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WO2025168812A1 - Compositions comprenant une endoribonucléase spécifique de séquence et procédés d'utilisation - Google Patents

Compositions comprenant une endoribonucléase spécifique de séquence et procédés d'utilisation

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Publication number
WO2025168812A1
WO2025168812A1 PCT/EP2025/053315 EP2025053315W WO2025168812A1 WO 2025168812 A1 WO2025168812 A1 WO 2025168812A1 EP 2025053315 W EP2025053315 W EP 2025053315W WO 2025168812 A1 WO2025168812 A1 WO 2025168812A1
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rna
endoribonuclease
sample
type iii
composition
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Bernd KETELSEN STRIBERNY
Ulli ROTHWEILER
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Arcticzymes ASA
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Arcticzymes ASA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • TITLE Compositions comprising a sequence specific endoribonuclease and methods of use
  • compositions comprising sequence specific endoribonucleases and methods of their use in RNA analysis and RNA synthesis.
  • the present disclosure relates to compositions and samples comprising isolated endoribonuclease of Type III toxin-antitoxin systems preferably endoribonucleases of subfamily CptN and subfamily TenpN.
  • Endoribonucleases are a group of enzymes that cleaves internal phosphodiester bonds between adjacent nucleotides of RNA in either single-stranded RNAs or double-stranded RNAs depending on the enzyme.
  • the endoribonuclease may be sequence specific (e.g. restriction endoribonucleases) or sequence independent.
  • Endoribonuclease enzymes have several applications within molecular biology research. For instance, removal of RNA in DNA extraction processes and recombinant protein purifications, cDNA synthesis, RNA fingerprinting, and detection of RNA modifications, e.g. 5 ’capping of mRNA.
  • endoribonucleases Despite the existence of endoribonucleases there is a continued need for providing further endoribonucleases which permit efficient and simplified methods for RNA analysis or RNA synthesis that overcome one or more of the disadvantages of the endoribonucleases and the methods of prior art.
  • cleaving RNA at specifically recognized sequence motifs can be achieved when the aqueous compositions comprise monovalent cations in a suitable concentration. It is also found that presence of divalent cations at certain concentration (> 3mM) is beneficial with respect to sequence motif specificity and catalytic activity.
  • Said aqueous solutions can have a pH in the range 7.0 to 8.5.
  • Said aqueous solutions can have a pH in the range 7.5 to 8.0.
  • Said aqueous solutions can comprise an endoribonuclease represented by SEQ ID NO: 1 or SEQ ID NO: 2, or an endonuclease with more than 90% sequence identity thereto.
  • the composition comprises a concentration of an alkali metal salt or ammonium salt about ⁇ 300 mM, such as about ⁇ 200 mM, such as from about 10 mM to about 400 mM, such as from about 20 mM to about 300 mM, such as from about 50 mM to about 200 mM.
  • composition is a solution for application to a sample comprising at least one polyribonucleic acid (RNA) molecule.
  • RNA polyribonucleic acid
  • said sample has a volume from 0.1 pl about to about 500pl, preferably from about 0.1 pl to about 300 pl, preferably from about 0.1 pl to about 250 pl, preferably from about 0.1 pl to about 200 pl, more preferably from about 0.1 pl to about 150 pl, more preferably from about 0.1 pl to about 100 pl, more preferably from about 0.1 pl to about 75 pl, more preferably from about 0.1 pl to about 50 pl.
  • the monovalent salt of the composition or sample is an inorganic salt comprising alkali metal or ammonium ions.
  • the monovalent salt is preferably an alkali metal or ammonium salt.
  • the alkali metal ions of the salt are selected from Na+, K+, Li+, Rb+, Cs+ and Fr+ or any combinations thereof.
  • the alkali metal ions are selected from Na+, K+, Li+ and Rb+.
  • ammonium (NH4+) is selected as cation.
  • anions of the salts comprising alkali metals or ammonium ions are preferably selected from fluorine (F), chlorine (Cl), bromine (Br), iodine (I), sulphates, phosphates or hydroxides, acetates, citrate, carbonate or any suitable combinations thereof.
  • the alkali metal salt is selected from NaCl, KC1, Na2SO4, K2SO4, KOH, NaOH, Na-Phosphates, K-Phopshates, Ammonium Acetate, Ammonium Cloride, or any suitable combinations.
  • the composition comprises a concentration of a divalent metal ions of about ⁇ 20 mM, such as about ⁇ 18 mM, such as from about 0.1 mM to about 20 mM, such as from about 1 mM to about 18 mM, such as from about 3 mM to about 15 mM.
  • the divalent metal cations are preferably Mg 2+ or Mn 2+ .
  • composition or sample is essentially without divalent metal cations.
  • the divalent metal cations are preferably Mg 2+ or Mn 2+ .
  • the composition or sample is essentially without divalent metal cations, i.e. comprises ratio of concentration of a divalent metal cation to the concentration of divalent ion chelating agent in the composition providing that the concentration of free divalent metal cation present in the composition is about ⁇ 3 mM, preferably about ⁇ 2mM, more preferably about ⁇ ImM.
  • composition or sample comprises a concentration of a divalent ion chelating agent of about ⁇ lOmM.
