EP2929019A1

EP2929019A1 - Agent and method for modifying the 5' cap of rna

Info

Publication number: EP2929019A1
Application number: EP13826999.8A
Authority: EP
Inventors: Andrea Rentmeister; Daniela STUMMER
Original assignee: Universitaet Hamburg
Current assignee: Universitaet Hamburg
Priority date: 2012-12-10
Filing date: 2013-12-06
Publication date: 2015-10-14
Also published as: DE102012222675A1; JP2016501879A; WO2014090246A1; US20160032257A1

Abstract

The invention provides an agent and method for modifying the 5' cap of RNA, for example for the purposes of isolation and analysis. According to one aspect the invention provides modified enzymes, namely modified trimethylguanosine synthases 2 from Giardia lamblia (GlaTGS2), the enzymatic activity of which is changed such that as compared to wild type enzymes the former can use AdoMet analogues better as cofactors.

Description

MEDIUM AND METHOD FOR MODIFYING THE 5 'CAP OF RNA

The invention relates to means and methods for modifying the 5 'cap of RNA. By examining the genome of a cell, important information about its

State, for example, whether the cell's own regulatory processes are correct or whether there are changes compared to the normal state. This information can then be used, for example, to determine if a cell is degenerate, has been infected by viruses, or is in an irregular state. In this way, for example, conclusions about any

present diseases are drawn.

For this purpose, it is known, for example, to study the expression of genes by means of isolation and analysis of the mRNA molecules present in the cells. To be reliable

To obtain information, the quality of the mRNA preparation is of particular importance

Importance. However, since numerous other molecules and other RNA species, in particular non-coding RNAs (for example sRNAs, tRNA, rRNA, ncRNA, miRNA, etc.) are present in the cell in addition to mRNAs, selective enrichment must take place on the basis of specific features. For the specific isolation of mRNA from eukaryotes one uses their

characteristic features, namely the so-called poly (A) tail at the 3 'end and the cap structure at the 5' end (J. Pease, R. Sooknanan, Nat Meth 2012, 9; JS Marcus, WF Anderson, SR Quake, Analytical Chemistry 2006, 78, 3084-3089; ZY Yang, HJ

Edenberg, R.L. Davis, Nucleic Acids Res 2005, 33; M.E. Folkers, D.A. Deiker, C.I.

Maxwell, C.A. Nelson, J.J. Schwartz, D.A. Nix, C.H. Hagedorn, Plos One 2011, 6; Z. Gao, Q. Zhang, Y. Cao, P. Pan, F. Bai, G. Bai, Journal of Chromatography A 2009, 1216, 7670-

7676; U. Schibler, D. Rifat, DJ Lavery, Methods 2001, 24, 3-14; EZ Bajak, CH Hagedorn, Methods in Molecular Biology (Clifton, NJ) 2008, 419, 147-160; AK Shukla, AK Shasany, SPS Khanuja, Indian Journal of Experimental Biology 2005, 43, 197-201). These features usually bind the mRNA non-covalently to other molecules immobilized, for example, on appropriate columnar materials (eg, binding via the poly (A) tail to oligo-dT columns or via the 5 'cap to the protein eIF4E ). For the isolation of mRNAs currently mainly the included poly (A) tail is used. This, like the cap, is a characteristic structure of mature rnRNA molecules and miRNA precursors (pri-miRNA). Through this region, the molecules can hybridize to immobilized, complementary deoxynucleotide (01igo-dT) probes and thus be isolated from complex samples. The required column materials ("beads") are known in the art and are used by default.

