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WO2019134791A1 - Circularisation contrôlée d'arn - Google Patents

Circularisation contrôlée d'arn Download PDF

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Publication number
WO2019134791A1
WO2019134791A1 PCT/EP2018/084289 EP2018084289W WO2019134791A1 WO 2019134791 A1 WO2019134791 A1 WO 2019134791A1 EP 2018084289 W EP2018084289 W EP 2018084289W WO 2019134791 A1 WO2019134791 A1 WO 2019134791A1
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Prior art keywords
helix
rna
loop
activatable
circularization
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Inventor
Sonja PETKOVIC
Sabine MÜLLER
Cathrin HANSEN
Georg Sczakiel
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Universitaet zu Luebeck
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Universitaet zu Luebeck
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/122—Hairpin
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the present invention relates to a method for controlled circularization of RNA comprising the steps of providing an activatable ribozyme structure and performing a ribozyme reaction, to circularized RNA prepared by the method and to an activatable ribozyme structure used in the method.
  • Ribozymes are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. According to their function, they can be classified in different groups.
  • the hammerhead ribozyme and the hairpin ribozyme for example, belong to the group of small ribozymes and can catalyze the cleavage and ligation of nucleotide strands.
  • the secondary structure of the minimal catalytic motif of the hairpin ribozyme consists of four Watson-Crick base paired helices separated by two internal loops A and B. Close association of loops A and B generates the local environment in which catalysis occurs (tertiary structure). The tertiary structure is further stabilized by a Watson- Crick base pair between a loop A guanosine and a loop B cytidine.
  • the cleavage reaction of the hairpin ribozyme generates RNA segments with termini consisting of a 2’,3’-cyclic phosphate and a 5’-hydroxyl group.
  • the ligation reaction of the hairpin ribozyme is a reversal of the cleavage reaction, i.e. covalent joining of RNA segments with termini consisting of a 2’,3’-cyclic phosphate and a 5’-hydroxyl group, resulting in a 3’,5’-phosphodiester linkage common in both RNA and DNA.
  • a circular RNA strand is produced.
  • the naturally occurring hairpin ribozyme is not controllable and results in a multitude of different cleavage/ligation products.
  • Circularized RNA generally exists as a covalently closed single stranded RNA-molecule.
  • the biological functions of circRNA are not fully known to this day, however, it is known that circRNA is capable of regulating splicing processes and it is possible that circRNA fulfills functions in brain tissue, in neural aging or in the context of disease risks, for example the risk of arteriosclerosis.
  • RNA synthesis includes a permuted intron- exon method, a chemical circularization, an enzymatic circularization and an uncontrolled ribozyme reaction.
  • all of these techniques share the disadvantages of a low yield and the presence of side products, like multimerization products.
  • the permuted intron-exon method is very time intensive and the chemical circularization relies on in vitro (click-)chemistry and therefore produces circularized RNA with unphysiological ligation sites and protective groups, which have to be removed after the circularization reaction.
  • the enzymatic circularization of RNA is only possible in vitro and often results in a 3 terminus with non-coding adenines.
  • the enzymatic circularization requires a subsequent ligation step after the RNA synthesis and is expensive.
  • the technical problem underlying the present invention is to provide a method for controlled circularization of RNA of a predefined length in a high yield, which is physiologically ligated, using an activatable ribozyme structure.
  • the present invention provides a method for controlled circularization of RNA comprising the steps of:
  • activatable ribozyme structure comprises two catalytic subdomains, separated by a RNA sequence to be circularized comprising at least 3 nucleotides, wherein the secondary structure of the activatable ribozyme structure comprises the following elements in this order:
  • the spacer sequence may be replaced to facilitate circularization to form any four- way junction RNA by a skilled person by methods known in the art.
  • the activatable ribozyme structure of step a) is a ribozyme structure which exhibits two or more structural conformations.
  • the activatable ribozyme structure exhibits a catalytically active conformation and a conformation where the catalytic activity is inhibited.
  • the transition from one conformation to an other is caused by at least one agent.
  • the activatable ribozyme structure When the catalytic activity is activated, the activatable ribozyme structure is capable of catalyzing specific biochemical reactions. These biochemical reactions can be, for example, cleavage of nucleotide strands, inter- and intramolecular ligation of nucleotide strands, peptide bond formation, phosphate-group transfer or splicing. In a preferred embodiment, the activatable ribozyme structure catalyzes cleavage and ligation of nucleotide strands, wherein the ligation is preferably intramolecular. These reactions may be performed in cis and in trans.
  • the catalytic activity of the activatable ribozyme structure in its catalytically active conformation is caused by a specific arrangement in the tertiary structure of the activatable ribozyme structure.
  • the tertiary structure of the activatable ribozyme structure forms a catalytic site upon activation by the at least one agent.