  • the isolated CptN endoribonuclease or an enzymatically active fragment thereof comprises amino acid sequence of SEQ ID No.l or comprising an amino acid sequence which is at least 30% identical to SEQ ID No. l.
  • the isolated CptN endoribonuclease or an enzymatically active fragment thereof comprises amino acid sequence of SEQ ID No.l or comprising an amino acid sequence which is at least 70% identical to SEQ ID No. l.
  • the isolated CptN endoribonuclease or an enzymatically active fragment thereof comprises an amino acid sequence of SEQ ID No. l or comprises an amino acid sequence which is at least 75%, 80%, 85%, 90%, 92%, 94%, 95%, 98% or 99% identical to SEQ ID No. l.
  • the isolated TenpN endoribonuclease or an enzymatically active fragment thereof comprises amino acid sequence of SEQ ID No.2 or comprising an amino acid sequence which is at least 30% identical to SEQ ID No.2.
  • the isolated TenpN endoribonuclease or an enzymatically active fragment thereof comprises amino acid sequence of SEQ ID No.2 or comprising an amino acid sequence which is at least 70% identical to SEQ ID No.2.
  • the isolated TenpN endoribonuclease or an enzymatically active fragment thereof comprises an amino acid sequence of SEQ ID No. l or comprises an amino acid sequence which is at least 75%, 80%, 85%, 90%, 92%, 94%, 95%, 98% or 99% identical to SEQ ID No.2.
  • the isolated CptN endoribonucleases disclosed herein is not in a complex with ToxI RNA.
  • the isolated TenpN endoribonucleases disclosed herein is not in a complex with ToxI RNA.
  • composition or sample comprises a ToxN endoribonuclease or an enzymatically active fragment thereof, said CptN endoribonuclease comprises amino acid sequence of SEQ ID No. l or comprising an amino acid sequence which is at least 30% identical, such as at least 70% identical to SEQ ID No.1 wherein
  • the composition or sample comprises ratio of concentration of a divalent metal cation to the concentration of divalent ion chelating agent in the composition or sample such that the concentration of free divalent metal cation present in the sample or composition is about ⁇ 1 mM and the concentration of divalent ion chelator is about ⁇ 10 mM
  • the divalent metal cations are preferably Mg 2+ or Mn 2+ and provided as inorganic salts such as MgCE or MnCE and
  • composition or sample comprises a TenpN endoribonuclease or an enzymatically active fragment thereof, said TenpN endoribonuclease comprises amino acid sequence of SEQ ID No.2 or comprising an amino acid sequence which is at least 30% identical such as at least 70% identical to SEQ ID No.2 wherein
  • -concentration of monovalent salt in the composition or sample preferably about ⁇ 100 mM, about ⁇ 75 mM, about ⁇ 55 mM, more preferably from about 20 mM to about 75 mM, more preferably from about 20 mM to about 55 mM; and wherein
  • the divalent metal cations are preferably Mg 2+ or Mn 2+ and wherein the divalent metal cations are provided as inorganic salts such as MgCE or MnCE.
  • the composition or sample comprises a ToxN endoribonuclease or an enzymatically active fragment thereof
  • said TenpN endoribonuclease comprises amino acid sequence of SEQ ID No.2 or comprising an amino acid sequence which is at least 30% identical, such as at least 70% identical to SEQ ID No.2 wherein -concentration of monovalent salt in the composition or sample preferably about ⁇ 100 mM, about ⁇ 75 mM, about ⁇ 55 mM, more preferably from about 20 mM to about 75 mM, more preferably from about 20 mM to about 55 mM; and wherein
  • the composition or sample comprises ratio of concentration of a divalent metal cation to the concentration of divalent ion chelating agent in the composition or sample such that the concentration of free divalent metal cation present in the sample or composition is about ⁇ 1 mM and the concentration of divalent ion chelator is about ⁇ 10 mM
  • the divalent metal cations are preferably Mg 2+ or Mn 2+ and provided as inorganic salts such as MgCh or MnCh and
  • the divalent ion chelating agent is preferably EDTA or EGTA.
  • a method of cleaving single stranded RNA molecules in a sample comprising the steps: a. providing a sample comprising at least one single stranded RNA molecule comprising a cleavage site for an isolated endoribonuclease of the Type III toxin-antitoxin systems; and b.
  • the cleavage step will typically be incubation which permits cleavage of at least a portion said RNA molecule present in the sample.
  • composition and sample comprising an endoribonuclease of the Type III toxin-antitoxin systems is the composition and sample as described in the first aspect and embodiments thereof.
  • at least one single stranded RNA molecule comprising a cleavage site for an isolated CptN endoribonuclease.
  • the incubation takes place at around 10°C to around 60°C, such as around 15°C to 55°C, preferably in the range 35 to 45°C such as around 45°C.
  • the incubation time which permits cleavage of at least a portion said RNA molecule present in the sample is from about 1 minute to about 2 hours, such as from about 5 minutes to about 1.5 hours, such as from about 10 minutes to about 1 hour.
  • RNA molecule comprising a 3'- PO4 end and a 5'-OH end
  • concentration of a monovalent salt in the sample is about ⁇ 400 mM, such as about ⁇ 300 mM and wherein the monovalent salt is preferably an alkali metal salt or an ammonium salt
  • concentration of a divalent metal ion in the sample is about ⁇ 20 mM, such as about ⁇ 10 mM and wherein the divalent metal ion is preferably Mg2+ or Mn2+ or a combination thereof and c.