Bajak and Hagedorn (E.Z. Bajak, C.H. Hagedorn, Methods in Molecular Biology (Clifton, N.J.) 2008, 419, 147-160) have established a method in which a variant of the

Translational initiation factor eIF4E is used to isolate RNA via its cap structure (see also US 6841363 B2, Gowda, Nucleic Acids Research, 2010, 38, 21, 7558-7569). One advantage of this is that RNA molecules are identified and enriched, regardless of the length of their poly (A) tail, solely because of the cap structure involved, thus also short poly (A) tail RNAs (both mRNAs and

Precursors of miRNAs) for subsequent analyzes. To carry out the assay, an eIF4E variant which has up to 10-fold higher affinity for the target molecules than the wild-type protein is immobilized on glutathione beads via a protein affinity tag (in particular glutathione-S-transferase, GST) the sample is incubated. The RNA with a cap structure binds non-covalently to the immobilized protein and can thus be isolated from the complex sample with the help of the beads. In this way, regardless of the varying motif of the poly (A) tail, RNA molecules of hepatitis C-infected cells could be isolated. After carrying out a next-generation sequencing experiment, new statements could be made regarding changes in the

Gene regulation within the host cell after infection by the virus are made (Folkers M., PLOS one, 2011, 6, 2, el4697, Papic, 2012, Viruses, 2012, 4, 581.612).

A disadvantage of these methods is the inability to covalently attach RNA molecules directly to a support, which limits the choice of wash conditions in the removal of impurities. In addition, the common 1: 1 relationship between the binding molecule and the RNA molecule limits the use of these methods. Dalhoff et al. (Dalhoff C, Lukinavicius G, Klimasäuskas S, Weinhold E., 2006, Nat. Chem. Biol. 2 (1): 31-32) describe the direct transfer of an ethyl, propyl, propenyl and 2-butynyl group to 2'- Deoxycytidine and 2'-deoxyadenosine by three S-adenosyl-L-methionine (AdoMet) -dependent DNA methyltransferases via the use of corresponding analogues of this cofactor. The AdoMet analog carries the corresponding group instead of a methyl group on the sulfur atom. Lukinavicius et al. (G. Lukinavicius, V. Lapiene, Z. Stasevskij, C. Dalhoff, E. Weinhold, S. Klimasäuskas, J. Am. Chem. Soc., 2007, 129, 2758-2759) used another, an NH ₂ group containing, AdoMet analogue for the transmission of this group by means of suitable DNA methyltransferases on DNA nucleosides. A similar approach is also used by Motorin et al. which describe the use of a combination of enzymatic transfer and click chemistry for site-specific labeling of tRNA molecules for biophysical studies (Y. Motorin, J. Burhenne, R. Teimer, K. Koynov, S. Willnow, E. Weinhold, M. Helm, Nucleic Acids Research, 2010, 1-10,

doi: 10.1093 / nar / gkq825). AdoEnYn, also an analogue of the cosubstrate S-adenosyl-L-methionine, was used to enzymatically label the pentenine residue CH = C-CH = CH-CH ₂ - by tRNA: methyltransferase Trml on the exocyclic N2 atom of guanosine at position 26 to transfer a tRNA ^phe . Subsequently, a fluorophore was bound to the modified tRNA ^phe by Cu (I) - catalyzed azide-alkyne-1,3-dipolar cycloaddition (CuAAC). Also, sequence-specific click labeling of RNA was achieved using Box C / D RNP methyltransferases (M. Tomkuviene, B. Clouet-d'Orval, I.

Cerniauskas, E. Weinhold, S. Klimasäuskas, Nucleic Acids Res 2012).

However, there is still a need for further possibilities to isolate certain RNA molecules, in particular mRNA molecules, from cells and to supply them to an analysis.

The present invention therefore has for its object to provide such a further possibility. This problem is solved by the subjects of the independent claims.

Advantageous embodiments of the invention are specified in the subclaims. It has surprisingly been found that a modified at a certain position enzyme, namely the Trimethylguanosinsynthase 2 from Giardia lamblia (hereinafter abbreviated to "GlaTgs2"), whose wild-type sequence is shown in SEQ ID NO: 1, new possibilities for labeling and and / or isolation of RNA species having a 5 'm ⁷ GpppN cap, this m ⁷ GpppN cap (engl., "m ⁷ GpppN cap") is a guanosine residue methylated at position N7. at position 5 'over a

Triphosphorsäureesterbrücke is bound to the 5 'end of an RNA molecule (5'-5' linkage), as shown in the following formula (II):

R ¹ is OH or OCH ₃ . The B in the above formula stands for any nucleobase. The N in the expression m ⁷ GpppN stands for nucleoside, nucleotide, nucleoside or nucleotide analogue, ppp for the triphosphate bridge, G for guanosine and m ⁷ for the methyl group at N7.