  • the catalytic activity of the activatable ribozyme structure is inactivated, when the tertiary structure of the activatable ribozyme structure is distorted in a way that the catalytic site cannot be formed.
  • the inactivation can be caused by, for example, chemical modifications to one or more nucleotides, by inhibitors, interfering with the formation of the tertiary structure or with the catalyzed reaction itself, or by exchange of one or more nucleobases in the activatable ribozyme structure compared to an always active variant.
  • the inactivation of the catalytic activity is caused by a nucleobase exchange.
  • a nucleobase exchange according to the present invention is the replacement of at least one nucleobase of an initial nucleotide sequence by at least one other nucleobase, different from the original at least one nucleobase.
  • the at least one nucleobase can be any naturally occurring or artificial nucleobase.
  • Artificial nucleobases include modified naturally occurring nucleobases, for example chemically modified nucleobases, as well as specifically designed nucleobases.
  • the at least one nucleobase can, for example, be adenine, guanine, cytosine, thymine, uracil, hypoxanthine, xanthine, 7-methylguanine, 5,6- dihydrouracil, 5-methylcytosine, 5-hyd roxymethylcytosi ne , iso-guanine and isocytosine.
  • the exchanged nucleobases can be consecutive nucleobases neighboring each other or they can be separated by one or more not exchanged (or unmodified) nucleobases or a combination thereof.
  • an activatable ribozyme structure which is initially inactivated and can be reactivated by at least one agent, it is possible to perform the catalyzed reactions in a controlled manner.
  • the amount of side products can be reduced by employing an activatable ribozyme structure.
  • Side products include, for example, products of multiple cleaving and ligating steps, i.e. multimers of different size.
  • the agents, which activate the catalytic activity of the activatable ribozyme structure of the present invention are not particularly restricted and depend on the type of inactivation.
  • the catalytic activity can, for example, be reactivated by a chemical reaction, by removal of an inhibitor, or by addition of one or more effectors.
  • the one or more effectors are not particularly limited.
  • the effectors are oligonucleotides, which can interact with the activatable ribozyme structure.
  • the effectors preferably intercalate in the tertiary structure of the activatable ribozyme structure and thereby enable the formation of the catalytic site.
  • This intercalation may be reversible or irreversible, preferably it is reversible.
  • the intercalation of the effector is reversible, it is possible to deactivate the catalytic activity of the activatable ribozyme structure by removing the effector.
  • the effectors can be between 1 and 100 nucleotides long, preferably between 5 and 75, more preferably between 10 and 50 and most preferably between 15 and 30.
  • the sequence of the at least one effector is not particularly limited, as long as it serves to activate the catalytic activity of the activatable ribozyme structure of the present invention.
  • the sequence of the at least one effector is preferably chosen to provide, upon intercalation in the tertiary structure of the activatable ribozyme structure of the present invention, the at least one nucleobase which has been exchanged for inactivation of the activatable ribozyme structure, at a position which allows for formation of the catalytic site.
  • the RNA sequence to be circularized comprises at least three nucleotides.
  • the upper limit of the RNA sequence to be circularized is not particularly limited and can be adjusted by a person skilled in the art depending on the circumstances.
  • the RNA sequence to be circularized may contain any natural occurring or artificial RNA or DNA nucleotide.
  • the RNA sequence to be circularized may comprise non-nucleotide elements, like peptides, which can be introduced into the RNA sequence to be circularized by methods known in the art.
  • the secondary structure of the activatable ribozyme structure comprises structural elements including helices, loops and stem-loops.
  • the type, number and arrangement of these structural elements is not particularly limited, as long as they can arrange in the tertiary structure of the activatable ribozyme structure.
  • a helix describes a part of a secondary structure, where a single-stranded RNA strand hybridizes with another single-stranded RNA or with itself and forms a double stranded structure. This hybridization can be supported by Watson-Crick base pairing and/or wobble base pairing.
  • a loop describes a part of a secondary structure, where, due to a lack of base pairing, a RNA strand does not hybridize.
  • a stem-loop describes a part of a secondary structure, where a RNA strand forms a loop to fold back on itself, thereby, for example, closing the open ends of a helix structure.
  • the secondary structure of the activatable ribozyme structure comprises the structural elements Stem-loop 1 - Helix 1 - Loop A - Helix 2 - Helix 3 - Loop B - Helix 4 - Stem-loop 2, wherein Helix 2 and Helix 3 are separated on at least one strand by a spacer sequence.
  • the helical structures of the activatable ribozyme structure may be present in the inactivated form of the activatable ribozyme structure or one or more of the helical structures may only be formed upon activation of the activatable ribozyme structure.
  • the formation of the helical structures upon activation may be initiated by the activating agent or the helical structures may be formed upon hybridization with an effector.
  • Helix 3 and Helix 4 are formed upon activation of the activatable ribozyme structure.