  • the sample comprising an endoribonuclease of the Type III toxin-antitoxin systems is the sample as described in the first aspect and embodiments thereof.
  • Figure 9b depicts a capping analysis of an IVT (In Vitro Translated) construct of 40 bases made with the commercial kit “CleanCap”.
  • the dark grey triangles show the 13 base (uncapped) and 14 base (capped) fragments after cleavage reaction with E.coli ToxNl.
  • the addition bands marked with white triangles are caused by RNA initiation slippage.
  • Lane 1 DNA Ladder: bands 50, 20, 15, 8, 6 bases; Lane 2: Capped IVT cut with E.coli ToxNl; Lane 3: uncapped IVT cut with E.coli ToxNl; Lane 4: capped IVT uncut; Lane 5: uncapped IVT uncut.
  • the plasmid was linearized with Hindlll.
  • Figure 14 profiling EcoToxNl (ET-N1), EcoToxN5 (ET-N5) and BthToxN (BT- Nl) endoribonuclease activity.
  • Lane 1 RNA oligo ladder;
  • lane 2 ET-N1 and QI RNA oligo comprising ET-N1 recognition site;
  • lane 3 QI RNA oligo w/o enzyme;
  • lane 4 ET-N5 and RS2 RNA oligo comprising ET-N5 recognition site;
  • lane 5 RS2 RNA oligo w/o enzyme;
  • lane 6 ET-N5 and RS3 RNA oligo comprising ET-N5 recognition site;
  • lane 7 RS3 RNA oligo w/o enzyme;
  • lane 8 UTR-sequence with no recognition site for ET-N5;
  • lane 9 UTR-sequence w/o enzyme;
  • lane 10 RS3 RNA oligo with recognition site for BT-
  • Substrate AC2 GCCGAAAGUCUGACCCUGUA
  • a polyribonucleotide refers to a polymeric form of ribonucleotides.
  • a polyribonucleotide consists of ribonucleotides only.
  • a polyribonucleotide comprises ribonucleotides, and one or more modified ribonucleotides, but does not include any deoxyribonucleotides.
  • a polyribonucleotide comprises ribonucleotides, and may comprise one or more modified ribonucleotides, and one or more deoxyribonucleotides (including modified deoxyribonucleotides).
  • ribonucleic acid RNA
  • polyribonucleotide are used interchangeably and refer to a polymeric form of ribonucleotides of any length.
  • isolated refers to a protein or nucleic acid that, if naturally occurring, is in an environment different from that in which it may naturally occur. “Isolated” is meant to include proteins or nucleic acids that are within samples that are substantially enriched for the protein or nucleic acid of interest and/or in which the protein or nucleic acid of interest is partially or substantially purified. Where the protein or nucleic acid is not naturally occurring, “isolated” indicates the protein or nucleic acid has been separated from an environment in which it was made by either synthetic or recombinant means.
  • the method of RNA digestion involves contacting a sample comprising single stranded RNA with a endoribonuclease of the Type III toxin-antitoxin systems under conditions which permit the digestion of at least a portion of the single stranded RNA present in the sample.
  • the endoribonuclease of the Type III toxinantitoxin systems are sequence specific and will completely digest a polyribonucleotide at their target site under sufficient time and reaction conditions that permit enzyme function.
  • a digestion of at least a portion of the single stranded RNA present in the sample may numerically be expressed as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%.
  • 100% of the single stranded RNA present in the sample is digested, i.e. a complete digestion at their target site of all the RNA molecules present in the sample.
  • RNA antitoxin binds tightly to the toxin to form a hetero tetrameric or hetero hexameric cyclic, unique self-closing RNA-protein complexes, in which the toxin and antitoxin are arranged alternately in a 1 : 1 ratio, ToxIN complex.
  • Identification and functional characterization of the structure of the ToxN-an titoxin (RNA) complex from E.coli is described in Manikandan, P. et al., Identification, functional characterization, assembly and structure of ToxIN type III toxin-antitoxin complex from E.coli, Nucleic acid research, 2022, vol.50, no.3, p.1687-1700.
  • CptN endoribonuclease
  • CptI antagonist RNA molecule/inhibitor of CptN
  • CptIN heteromeric protein/RNA complex of CptN with CptI
  • the Type III toxin-antitoxin systems comprises at least three sub families of enzymes, the ToxN subfamily, the CptIN subfamily and the TenpIN subfamily, cf. Blower, T.R. et al., “identification and classification of bacterial Type III toxinantitoxin systems encoding in chromosomal and plasmid genomes”, Nucleic acid research, 2012, Vol. 40, No. 13, p. 6158-6173.
  • the Type III toxin-antitoxin systems comprise at least three sub families of enzymes, the ToxIN, CptIN and TenpIN, cf. Blower, T.R. et al., “identification and classification of bacterial Type III toxin-antitoxin systems encoding in chromosomal and plasmid genomes”, Nucleic acid research, 2012, Vol. 40, No. 13, p. 6158-6173.
  • the unspecific catalytic i.e. its staractivity of the CptN- and TenpN endoribonucleases are absent inabsence of a divalent metal cation or high concentrations of divalent metal cations such as >5mM.