Such a cap includes, for example, eukaryotic mRNA molecules, but also certain noncoding RNA species, e.g. snRNAs, snoRNAs and telomerase RNAs.

Wild-type GlaTgs2 (see SEQ ID NO: 1) has 258 amino acids and catalyzes the further methylation (hypermethylation) of capping guanosine at position N2 with S-adenosyl-L-methionine (AdoMet) as cofactor (S. Hausmann et al, J. Biol. Chem. 2008, 283, 31706-31718). Unlike human trimethylguanosine synthase hTgs, which can catalyze the transfer of two methyl residues to N2, the enzyme from Giardia lamblia does not seem to accept dimethylated nucleotides as a substrate, leaving the enzyme to transfer only a single methyl residue to N2. Thus, it is actually supposed to be a dimethylguanosine synthase and not a trimethylguanosine synthase act. However, to avoid misunderstandings, the term trimethylguanosine synthase or Tgs for this enzyme is used here nevertheless.

AdoMet is also abbreviated to "SAM" and serves as a cofactor for various enzymes to transfer the methyl group at the sulfur atom, leaving behind the CH ₃ group and leaving S-adenosyl-L-homocysteine, which is also abbreviated as "AdoHcy" or "SAH" becomes.

It has now surprisingly been found that an amino acid exchange at position 34 of the wild-type GlaTgs2 according to SEQ ID NO: 1, which results in that the amino acid valine located at this position is exchanged for another amino acid, preferably against a non-polar / hydrophobic or polar / neutral amino acid, and most preferably alanine, glycine or methionine, which alters enzymatic activity of the resulting enzyme such that it can use AdoMet analogues as cofactors or as compared to

Wildtype enzyme can better use. Preferably, the amino acid introduced instead of valine is not tryptophan or leucine. This opens up the

Possibility of a targeted modification of the 5 'end of RNA species carrying a m ⁷ GpppN cap, where appropriate, in subsequent steps to attach reporter groups and / or to achieve a specific immobilization of these RNA species.

Examples of nonpolar / hydrophobic amino acids are alanine, valine, methionine, leucine, isoleucine, proline, tryptophan and phenylalanine. Examples of polar / neutral amino acids are tyrosine, threonine, glutamine, glycine, serine, cysteine and asparagine.

By the term "AdoMet analog" herein is meant a compound of the following formula (I)

understood that carries a radical R on the sulfur atom, which is not a methyl group. It is thus a compound with the same basic skeleton as AdoMet (S-adenosyl-L-methionine), wherein the sulfur atom instead of the methyl group, another radical is attached. AdoMet analogs are described, for example, in WO 2006/108678 A2.

An example of such a radical is propenyl CH ₂ = CH-CH ₂ -. The corresponding AdoMet analogue (5 ^, - [(S) - [(3S) -3-amino-3-carboxypropyl] prop-2-enylsulfonio] -5'-deoxyadenosine) with this residue instead of methyl is called "AdoPropen" and has the following formula (Ia):

Further examples of radicals R are propynyl CH = C-CH ₂ -, butinyl CH = C-CH ₂ -CH ₂ -, pent-2-en-4-ynyl CH = C-CH = CH-CH ₂ -, benzyl Ph -CH ₂ - (Ph = C ₆ H ₅ ) and azidobutenyl N ₃ -CH ₂ - CH = CH-CH ₂ -. In the case of the propynyl radical CH = C-CH ₂ - the AdoMet analog here is referred to as "AdoPropin", in the case of the butinyl radical CH ^ C-CH ₂ -CH ₂ - as "AdoButin", in the case of pent-2-ene -4-inyl radical CH = C-CH = CH-CH ₂ - as "AdoEnYn", in the case of the benzyl radical "AdoBenzyl" and in the case of the azidobutenyl radical N ₃ -CH ₂ -CH = CH-CH ₂ - as "AdoAzid For the pent-2-en-4-inyl radical, the term "penteninyl radical" is also used here.