  • Helix 3 and Helix 4 are formed upon intercalation of an effector.
  • Helix 1 of the activatable ribozyme structure comprises from 2 to 100 base pairs, preferably from 3 to 75 base pairs, more preferably from 4 to 50 base pairs, even more preferably from 5 to 25 base pairs. In a specific embodiment, Helix 1 of the activatable ribozyme structure comprises 10 base pairs. Helix 2 of the activatable ribozyme structure comprises from 2 to 100 base pairs, preferably from 3 to 75 base pairs, more preferably from 4 to 50 base pairs, more preferably from 4 to 25 base pairs, even more preferably from 4 to 10 base pairs. In a specific embodiment, Helix 2 of the activatable ribozyme structure comprises 4 base pairs.
  • Helix 3 of the activatable ribozyme structure comprises from 2 to 100 base pairs, preferably from 3 to 75 base pairs, more preferably from 4 to 50 base pairs, more preferably from 5 to 25 base pairs, even more preferably from 5 to 10 base pairs. In a specific embodiment, Helix 3 of the activatable ribozyme structure comprises 5 base pairs. Helix 4 of the activatable ribozyme structure comprises from 2 to 100 base pairs, preferably from 3 to 75 base pairs, more preferably from 4 to 50 base pairs, more preferably from 5 to 25 base pairs, even more preferably from 5 to 10 base pairs. In a specific embodiment, Helix 4 of the activatable ribozyme structure comprises 5 base pairs.
  • Loop A of the activatable ribozyme structure comprises from 2 to 100 nucleotides, preferably from 3 to 75 nucleotides, more preferably from 4 to 50 nucleotides, more preferably from 5 to 25 nucleotides, even more preferably from 6 to 10 nucleotides In a specific embodiment, Loop A of the activatable ribozyme structure comprises 8 nucleotides.
  • Loop B comprises from 2 to 100 nucleotides, preferably from 5 to 75 nucleotides, more preferably from 10 to 50 nucleotides, even more preferably from 15 to 25 nucleotides. In a specific embodiment, Loop B of the activatable ribozyme structure comprises 16 nucleotides.
  • Helix 2 and Helix 3 are separated by one or more spacer sequences on either one or both strands of the double stranded Helix element.
  • the one or more spacer sequence(s) is not particularly limited, as long as it does not negatively interfere with the catalytic activity of the activatable ribozyme structure of the present invention.
  • the spacer sequence is located at the junction of Helix 2 and Helix 3.
  • the at least one spacer sequence comprises from 1 to 100 nucleotides, preferably from 2 to 75, more preferably from 3 to 50, even more preferably from 4 to 25 and most preferably 5 nucleotides. In a case where more than one spacer sequence is present, the spacer sequences can be the same or different.
  • the at least one spacer sequence can consist of different types of nucleotides or be a sequence consisting of just one type of nucleotide. In a specific embodiment, the at least one spacer sequence consists of just one type of nucleotide, preferably the at least one spacer sequence consists of cytosine.
  • the at least one spacer sequence comprises at least 10 nucleotides and forms one or more double stranded regions.
  • These double stranded regions can, for example, be arranged in one or more helices.
  • the one or more double stranded regions may be directly adjacent to each other or they may be separated by loops, stem-loops or other non-hybridized regions.
  • the activatable ribozyme structure forms a 4-way junction as known in the art, which may be similar to a Holliday junction known from DMA.
  • the four-way junction comprises 4 helices (arms) in total, originating from a single center, in this specific embodiment the origin is between helix 2 and 3.
  • the two additional helices (arms) that facilitate ligation reaction comprise at least 4 to 5 base pairs to ensure helix formation at the given reaction parameters e.g. at 37 °C.
  • this one or more modification can be located in any one or more of Helices 1 to 4, Loop A and Loop B.
  • the one or more modification is located in Loop A or Loop B, most preferably in Loop B.
  • the type of inactivation is not particularly limited, as long as it is reversible or can be overcome to reactivate the catalytic activity.
  • the inactivation can, for example, be caused by chemical modification of the primary structure of the activatable ribozyme structure, by exchanging one or more nucleobases of the primary structure of the activatable ribozyme structure or by any external inhibitor suitable to suppress folding into the catalytically active tertiary structure.
  • the inactivation is achieved by the exchange of a nucleobase in a catalytically relevant position for a base which does not confer the catalytic activity to the activatable ribozyme structure of the present application.
  • Stem-loops 1 and 2 are not particularly limited in their size or nucleotide sequence, as long as they do not interfere with the catalytic activity of the activatable ribozyme structure of the present invention.
  • the stem loop of Helix 1 (stem loop 1 ) and the stem loop of Helix 4 (stem loop 2) may be the same or different.