  • the family of ToxN endoribonucleases are thus not dependent on divalent metal cations for their catalytic activity but increased levels of metal ions improve sequence motif specificity.
  • Divalent metal cations are known to stabilise RNA and protein three dimensional structures.
  • cleavage of RNA in the absence or at high concentrations of divalent metal cations may be more complete as such reaction conditions destabilizes the RNA three dimensional structures thereby improving the ToxN endoribonucleases’ accessibility to its target site in the RNA molecule thereby decreasing the amount of enzyme needed for a complete digestion.
  • a sequence specific endoribonuclease of the Type III toxin-antitoxin systems cleaves single stranded RNA specifically at its recognition site in the presence of particular concentrations of a monovalent salt i.e. unspecific catalytic activity (also called star-activity”) of the enzyme is reduced or absent at particular concentrations of a monovalent salt.
  • the inventors have also for the first time determined that, surprisingly, the unspecific catalytic, i.e. its star-activity of endoribonuclease of the Type III toxinantitoxin systems is reduced or absent at high concentrations or without divalent metal cations, preferably Mg 2+ or Mn 2+ , present in the composition or sample.
  • the inventors have also shown that, surprisingly, it is not necessary to remove divalent metal cations from a composition or sample comprising a endoribonuclease of the Type III toxin-antitoxin systems endoribonuclease, rather the sequence specific catalytic activity may be maintained by a divalent cation chelator such as EDTA or EGTA.
  • a divalent cation chelator such as EDTA or EGTA.
  • essentially free of divalent metal cations in the context of a divalent ion chelating agent is meant that the ratio of the concentration of a divalent cation, preferably Mg2+ or Mn2+ to the concentration of divalent ion chelating agent, e.g. EDTA or EGTA, in the sample is from 3 : 1 to 1 : 10, such as (e.g. Mg 2+ or Mn 2+ : EDTA or EGTA).
  • the ToxN enzyme tolerates a low concentration of a divalent metal cation present in the sample that is not bound to a divalent ion chelator without losing its catalytic activity.
  • composition or sample comprising an isolated endoribonuclease of the Type III toxin-antitoxin systems or an enzymatically active fragment of the ToxN may not comprise free divalent metal cations.
  • the CptN endoribonucleases may be the CptN with an amino acid sequence of SEQ ID NO: 1 (NCBI Acc. No.: PDB: 7D8O A) or an amino acid sequence which is at least about 70% identical to SEQ ID NO: 1.
  • SEQ ID NO: 1 NCBI Acc. No.: PDB: 7D8O A
  • the CptN endoribonuclease may be a CptN endoribonuclease or an enzymatically active fragment thereof wherein the CptN comprises an amino acid sequence which is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 98% or 99% identical to SEQ ID No. l.
  • the TenpN endoribonucleases may be the TenpN with an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence which is at least about 70% identical to SEQ ID NO: 2.
  • Example of sequences with least 70% sequence identity to SEQ ID NO: 2 is the TenpN with an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence which is at least about 70% identical to SEQ ID NO: 2.
  • the TenpN endoribonuclease may be a TenpN endoribonuclease or an enzymatically active fragment thereof wherein the TenpN comprises an amino acid sequence which is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 98% or 99% identical to SEQ ID No.2.
  • sequence identity may be at least 69%, 69.5% or 69.9% to SEQ ID No. l or SEQ ID No.2.
  • the solution is a reagent for application to a sample comprising one or more RNA molecules.
  • a reagent is applied to a sample in order for the endoribonuclease of the Type III toxin-antitoxin systems in said reagent to digest said one or more ribonucleotides present in the sample.
  • the sample comprises multiple ribonucleotides.
  • the solution comprises an endoribonuclease of the Type III toxin-antitoxin systems or enzymatically active fragment thereof.
  • sample refers to a composition comprising single stranded RNA molecules.
  • the skilled person is able to determine the appropriate concentration of the enzyme to include in the sample and the reaction mixture in order to obtain digestion of preferably all RNA molecules in the sample and at the same time avoiding unspecific digestion and star activity.
  • the alkali metal ions of the salt are selected from Na+, K+, Li+ and Rb+.
  • alkali metal salts are NaCl, KC1, Na2SO4, K2SO4, KOH, NaOH, Na- Phosphates, K-Phopshates or any suitable combinations.
  • the composition or sample comprising an endoribonuclease of the Type III toxin-antitoxin systems or an enzymatic active fragment thereof comprises a concentration of alkali metal salt in the composition or sample ⁇ 400 mM, about ⁇ 300 mM, about ⁇ 200 mM, about ⁇ 100 mM, from about 10 mM to about 400 mM, from about 20 mM to about 300 mM.
  • composition or samples may be without free Mn 2+ .
  • nucleic acid molecule encoding a ToxN endoribonuclease or encoding an enzymatic active fragment thereof may be inserted in a suitable expression vector comprising necessary transcriptional and translational elements for expression that are appropriate for the chosen host cell.
  • suitable expression vector comprising necessary transcriptional and translational elements for expression that are appropriate for the chosen host cell.
  • Examples of commonly used expression vectors are plasmids or viruses.