In a first aspect, therefore, the invention provides an isolated or synthetic protein which

a. has or comprises an amino acid sequence according to SEQ ID NO: 2, or b. has or comprises an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 2, provided that valine is not present in the homologous amino acid sequence at the position corresponding to position 34 of SEQ ID NO: 2, or c , has or comprises an amino acid sequence which is homologous to the amino acid sequence according to SEQ ID NO: 2, wherein the amino acid sequence to the amino acid sequence according to SEQ

ID NO: 2 is more than 85%, preferably at least 90%, more preferably at least 95% identical, with the proviso that in the homologous amino acid sequence at the

Position corresponding to position 34 according to SEQ ID NO: 2, not valine is, or d. a contiguous partial sequence of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 80, 90 or at least 100 amino acids, preferably at least 1 10, 120, 130, 140,

150, 160, 170, 180, 190 or at least 200 amino acids, more preferably at least 210, 220, 230, 240 or at least 250 amino acids of the amino acid sequence according to a, b or c, or comprises, with the proviso that the partial sequence the amino acid Position 34 according to SEQ ID NO: 2 or the corresponding homologous amino acid, and

e. does not have the amino acid sequence according to SEQ ID NO: 1 (GL50581 2635 from Giardia intestinalis ATCC 50581, GenBank No. EET00120.1).

The formulation that the protein "has an amino acid sequence" means that the protein consists of the sequence, ie no further amino acids are present at the C- and / or N-terminus The formulation that the protein "comprises an amino acid sequence", means that the protein contains the sequence, without being limited to proteins having no further amino acids at the C and / or N-terminus, but this term also encompasses the formulation that the protein has an amino acid sequence ", ie consists only of the amino acids listed in the sequence and in the order given in the sequence.

The term "protein" as used herein refers to polymers of any number of amino acids linked together by peptide bonding and includes the terms "peptide" and "polypeptide." The linear sequence of amino acids in a protein is referred to as "amino acid sequence." The term "synthetic" as used herein means "artificially produced" and includes proteins that do not occur naturally, ie, with the particular amino acid sequence. "Isolated" here means that a protein from its original or natural environment, for example from a

Eukaryotic or prokaryotic cell has been liberated.

The term "homologous" in relation to a protein means that a protein is in its

Amino acid sequence largely coincides with a different protein compared with it, without being completely identical. For example, homolog may mean that a protein other than an amino acid has an amino acid sequence identical to the trimethylguanosine synthase of Giardia lamblia. The presence of homology between two proteins can be determined by comparing each one position in one sequence with the corresponding position in the other sequence and determining whether identical or similar residues are present. Two compared sequences are homologous if there is a certain minimum level of identical or similar amino acids. Identity means that when comparing two sequences at equivalent positions in each case the same amino acid. If necessary, it may be necessary to take account of sequence gaps in order to produce the best possible alination of the comparison sequences. Similar amino acids are amino acids with the same or equivalent chemical and physical properties. When one amino acid is replaced by another

Amino acid with the same or equivalent chemical-physical properties is called a "conservative exchange." Examples of physicochemical

Properties of an amino acid are, for example, the hydrophobicity or the charge. Particular examples of similar amino acids are nonidentical amino acids which have been synthesized using the computer program "Basic Local Alignment Search Tool", abbreviated BLAST, (SF Altschul et al., 1990, Basic Local Alignment search tool, J. Mol BLOSUM62 substitution matrix (Henikoff, S., and Henikoff, J., Amino Acid Substitution Matrices from Protein Blocks, Proc. Natl Acad., USA, 89: 10915-10919, 1992) are reported as "positive," ie, have a positive score in the BLOSUM62 substitution matrix. For the purposes of the present invention, it is anticipated that there will be homology between two sequences if at least 45%, preferably at least 50%, at least 55%>, at least 60%>, at least 65%>, at least 70%>, at least 75%>, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the amino acids are identical or similar, preferably identical. In particular, the presence of homology between two sequences is assumed when using the computer program BLAST (SF