  • Stem loop 1 is at least 3 nucleotides long, preferably at least 4 nucleotides long.
  • the upper limit of the length of Stem-loop 1 is not particularly limited. In case Stem-loop 1 contains the RNA to be circularized, the length of Stem-loop 1 depends on the length of the RNA to be circularized.
  • Stem-loop 1 does not contain the RNA to be circularized
  • the upper limit of the length of Stem loop 1 is not particularly limited.
  • Stem loop 2 may be from 3 to 1000 nucleotides long, preferably from 4 to 750, more preferably from 5 to 500. In a specific embodiment, Stem loop 2 is 6 nucleotides long.
  • the secondary structure of the activatable ribozyme structure further comprises loose (“un-paired”) ends. This means that at the 5’ and at the 3’ end of the activatable ribozyme structure, there are nucleotide sequences, which are not permanently part of a structural element. In a preferred embodiment, the 5’ and the 3 loose ends competitively alternate in being part of the Helix 1 - Loop A - Helix 2 motif.
  • the at least one effector can intercalate in the Helix 1 - Loop A - Helix 2 motif or the Helix 3 - Loop B - Helix 4 motif or both.
  • the at least one effector intercalates in the Helix - Loop - Helix motif which carries the cause of the inactivation of the catalytic activity of the activatable ribozyme structure.
  • the double stranded regions i.e.
  • helices 1 to 4 have to be stable enough to ensure formation of the secondary and tertiary structure and at the same time instable enough to allow for intercalation of the effector.
  • the stability of a helix can be adjusted by controlling the number and ratio of Watson- Crick base pairs, Wobble base pairs and unbinding base pairs in the helix.
  • the primary structure of the activatable ribozyme structure is not particularly limited, as long as it folds into the secondary and/or tertiary structure of the activatable ribozyme structure as described above.
  • the primary structure of the activatable ribozyme structure according to the present invention comprises two catalytic subdomains SD1 and SD2, which are separated by the RNA sequence to be circularized. Thereby, it is possible to circularize any RNA sequence using the activatable ribozyme structure by inserting the RNA sequence to be circularized between SD1 and SD2.
  • SD1 and SD2 together form the Helix 1 - Loop A - Helix 2 motif
  • SD 2 forms the Helix 3 - Loop B - Helix 4 motif
  • Stem-loop 2 and the RNA sequence to be circularized forms Stem-loop 1 , when the primary structure of the activatable ribozyme structure is allowed to fold into the respective secondary structure.
  • the primary structures of Helix 1 and Helix 2 are not particularly limited and can be chosen by a person skilled in the art according to the circumstances, as long as the Helix structures 1 and 2 are formed upon folding of the RNA strand or upon activation.
  • the primary structure of the helices may comprise sequences which form fully Watson-Crick base paired helices or they may comprise sequences which cause mispairing and/or Wobble-base pairing, as long as the resulting helices are stable and formed upon folding of the primary structure.
  • Helix 1 and Helix 2 each comprise at least 8 nucleotides.
  • Helix 3 and Helix 4 The primary structure of Helix 3 and Helix 4 is not particularly limited and can be chosen by a person skilled in the art according to the circumstances, as long as the Helix structures 3 and 4 are formed upon folding of the RNA strand or upon activation.
  • the Helix 3 - Loop B- Helix 4 structure preferably allows an efficient intercalation of an effector.
  • Helix 3 and Helix 4 comprises a ratio of Watson-Crick base pairs to Wobble-base pairs and unbinding base pairs which makes them flexible enough to allow intercalation of an effector.
  • the ratio of Watson-Crick base pairs to Wobble-base pairs and unbinding base pairs is from 3:2 to 1 :4.
  • sequences of the parts of the primary structure of the activatable ribozyme structure which form the Loop A and Loop B elements in the respective secondary structure contain the consensus sequence of the hairpin ribozyme, which has been altered by a nucleobase exchange to inactivate the catalytic activity.
  • This nucleobase exchange can be located in Loop A, Loop B, or both.
  • the nucleobase exchange is located in Loop B.
  • the nucleobase which is exchanged is preferably essential for the catalytic activity.
  • the nucleobase which is exchanged is C25.
  • the C25 base is preferably exchanged for G or A, more preferably G.
  • the primary structure of the activatable ribozyme reaction has the following nucleotide sequence:
  • variable sequence is not particularly limited and comprises at least 3 nucleotides, more preferably at least 4 nucleotides.
  • the upper limit of the length of the variable sequence is not particularly limited. In view of a facile preparation of the nucleotide sequence, the upper limit is preferably 1000 nucleotides, more preferably 750 nucleotides.
  • the effector preferably is SEQ ID NO: 3.