  • the endoribonuclease of the Type III toxin-antitoxin systems may be synthesized using recombinant DNA technology.
  • the endoribonuclease may be produced using a cell-free expression system or chemical synthesis of a endoribonuclease of the Type III toxin-antitoxin systems.
  • a endoribonuclease of the Type III toxin-antitoxin systems enzyme comprising a signaling peptide for secretion into cell culture media may be isolated and purified from the host cell culture media using any technique known in the art and well described in literature. Examples of such techniques or any combination may include precipitation, ultrafiltration, different chromatographic techniques e.g. sizeexclusion chromatography, immobilized metal affinity column chromatography and/or immunoadsorption chromatography.
  • RNA digestion buffers Suitable RNA digestion buffers and reaction conditions for carry out cleavage of RNA is described in detail above.
  • the enzymatic activity of the endoribonuclease of the Type III toxin-antitoxin systems makes such enzymes especially suited for use in methods that involves cleavage of single stranded RNA.
  • the RNA polymerase will continue to amplify RNA into long chains with multiple copies of the RNA of interest;
  • siRNA Small interfering RNA
  • siRNA is a class of double-stranded RNA typically 20-24, normally around 21 base pairs in length, i.e. similar to miRNA.
  • siRNAs operate within the RNA interference (RNAi) pathway and it interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation.
  • RNAi RNA interference
  • mRNA capping structures are well known to a skilled person and Chan, S.H. et al., RNase H-based analysis of synthetic mRNA 5’cap incorporation, RNA 2022, vol.28, p.l 144-1155 discloses several examples of 5’mRNA capping structures.
  • Chan, S.H. et al. 2022 also discloses analysis of 5’capping efficiency of mRNA using DNA-RNA chimera-guided RNaseH cleavage of newly synthetically produced mRNA.
  • a problem with RNaseH in this method is that it is difficult to get uniform cuts as the reaction has to be optimized both with regard to optimal DNA-RNA hybridization and the enzymes ability to provide a uniform cut.
  • Another advantage with ToxN endoribonucleases is that they only require a pentamer as recognition site.
  • ribozymes instead of RNase H has been suggested, EP3183340, however ribozymes require excess amount of the enzyme and the enzyme is highly dependent on a 3D-structure of the RNA for cleavage.
  • step c) of the above method is based on different molecular weight, charge or length of the generated RNA fragments.
  • the 3’ terminal RNA fragments comprising poly(A)-tails may be enriched by using an oligo-dT based enrichment step thereby removing the non-Poly(A) containing fraction.
  • the endoribonuclease of the Type III toxin-antitoxin systems are the CptN endoribonucleases or TenpN endoribonucleases as defined above.
  • separation and detection of the RNA fragments is selected from gel electrophoresis, capillary electrophoresis, high pressure liquid chromatography (HPLC), mass spectrometry (MS) or LC-MS, LC-UV.
  • HPLC high pressure liquid chromatography
  • MS mass spectrometry
  • LC-MS LC-UV
  • capping analysis after cleavage with an endoribonuclease of the Type III toxin-antitoxin systems can be performed the same way as the other methods with LC-MS, FFF (Fastflow Fractionation) or similar techniques.
  • the pentamer recognition site is quite versatile and can be placed anywhere in the 5’UTR.
  • the enzyme does not require an overhang and cuts ssRNA that starts with the cleavage sites.
  • Example lOa-c and figure 9b-d shows that the endoribonuclease of the Type III toxin-antitoxin systems may be used in a method for analysing RNA modifications such as for example 5’ capping of RNA.
  • RNA restriction enzymes may be used to exploit the different location of cleavage sites in a ssRNA.
  • RNA fingerprinting is an important tool within diagnostics and therapeutics, endoribonuclease of the Type III toxin-antitoxin systems for use in analytically RNA fingerprinting has several advantages including a fast and reliable fragmentation of single stranded RNA molecules of defined length that can be analysed by gel electrophoresis, capillary electrophoresis, high pressure liquid chromatography (HPLC), mass spectrometry (MS) or LC-MS, LC-UV.
  • HPLC high pressure liquid chromatography
  • MS mass spectrometry
  • LC-MS LC-MS
  • RNAs can be digested to release RNA fragments comprising the IDR sequence, and analytical methods can be used to quantify the types and amounts of RNA fragments containing each IDR, to generate a profile of the types and/or amounts of each RNA species in a RNA composition.
  • IDR sequences for analysis allows characterization of multivalent RNA compositions comprising several distinct RNA species, even if multiple RNA species are difficult to distinguish by length or coding sequence.
  • a multivalent RNA composition comprising eight RNA species, each encoding a different serotype of the same protein, may have similar lengths and coding sequences, but each RNA species may comprise a different IDR pattern in a coding or non-coding region. Because each IDR sequence unambiguously identifies a particular RNA species, the abundance of IDR sequences may be measured to determine the abundance of RNAs encoding each serotype.
  • separation and detection of the RNA fragments is selected from gel electrophoresis, capillary electrophoresis, high pressure liquid chromatography (HPLC), mass spectrometry (MS) or LC-MS, LC-UV.
  • HPLC high pressure liquid chromatography
  • MS mass spectrometry
  • LC-MS LC-UV
  • Example 11 and figures 10b and 10c shows that endoribonucleases from Type III toxin-antitoxin (TA) system such as endoribonucleases from the ToxN subfamily may be used in a method for RNA fingerprinting.
