example 1 using rhIFIT1 and total RNA from yeast as well as rhIFIT5 and total RNA from bacteria were used to prepare RNA for direct nanopore sequencing. RNA released from complexes with the IFIT proteins were polyadenylated (Poly(A) (using the Tailing Kit, Invitrogen according to the manufacturer's protocol). Then high-throughput sequencing was performed using the MinION device (Oxford Nanopore, according to the manufacturer's protocol).

[0094] Preliminary analysis of the obtained results showed that the RNA sample after pull-down experiments was enriched with mRNA molecules, while the rRNA and tRNA fraction decreased.

Example 8

Enrichment of RNA Material by the Method of the Invention for Detecting Pathogen RNA in Diagnostic Tests

[0095] The method for selection and/or enrichment of RNA according to the invention, including pull-down experiments, was carried out analogous to that described in the Example 3. Then, the enriched RNA was further analyzed for the presence of specific RNA derived from the Listeria monocytogenes pathogen in the sample tested by a colorimetric test using a commercial RT-LAMP kit (e.g. WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA), New England Biolabs, as per manufacturer's recommendations). The RT-LAMP assay combines reverse transcription, isothermal nucleic acid amplification and reading by changing the color of the reaction.

[0096] Preliminary results showed enrichment of the RNA obtained by the method of the invention compared to the initial sample as well as increased test sensitivity resulting from the method. It has been shown that the enrichment of RNA molecules can be a diagnostic test step for detecting the pathogen presence. Enrichment of samples taken from patients with suspected infection with pathogen RNA material increases the sensitivity and selectivity of diagnostic tests.

TABLE-US-00001 Sequence listing amino acid sequence of human protein IFIT1 SEQ ID NO: 1 MSTNGDDHQVKDSLEQLRCHFTWELSIDDDEMPDLENRVLDQIEFLDTKYS VGIHNLLAYVKHLKGQNEEALKSLKEAENLMQEEHDNQANVRSLVTWGNFA WMYYHMGRLAEAQTYLDKVENICKKLSNPFRYRMECPEIDCEEGWALLKCG GKNYERAKACFEKVLEVDPENPESSAGYAISAYRLDGFKLATKNHKPFSLL PLRQAVRLNPDNGYIKVLLALKLQDEGQEAEGEKYIEEALANMSSQTYVFR YAAKFYRRKGSVDKALELLKKALQETPTSVLLHHQIGLCYKAQMIQIKEAT KGQPRGQNREKLDKMIRSAIFHFESAVEKKPTFEVAHLDLARMYIEAGNHR KAEENFQKLLCMKPVVEETMQDIHFHYGRFQEFQKKSDVNAIIHYLKAIKI EQASLTRDKSINSLKKLVLRKLRRKALDLESLSLLGFVYKLEGNMNEALEY YERALRLAADFENSVRQGP amino acid sequence of human protein IFIT5 SEQ ID NO: 2 MSEIRKDTLKAILLELECHFTWNLLKEDIDLFEVEDTIGQQLEFLTTKSRL ALYNLLAYVKHLKGQNKDALECLEQAEEIIQQEHSDKEEVRSLVTWGNYAW VYYHMDQLEEAQKYTGKIGNVCKKLSSPSNYKLECPETDCEKGWALLKFGG KYYQKAKAAFEKALEVEPDNPEFNIGYAITVYRLDDSDREGSVKSFSLGPL RKAVTLNPDNSYIKVFLALKLQDVHAEAEGEKYIEEILDQISSQPYVLRYA AKFYRRKNSWNKALELLKKALEVTPTSSFLHHQMGLCYRAQMIQIKKATHN RPKGKDKLKVDELISSAIFHFKAAMERDSMFAFAYTDLANMYAEGGQYSNA EDIFRKALRLENITDDHKHQIHYHYGRFQEFHRKSENTAIHHYLEALKVKD RSPLRTKLTSALKKLSTKRLCHNALDVQSLSALGFVYKLEGEKRQAAEYYE KAQKIDPENAEFLTALCELRLSI amino acid sequence of rabbit protein IFIT1 SEQ ID NO: 3 MWNPQRTRARSQRAQSGSGNLQTSASFSATMSECAEEHPLKDRLQKLRCHF TWGLLIEDTGLPDLEDRILEEIQFLDTENKVGYNLLAYVKHLQGKHEDALE NLKEAEEVVQGDQADHSDVRSLVTWGNYAWVHYHMGRLADAQTYLDKVENT CQKSADPTRYSTQCPEMDCEEGWALLKCGGKNYERAKACFEKALEADPENP EFNTGYAITVYRLDYPAKRPCDVSDAFSLQPLRKAIRLNPQDAYLKALLAL KLQDVGEEAEGRECLEEALAHTSSQTYVFRYAAKFFRRQGRVDEALKYLKM ALKATPSSAFLHQQIGLCYKKKTNQIMNATHMQPTRQDRENVDRLIQLAIF HFEYAVKQKPTFEVAYVDLARMYITAGDHEKAEDTFQKVLCMTPLQEHIQQ NIHFSYGQFQQFQKKSEVDAITHYLQAVTIRKDSYARDKSIKALEQLVSWK LERNPLDQEALSLREVLHRLVGGRDEALECSEQDLRLAADSGNWVGSSL amino acid sequence of fish protein IFIT12B of Danio rerio SEQ ID NO: 4 MTSDVSSMEADRALRTKLHQLECHFTWALIKDDIDINDLLNRLEEQINLDL EKKERLARTYSALAYVQYLLGFHEKAHQSLMTSKKLHIESHGDEFYRTLIV TYGNLAWLNYHMKNYTECESYLNSLQRINETSPAEFSSIPEVLGEKGWTFL KFSRKYYDGAKECFRKAVELEPEEPEWHTGYAIALYRTEFESTVLEDSATV KQLRLAIEMNPDDDVLKVLLSLRLIVYKRYGEAESWVEKALEKSPDHPHVM RYVGKFFRNKGCVDRSIDLLKRALERSPNSSFIHHQLALCYKYKKIQVLQE QSHHARGSRVQQLRDQCIFHLEKATSLTTSFISAMSDLALQYGENGDIPRA EELFQVTFKIAKEKNDGLHVVNYYYAEYQLYCHRCEPLAVQHYMECLKMCP KSVEGRISSTRMKKTAEKWIDRKSQEGKAYGMLAFLHKVKGEIAQAIECYE KALSYEDNNEFLRNLRELRLSLL