  • the method for controlled circularization of RNA according to the present invention comprises the steps of:
  • a) providing an activatable ribozyme structure wherein the activatable ribozyme structure comprises two catalytic subdomains, separated by a RNA sequence to be circularized comprising at least 3 nucleotides, wherein the secondary structure of the activatable ribozyme structure comprises the following elements in this order: Stem-loop 1 - Helix 1 - Loop A - Helix 2 - Helix 3 - Loop B - Helix 4 - Stem- loop 2, wherein Helix 2 and Helix 3 are separated on at least one strand by a spacer sequence, the RNA sequence to be circularized is part of one or more of Stem- loop 1 , Helix 2, Helix 3 and the spacer sequence separating Helix 2 and Helix 3, and Loop A and Loop B represent the consensus sequence of the hairpin ribozyme, which has been inactivated by a nucleobase exchange; and b) performing a ribozyme reaction.
  • the activatable ribozyme structure may be prepared by any technique known in the art. It can, for example be prepared by in vivo or in vitro transcription of a DMA template into RNA or by direct synthesis of the nucleotide sequence.
  • the activatable ribozyme structure is prepared from a DNA template according to a method comprising the following steps: a1 ) providing a double stranded DNA template;
  • the double stranded DNA template provided in step a1) can be prepared by any technique known in the art, for example by in vivo replication or in vitro by direct synthesis of the nucleotide sequence.
  • the DNA template is prepared via Klenow reaction and optional amplified by polymerase chain reaction (PCR) or molecular cloning.
  • PCR polymerase chain reaction
  • the Klenow reaction is performed using the Klenow primers with the sequences SEQ ID NO: 4 and SEQ ID NO: 5.
  • the DNA template is preferably provided with specific cutting sites for restriction endonucleases and is amplified by inserting the DNA template into a plasmid.
  • This recombinant plasmid can in turn be introduced into bacteria to be amplified.
  • the in vitro transcription of the DNA template of step a1 ) into a single stranded RNA in step a2) can be performed by techniques known in the art, which will be adjusted by a person skilled in the art according to the circumstances.
  • the in vitro transcription can, for example, be achieved using T7 RNA polymerase.
  • the RNA is combined with a buffer solution and, optionally, an agent which supports the folding of the RNA strand.
  • the buffer solution is not particularly limited, as long as it is a suitable buffer for RNA storage and incubation.
  • the buffer solution preferably has an effective pH range between 4.0 and 10.0.
  • the buffer solution is preferably free of components interfering with ribozyme activity, such as ions competing for Mg 2+ - binding sites in the ribozyme structure.
  • a buffer solution substantially free of components interfering with ribozyme activity is preferably a buffer solution comprising components interfering with ribozyme activity in a concentration of less than 100 mM, more preferably 10 mM.
  • the buffer solution is preferably Tris-HCI buffer.
  • the mixture is incubated at a temperature and for a time suitable for denaturing the RNA, i.e. to break all base pairings.
  • the temperature can, for example, be from 50 to 150°C, preferably from 60 to 130°C, more preferably from 70 to 110°C, even more preferably from 80 to 00°C and most preferably 90°C.
  • the incubation time can, for example, be 10 seconds to 5 minutes, preferably 30 seconds to 2 minutes and most preferably 1 minute.
  • the RNA segment is allowed to fold into the secondary structure at a temperature suitable for folding, for example at a temperature of 15 to 50°C, preferably 20 to 45°C, more preferably 30 to 40°C and most preferably 37°C for a time of 30 seconds to 1 hour, preferably 1 minute to 30 minutes, more preferably 2 minutes to 10 minutes and most preferably for 5 minutes.
  • a temperature suitable for folding for example at a temperature of 15 to 50°C, preferably 20 to 45°C, more preferably 30 to 40°C and most preferably 37°C for a time of 30 seconds to 1 hour, preferably 1 minute to 30 minutes, more preferably 2 minutes to 10 minutes and most preferably for 5 minutes.
  • the folding can optionally be supported by the addition of a folding agent which stabilizes the folded structure by compensating for charges occurring on the RNA.
  • the folding agent is, for example, a polyelectrolyte, a salt or a mixture thereof.
  • the folding agent is a salt, it preferably comprises a divalent cation.
  • the salt is preferably MgC .
  • the folding agent may be added before the denaturing of the RNA, i.e. before heating the mixture to a temperature of 50 to 150°C, preferably from 60 to 130°C, more preferably from 70 to 110°C, even more preferably from 80 to 100°C and most preferably to 90°C or after the mixture has cooled down to a temperature suitable for folding of the RNA.
  • the folding agent is preferably added before the denaturing step.
  • it is preferably added in a concentration of 0.1 mM to 200 mM, preferably 1 mM to 100 mM, more preferably 5mM to 50 mM, even more preferably 7mM to 20 mM and most preferably 10 mM.
  • the folding agent when added, supports the folding of the RNA into the secondary and tertiary structure of the activatable ribozyme structure.