  • Figure 10b depicts two synthetic RNA oligoes of identical length of 20 bases that migrate equally in the urea/acrylamide gel (lanes 1 and 8). However, each RNA oligo has the E.coli ToxNl (ET-N1) cleavage site located at different position in the RNA sequence. Thereby, the RNA molecules present in the mixture can be identified by their unique pattern of RNA fragments. Furthermore, the band intensity can be used to estimate the ratio of the two RNA molecules in the mixture, cf. figure 10c.
  • E.coli ToxNl E.coli ToxNl
  • a composition comprising an isolated endoribonuclease of the Type III toxinantitoxin systems or an enzymatic active fragment thereof, wherein a concentration of a monovalent salt in the composition is ⁇ 150 mM, such as about ⁇ 100 mM and wherein the monovalent salt is preferably an alkali metal salt.
  • a sample comprising at least one polyribonucleotide and an isolated endoribonuclease of the Type III toxin-antitoxin systems or an enzymatic active fragment thereof, wherein a concentration of a monovalent salt in the sample is about ⁇ 150 mM, such as about ⁇ 100 mM and wherein the monovalent salt is preferably an alkali metal salt.
  • a method of cleaving single stranded RNA molecules in a sample comprising the steps: a. providing a sample comprising at least one single stranded RNA molecule comprising a cleavage site for an endoribonuclease of the Type III toxin-antitoxin systems; and b.
  • a method of RNA fingerprinting comprising the steps: a. providing a sample comprising a at least one single stranded RNA molecule with unknown sequence; b. contacting said sample from step a) with an isolated endoribonuclease of the Type III toxin-antitoxin systems under conditions that permit cleavage of at least a portion of said RNA molecules thereby obtaining a plurality of RNA fragments, wherein concentration of a monovalent salt in the sample is about ⁇ 150 mM, such as about ⁇ 100 mM and wherein the monovalent salt is preferably an alkali metal salt; and c. separation and detection of the fragmented RNA molecule from step b, thereby obtaining a fingerprint of said RNA molecule with unknown sequence and compare the obtained fingerprint with fingerprints from RNA molecules with known sequence.
  • RNA molecule is selected from mRNA, RNA virus, an immunogenic RNA molecule, viroid, long non-coding RNA and ribozyme.
  • compositions, sample or method according to any one of items 1 to 6, wherein the composition or sample comprises ratio of concentration of a divalent metal cation to concentration of divalent ion chelating agent in the composition or sample providing that concentration of free divalent metal cation present in the composition is about ⁇ 3 mM, such as about ⁇ 2mM, such as about ⁇ ImM, wherein the divalent metal cations are preferably Mg 2+ or Mn 2+ .
  • composition, sample or method according to any one of items 1 to 6, wherein the isolated endoribonuclease of the Type III toxin-antitoxin systems is an TenpN endoribonuclease comprises amino acid sequence of SEQ ID No.2 or an enzymatically active fragment thereof or a TenpN endoribonuclease comprising an amino acid sequence which is at least 30% identical to SEQ ID No. l.
  • composition or sample according to item 1 or item 2 wherein the isolated endoribonuclease of the Type III toxin-antitoxin systems is not in a complex with ToxI RNA.
  • a kit comprising: a. A composition comprising an isolated endoribonuclease of the Type III toxin-antitoxin systems or an enzymatic active fragment thereof, wherein a concentration of a monovalent salt in the composition is ⁇ 150 mM, such as about ⁇ 100 mM and wherein the monovalent salt is preferably an alkali metal salt and; b.
  • the isolated endoribonuclease of the Type III toxin-antitoxin systems of a) is a CptN endoribonuclease or a TenpN endoribonuclease and the isolated endoribonuclease of the Type III toxinantitoxin systems of b) is one further CptN endoribonuclease or a TenpN endoribonuclease with a different recognition site from the endoribonuclease of the CptN endoribonuclease or the TenpN endoribonuclease of a).
  • Example 1 Cloning, expression and purification of E.coli ToxNl (ET-N1) endoribonuclease with SEQ ID NO: 3
  • E.coli ToxNl (ET-N1) enzyme toxin
  • toxin enzyme
  • RNA antagonistitoxin
  • the plasmids containing toxin and antitoxin were cotransformed into E. coli BL21(DE3) cells and grown overnight at 37°C, 180 rpm, followed by the secondary culture at 37°C, 180 rpm till OD600 ⁇ 0.5.
  • the culture was incubated at 15°C without shaking for 30 min and the toxin was induced by adding IPTG to a final concentration of 1 mM and incubated at 15°C, 180 rpm for 24 h.
  • the cells were harvested by centrifugation at 6000 rpm for 15 min.
  • the cells were resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 2 mM 2-mercaptoethanol pH 7.5 at 25°C) and lysed by sonication.
  • the lysate was centrifuged at 13 000 rpm for 30 min, and the supernatant was loaded on a Ni 2+ -NTA column.
  • the complex was eluted using elution buffer (lysis buffer + 200 mM imidazole).
  • Fractions containing the complex were dialyzed against ion-exchange buffer (50 mM NaCl, 50 mM Tris-HCl, 1 mM DTT pH 7.5) and purified using anion exchange chromatography by increasing gradient of NaCl from 50 to 1000 mM, over a volume of 100 ml, which yielded separate fractions of toxin, -E.coli ToxNl (ET-N1) (at ⁇ 300 mM NaCl), antitoxin, RNA (at ⁇ 600 mM NaCl) and complex of E.coli ToxNl-RNA (at ⁇ 500 mM NaCl). They were further purified by size exclusion chromatography (SEC) using an S200 column (GE).
  • SEC size exclusion chromatography
  • Toxin E . coli ToxNl _Fwd 5 ' -AGGTCATATGGCGAAATTTTTCACAATATCA-3 ' Toxin ( E . coli ToxNl ) _Rev 5 ' -GGAAATAGACGATCTCGAGTCTAGAGCAT-3 '
  • Example 2 in vitro transcription and production of RNA oligoes comprising a ToxN recognition and cleavage site
  • In vitro transcription was done in 50 pL reactions with 30U T7 RNA Polymerase (ThermoScientific, EP0111) using the included 5X reaction buffer (200 mM Tris- HCl pH 7.9; 30 mM MgC12, 50 mM DTT, 50 mM NaCl, 10 mM spermidine), 50U RiboLock RNase Inhibitor (ThermoScientific, EO0381), 2 mM NTPs (ThermoScientific, R1481), and 1 pg linearized (Seal digested) pGEMEX-1 template.
  • 5X reaction buffer 200 mM Tris- HCl pH 7.9; 30 mM MgC12, 50 mM DTT, 50 mM NaCl, 10 mM spermidine
  • 50U RiboLock RNase Inhibitor ThermoScientific, EO0381
  • 2 mM NTPs Ther
  • any linearized plasmids with a T7 promoter and ToxN cleavage sites or PCR products including a T7 promoter and ToxN cleavage sites can be used.
  • the transcription reaction lasted for 2h at 37°C and was inactivated by adding 10 pL 60 mM ETDA followed by a 10 min incubation at 65°C. Reaction cleanup was conducted using the RNeasy MiniElute Cleanup Kit (Qiagen, 74204) to elute the purified RNA in RNase free water.
  • B. Thuringiensis ToxN (BT-N1): AAA A AAA
  • the experiment was performed in order to verify that the E.coli ToxNl (NCBI Acc. No.: WP 059274511) of use according to the invention exhibit endoribonuclease activity on a single-stranded RNA substrate.
  • Assay conditions (25 pl) : 0 nM to 120 nM ToxN enzyme 50 mM Tris-HCl pH 7.5 50 mM NaCl
  • Example 4 Activity profiling of E.coli ToxNl (ET-N1) endoribonucleases: concentration of monovalent salt and pH
  • RNA single strand oligo Digestion of a 50 nucleotide RNA single strand oligo with ToxN.
  • the recognition site GAAAU is in the centre of the oligo and results in a product of 26 nucleotides (the sequence of the oligo is shown in Example 3).
  • the results are shown in figure 3 and demonstrates that the ToxN enzyme are able to cleave single stranded RNA at pH ranging from 7.0 to 9.0.
  • the results further demonstrate that the specificity of the enzyme is dependent on monovalent ion concentration and is optimal at NaCl concentrations of 50 mM and lower. Above 50 mM the cleavage of the RNA is less specific and the enzyme digests RNA at secondary closely related sequence recognition sites.
  • Example 5 Activity profiling of ToxN endoribonucleases: concentration of divalent salt a) Digestion of a 50 nucleotide RNA single strand oligo with E.coli ToxNl (ET-N1). The recognition site GAAAU is in the centre of the oligo and results in a product of 26 nucleotides (the sequence of the oligo is shown in Example 3). The results are shown in figure 4a. E.coli ToxNl (ET-N1) enzyme activity is extensively inhibited by concentrations of MgCh above ImM (lane E - lane J).
  • Example 6 Activity profiling of E.coli ToxNl (ET-N1) endoribonucleases: concentration of divalent salt, EDTA and DTT
  • Example 7 Activity profiling of E.coli ToxNl (ET-N1) endoribonucleases: incubation time at 15°C Digestion of a 50 nucleotide RNA single strand oligo with E.coli ToxNl (ET-Nl)at increasing incubation time.
  • the recognition site GAAAU is in the centre of the oligo and results in a product of 26 nucleotides (the sequence of the oligo is shown in Example 3). The results are shown in figure 6 and demonstrates that the enzyme is efficient. 50% of the RNA oligos are cleaved after 5 min at an optimal assay temperature of 15°C (lane C).
  • Example 8 Activity profiling of ToxN endoribonucleases: incubation temperature
  • RNA single strand oligo Digestion of a 50 nucleotide RNA single strand oligo with E.coli ToxNl (ET-N1).
  • the recognition site GAAAU is in the centre of the oligo and results in a product of 26 nucleotides (the sequence of the oligo is shown in Example 3).
  • the aim of the study was to profile the enzyme activity at different incubation temperatures. The results are shown in figure 8 and demonstrates that the enzyme specificity decreases at temperatures above 30°C.