  • the folding agent when added before the denaturing step, it can support the folding of the RNA during the cooling down to a temperature suitable for folding of the RNA.
  • the ribozyme reaction in step b) comprises the steps of: b1) cleaving the partially -hybridized single strands (5’ and 3’ loose ends) of the activatable ribozyme structure of step a3); and
  • the steps b1 ) and b2) are performed autocatalytically by the activatable ribozyme structure.
  • the catalytic activity of the activatable ribozyme structure is activated by the addition of an effector, which is preferably added between step a) and step b).
  • the molar amount of the effector added is between 0.1 and 80 times the molar amount of the RNA, preferably 1 to 60 times, more preferably 3 to 40 times, even more preferably 4 to 20 times and most preferably 5 to 10 times.
  • the cleaving of the un-hybrid ized single strands is performed at a temperature and for a time suitable to ensure complete or nearly complete cleaving of the unhybridized single strands in a sample.
  • the cleaving reaction is, for example, performed at a temperature of 4 to 60 °C, preferably 15 to 50°C, more preferably 20 to 45°C, even more preferably 30 to 40°C and most preferably 37°C for a time of at least 1 minute, preferably at least 5 minutes, more preferably at least 10 minutes and most preferably at least 15 minutes.
  • the upper time limit is not particularly restricted, but in view of cost and time efficiency, it is, for example, at most 6 hours, at most 4 hours, at most 2 hours, at most 1 hour or at most 30 minutes.
  • the cleaving efficiency i.e.
  • the ratio of un- or partially hybridized single strands where both the 5’ and 3’ loose ends have been cleaved off to un-hybrid ized single strands in the sample before the cleaving reaction is preferably in a range of 50% to 100%.
  • the lower value is preferably 75%, more preferably 85%, even more preferably 95% and most preferably 99%.
  • the cleaving efficiency can, for example, be determined by radioactive or fluorescent assays.
  • the cleaving efficiency is increased by adding a cleaving agent in addition to the effector.
  • This cleaving agent stabilizes double strand regions in the activatable ribozyme structure, thereby improving the intercalation of the effector into the activatable ribozyme structure and stabilizing the intercalated structure. This results in a higher cleaving efficiency of the activatable ribozyme structure.
  • Suitable cleaving agents can be chosen by a person skilled in the art according to the circumstances.
  • the cleaving agent is a polyelectrolyte, preferably a polycation.
  • the cleaving agent is a polyamine, for example spermine or spermidine. Most preferably, the polycation is spermine.
  • the cleaving agent is added, it is added in a concentration of 0.1 mM to 10 mM, more preferably from 1 to 7.5 mM even more preferably from 4 to 6 mM and most preferably from 4.25 to 5 mM.
  • RNAs with termini consisting of a 2’,3’-cyclic phosphate and a 5’-hydroxyl group. These termini can be ligated in step b2), resulting in a 3’,5’-phosphodiester linkage. In this way, the linkage between the 3’- and the 5’-terminus is a physiological bond.
  • the ligating reaction of step b2) is performed at a temperature and time suitable for ligating all or close to all termini.
  • the ligating reaction is, for example, performed at a temperature of 15 to 50°C, preferably 20 to 45°C, more preferably 30 to 40°C and most preferably 37°C for a time of 1 minute to 6 hours, preferably 5 minutes to 4 hours, more preferably 10 minutes to 2 hours, even more preferably 20 minutes to 1 hour and most preferably for 30 minutes.
  • step b2) of ligating the product of step b1 ) is preferably performed after step b1 ) and catalyzed by the same catalytic site of the activatable ribozyme structure.
  • the ligation can be performed intramolecular or intermolecular.
  • the ligation is performed predominately intermolecular and results in circular dimers of the ribozyme.
  • the ligation is predominately performed intramolecular, i.e. the 5’- and 3’-terminus of the same activatable ribozyme structure are ligated to form a circularized RNA monomer.
  • the efficiency of the ligating reaction can be improved by addition of a ligating agent.
  • the ligating agent may be, for example, a polyelectrolyte or a salt. When the ligating agent is a salt, it preferably comprises a divalent cation.
  • the ligating agent is preferably MgCb.
  • the ligating agent is preferably added before the ligating step b2). When the ligating agent is added, it is added in a concentration of 0.01 to 500 mM, preferably from 0.1 1 to 250 mM, more preferably from 1 to 150 mM, even more preferably from 10 to 100 mM and most preferably 50 mM.
  • the ligating agent when added, stabilizes the secondary and tertiary structure of the RNA.
  • the ligating agent when added before the ligating step, the 3’- and S’-termini of the respective activatable ribozyme structure after the cleaving reaction are in close proximity.
  • the circularizing efficiency i.e. the molar ratio of circularized RNA to ligated RNA in total can be improved.
  • the circularizing efficiency is preferably in a range of 50% to 100%.
  • the lower value is preferably 75%, more preferably 85%, even more preferably 95% and most preferably 99%.
  • the circularizing efficiency can, for example, be determined by quantitative real-time PCR (qRT-PCR).
  • qRT-PCR quantitative real-time PCR
  • a circularizing efficiency of 100% means that only circularized RNA can be detected e.g. in a polyacrylamide gel electrophoresis (PAGE) using SYBR® Gold.
  • the reaction is terminated.
  • the termination can, for example, be achieved by removing the effector from the activatable ribozyme structure, thereby deactivating the catalytic activity of the activatable ribozyme structure.
  • the effector can be removed by techniques known in the art, which will be applied by a person skilled in the art depending on the circumstances.
  • the effector can, for example be removed by denaturing the activatable ribozyme structure with the intercalated effector with increased temperature, a chemical reaction or by changing the chemical environment of the activatable ribozyme structure.
  • the effector is removed by addition of a denaturing agent.
  • the denaturing agent is not particularly restricted and can be selected by a person skilled in the art according to the circumstances.
  • the denaturing agent is selected from the group consisting of urea, formamide, EDTA, sodium dodecyl sulfate (SDS), Tween, Triton-X-100 or Nonidet P-40. More preferably, the denaturing agent is urea or formamide.
  • the present invention also relates to circularized RNA prepared by the method for circularizing RNA.
  • the present invention further relates to an activatable ribozyme structure, which can be used in the method for controlled circularization of RNA according to the present invention.
  • the present invention relates to an activatable ribozyme structure, wherein the activatable ribozyme structure comprises two catalytic subdomains, separated by a RNA sequence to be circularized comprising at least 3 nucleotides and wherein the secondary structure of the activatable ribozyme structure comprises the following elements in this order:
  • the Helix 3 - Loop B - Helix 4 structure of the activatable ribozyme structure is flexible to allow intercalation of an effector.
  • the intercalated effector presents the exchanged nucleobase, thereby activating the catalytic activity of the activatable ribozyme structure.
  • the present invention offers the following advantages:
  • RNA to be circularized has terminal catalytic RNA-domains (SD1 and SD2), which are derived from the hairpin- ribozyme.
  • SD1 and SD2 terminal catalytic RNA-domains
  • Fig. 2 Secondary structure of one possible activatable ribozyme structure: sequences (in bold and grey), which are cleaved to form a 2’,3’- cyclophosphate and a hydroxy group; areas which are free to insert RNA for circularization are indicated with black bold arrows; areas to insert helices for additional junctions are tagged with grey arrows, Watson-Crick- base pairs are indicated by solid lines, while wobble-base pairs are indicated by dotted lines.
  • loop B the critical position is marked in bold und underlined.
  • Fig. 3 General procedure of circularization: a-b: upon Effector (EF-01 ) intercalation, two cleaving reactions take place, depending on which terminus complements Helix 1 and Loop A; b-c: the final cleaved product is circularized; c-d: a stable RNA ring is formed by removal of the EF-01. All steps are reversible, depending on reaction parameters controlled ring formation or opening may be performed.
  • EF-01 Effector
  • Fig. 4 Scheme of a reverse transcriptase polymerase chain reaction (RT-PCR) for verifying circularity of the RNA prepared by the method according to the present invention.
  • the scheme shows the circular product (1 ) of the ribozyme reaction in comparison with linear products (2, 3), which were only cleaved but not ligated.
  • Two different primer types have been designed for the RT-PCR.
  • Reverse primers 1 and 2 bind in the center of the RNA, such that the resulting transcript is only complete (89 nucleotides) when the RNA is circularized (case (1 )).
  • the resulting cDNA is shorter (case (2)) and is lacking the binding site for the forward primer for a subsequent polymerase chain reaction (PCR) step, so if RNA was not circularized, the resulting cDNA cannot be amplified.
  • Reverse primer 3 binds terminally to the RNA, thereby resulting in the full cDNA transcript, independent of whether the RNA was circularized or not.
  • Fig. 5 Verification of the circularization of the RNA: the circularized RNA has been transcribed into cDNA by reverse transcription and amplified by PCR. Only in case of circularized RNA, the cDNA is 89 nucleotides long. Additionally, PCR is only possible if the cDNA is complete, such that the PCR product has 89 base pairs. Urea was added to the reverse transcription (+urea) to make sure that the RT-primer binds specifically. Shown are furthermore a comparative sample with a length of 89 bp and a non-template control (NTC), which did not contain any cDNA for subsequent PCR.
  • the Gene Ruler ULR is a commercially available size standard. The used gel is a denaturing polyacrylamide gel with 7M urea; the nucleic acids are stained with SYBR ® Gold.