  • IVTT In vitro translation
  • IVT RNA Digestion of the IVT RNA was performed at 37 °C. After stopping the IVT reaction with EDTA, a fraction of the IVT reaction was mixed with 10-90 nM E.coli ToxNl (ET-N1) for 10-20 min in IVT buffer. The reaction was stopped by adding urea gel loading buffer (95% Formamide, 0.25 M EDTA, Bromophenol blue) and the samples loaded on a 20 % urea/acrylamide gel. The samples were visualized with SYBRGold.
  • E.coli ToxNl E.coli ToxNl
  • Example 10b Analysis of Post -transcriptional Capping efficiency of mRNA pGEM3Zf-ETNl + 13 was linearized with HincII and used in a T7 RNAP IVT reaction. Half of the resulting GEM3Zf mRNA was then capped using a vaccinia capping enzyme and purified on a silica column. Next, half of the capped and uncapped mRNA was digested using the E.coli ToxNl (ET-N1) endoribonuclease and loaded on a 10% polyacrylamide gel containing 7M urea (TBE, Sybr Gold).
  • E.coli ToxNl E.coli ToxNl
  • TBE Sybr Gold
  • FIJI was used to quantify the relative band intensity of capped ( « pl) and uncapped (p2) mRNA fragments (averaged peak heights of three slices, indicated as “a, b, c” for capped mRNA and “d, e, f’ for uncapped mRNA).
  • About 150 ng mRNA was loaded per lane after 1 : 1 dilution in RNA loading buffer and heating 10 minutes at 65°C.
  • Example 10c - Analysis of co-transcriptional capping efficiency of mRNA pAZ Ol was linearized with PpuMI and used in a T7 RNAP IVT reaction with and without co-transcriptional capping with cap analogs.
  • Half of the capped and uncapped pAZ_01 mRNA was digested using the E.coli ToxNl (ET-N1) endoribonuclease and loaded on a 10% polyacrylamide gel containing 7M urea (stained with Sybr Gold). Uncapped mRNA and capped mRNA that was digested with ET-N1 shows one band of larger size thereby suggesting near to 100% capping efficiency.
  • About 150 ng mRNA or 15 ng RNA oligos were loaded per lane after 1 : 1 dilution in RNA loading buffer and heating 10 minutes at 65°C.
  • pAZ_01 TAATACGACTCACTATA AGGTCTTCTGGTCCCCACAGAA ⁇ ATCTCAGAGA GAACCCACCATGGAGGACGCAAAGAACATAAAAAAAG*GACCC Resulting in mRNA AZ_01 :
  • RNA Fingerprinting by digestion of two 20 nucleotide RNA substrates simultaneously (MOD-UTR: GGGAAJ.AUAAGAGAGAAAAGA-FAM and Distl : GCCGAA AUAGUGACCCUGCA-FAM).
  • the FAM labelled products differ by just one nucleotide resulting in product band of 15 and 14 nucleotides respectively. Reaction conditions: 10 min at 37 °C with 10 nM E.coli ToxNl (ET-N1), 500 nM substrate. Only the product with the FAM label is visible.
  • Figure 10c depicts the ratio of the two RNA molecules in the mixture calculated by measuring the band intensities.
  • Mango aptamer production An IVT produced concatemer of the Mango aptamer was digested with E.coli ToxNl (ET-N1), at decreasing enzyme concentrations in a buffer comprising 25 mM Tris/HCl, 25 mM NaCl pH 7.5, for 20 min at 37 degrees Celsius.
  • E.coli ToxNl E.coli ToxN1
  • the Mango aptamer is described in Dolgosheina et al., 2014; doi: 10.1021/cb500499x.
  • Example 13 stability of the E.coli ToxNl (ET-N1) enzyme in the presence of monovalent salt in the storage buffer 100 mm NaCl to 500 mM NaCl.
  • Example 14 Digestion of RNA by other ToxN endoribonucleases of the ToxIN family
  • BT-N1 recognizes and cleaves with good confidence AAAJ.AAA.
  • ET-N5 cleaves GAAAJ.AAC and AAAAJ.AUC with similar efficiency.
  • This example demonstrates that further members of endoribonucleases belonging to the family of ToxN enzymes according to pFam classification also cuts single stranded RNA under the same buffer conditions as ToxN (ET-N1) even though the protein sequence % identity between ET-N1 and ET-N5 is only 40.88 and protein sequence % identity between ET-N1 and BT-N1 is only 30.95%.
  • the % identity is calculated using Clustal Omega, default settings, multiple protein sequence alignment tool from EMBL-EBI.

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Abstract

La présente invention concerne des compositions comprenant une endoribonucléase spécifique de séquence et des procédés d'utilisation de celles-ci dans l'analyse d'ARN, la synthèse d'ARN et l'empreinte digitale de molécules d'ARN. En particulier, la présente invention concerne des compositions et des échantillons comprenant une endoribonucléase isolée de systèmes toxine-antitoxine de type III, de préférence des endoribonucléases de la sous-famille CptN et de la sous-famille TenpN.
PCT/EP2025/053315 2024-02-07 2025-02-07 Compositions comprenant une endoribonucléase spécifique de séquence et procédés d'utilisation Pending WO2025168812A1 (fr)

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AU2016297778A1 (en) 2015-07-24 2018-03-15 The Johns Hopkins University Compositions and methods of RNA analysis
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