  • the activatable ribozyme structure has been prepared in a two-step process, wherein a Klenow-reaction is used to prepare a double stranded DNA-matrix, which is translated into the ribozyme structure by a reverse transcription.
  • Klenow-primer forward
  • Klenow-primer reverse
  • the Klenow-primer were first denatured for 2 minutes at 90°C to avoid the formation of secondary structures or mispairing of the primer. Since the Klenow-fragment exo- is not thermally stable, it was added to the reaction mixture with the dNTP-mix after the reaction mixture was cooled down to 37°C. The polymerisation was performed for 30 minutes at 37°C to obtain the double stranded DNA matrix.
  • the thus prepared DNA matrix was transcribed in vitro into single- stranded RNA using GMP-priming.
  • 1 x HEPES buffer 1 pM DNA matrix, 0.625 mM GTP, 2 mM ATP, 2 mM CTP, 2 mM UTP and 3 mM GMP were used.
  • 1 U/pL of the RNase- inhibitor RiboLock were added to the reaction mixture. The final volume was 50 pL.
  • RNA synthesis was performed for 3 hours at 37°C, followed by a hydrolysis of the DNA double strand with 2 U DNase I.
  • the reaction product was isolated by precipitation with ethanol and purified by gel electrophoresis.
  • the reaction product was treated with 2 x denaturing running buffer and denatured for 2 minutes at 90°C.
  • Gel electrophoresis was performed for 4.5 hours at 200 V at room temperature with a 15% denaturing polyacrylamide gel at a field strength of 8.3 to 13.8 V/cm.
  • the ribozyme reaction consists of two steps. First, the 3’ and the 5’ loose ends of the ribozyme structure are cleaved off, followed by a ligation of the remaining RNA structure.
  • RNA 0.75 pmol RNA were mixed with 10 mM Tris-HCI buffer, 10 mM of MgC and water (final volume 9 pL) and denatured for 1 minute at 90°C. Afterwards, the RNA was allowed to fold into the secondary structure for 15 minutes at 37°C, followed by an addition 7.5 pmol of the effector and 5 mM spermine to induce the cleaving reaction. Cleaving was performed for 30 minutes at 37°C, followed by the addition of 50 mM MgCh and ligating at 37°C for 30 min to obtain circularized RNA.
  • RNA reverse transcriptase polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • PCR polymerase chain reaction
  • the reverse primer for RT-PCR (SEQ ID NO: 6) has been designed in a way that the resulting amplicon includes the ligation site of the RNA and that the full cDNA transcript (in this case 89 nucleotides) is only obtained when the ligation site is covalently closed (case 1 in Fig. 4). In a case, where the RNA is not circularized (case 2 of Fig. 4), a shorter cDNA is obtained.
  • the forward primer used for the PCR (SEQ ID NO: 7) has been designed to bind in a section of the cDNA, which is not present in the shorter transcript of the non-circularized RNA.
  • SEQ ID NO: 6 (RT-PCR reverse primer):
  • SEQ ID NO: 7 (PCR forward primer):
  • SEQ ID NO: 8 control RT-PCR reverse primer

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Abstract

La présente invention concerne un procédé de circularisation contrôlée d'ARN comprenant les étapes consistant à utiliser une structure de ribozyme activable et à effectuer une réaction de ribozyme, l'ARN circularisé préparé par le procédé et une structure de ribozyme activable utilisée dans le procédé.
PCT/EP2018/084289 2018-01-04 2018-12-11 Circularisation contrôlée d'arn Ceased WO2019134791A1 (fr)

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WO2025171319A1 (fr) * 2024-02-09 2025-08-14 Sail Biomedicines, Inc. Compositions photoréactives et procédés de production de polyribonucléotides circulaires
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
JP2023521290A (ja) * 2021-03-10 2023-05-24 アールズィーノミクス・インコーポレイテッド 自己環状化ｒｎａ構造体
JP7531237B2 (ja) 2021-03-10 2024-08-09 アールズィーノミクス・インコーポレイテッド 自己環状化ｒｎａ構造体
AU2022234607B2 (en) * 2021-03-10 2025-01-23 Rznomics Inc. Self-circularized RNA structure
WO2024054047A1 (fr) * 2022-09-06 2024-03-14 알지노믹스 주식회사 Structure d'arn à auto-circularisation
US12441997B2 (en) 2022-12-22 2025-10-14 Genscript Usa Inc. Method of preparing self-circularized RNA
WO2025105906A1 (fr) * 2023-11-17 2025-05-22 Green Cross Corporation Nouvelle construction d'arn et procédé de préparation d'arn circulaire l'utilisant
WO2025171319A1 (fr) * 2024-02-09 2025-08-14 Sail Biomedicines, Inc. Compositions photoréactives et procédés de production de polyribonucléotides circulaires

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