Altschul et al. (1990), Basic Local Alignment search tool, J. Mol. Biol. 215: 403-410; s. e.g. http://www.ncbi.nlm.nih.gov/BLAST/) using default default parameters ("Expect Threshold" = 10, "Word size" = 3, "Existence Gap Costs" = 11, "Extension Gap Costs = 1 and the BLOSUM62 substitution matrix (Henikoff, S., and Henikoff, J., Amino Acid Substitution Matrices from Protein Blocks, Proc Natl Acad., USA, 89: 10915-10919, 1992) have an identity or similarity (" Positive "), preferably identity, of at least 45%, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. Preferably, in this case, a minimum length of 20, preferably a minimum length of 25, 30, 35, 40, 45, 50, 60, 80 or 100, more preferably a minimum length of 120, 140, 160, 180 or 200

Amino acids, or assumed a minimum length of 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amino acids of the respective amino acid sequences. Particular preference is given to the full length of the respective protein. It will be readily apparent to those skilled in the art, from a knowledge of the art, which of the available BLAST programs, e.g. BLASTp, eligible for identification of homology. In addition, there are other programs known to those skilled in the art, and may be used in assessing the homology of two or more sequences to be compared if necessary. Such programs are, for example, on the websites of the European

Bioinformatics Institute (EMBL) available (see for example http://www.ebi.ac.uk/Tools/similarity.html). In particular, "homologous" in the present application means a match, ie identity in the amino acid sequence, of at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or at least 99%, more preferably at least 99.5%. In particular, "homologue" may also mean that one trimethylguanosine synthase contains no more than 60, preferably not more than 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11, particularly preferably not more than 9, 8, 7, 6, 5, 4 , 3, 2 or 1 position (s) has a different, missing or additional amino acid. The term "alkyl" includes saturated aliphatic (non-aromatic) groups,

including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl) and branched chain alkyl groups (e.g., isopropyl, tert -butyl, isobutyl). The term also includes O, N, S or P alkyl groups (e.g., -O-methyl), i. Alkyl groups which are bonded to a compound via an oxygen, nitrogen, sulfur or phosphorus atom.

The term "C _n -C _m ", wherein n and m are each positive integers and m is greater than n, means an area indicating the number of C atoms of a compound or a group Thus, for example, the term "Ci-Cio" (n = 1, m = 10) means a compound, a group or a residue with 1-10, ie 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. "Ci-Cio" therefore simultaneously also includes, for example, _"C2-C6", ie 2, 3, 4, 5 or 6 C Atoms, or "C1-C4", ie 1, 2, 3 or 4 C-atoms, or "C4-C9", ie 4, 5, 6, 7, 8 or 9 C-atoms. Accordingly, the term "C ₂ -C 10 -alkyl" means, for example, an alkyl group having 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms and includes all combinations of the values of n and m, the in the range from n = 2 to m = 10, eg "Cs-Cy-alkyl", ie an alkyl having 5, 6 or 7 C atoms.

The term "alkenyl" includes unsaturated aliphatic (non-aromatic) groups having at least one CC double bond, including straight-chain and branched-chain alkenyl groups The term also includes O, N, S or P alkenyl groups (eg, -O-propenyl). , ie alkenyl groups, which have an oxygen, nitrogen, sulfur or

Phosphorus atom are bound to a compound. The term "C ₂ -C 10 alkenyl" means an alkenyl group having 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms.

The term "alkynyl" includes unsaturated aliphatic (non-aromatic) groups having at least one C-C triple bond, including straight-chain and branched-chain

Alkenyl groups. The term also includes O-, N-, S- or P-alkynyl groups (eg -O-butynyl), ie alkynyl groups attached via an oxygen, nitrogen, sulfur or phosphorus atom to a Connection are bound. The term "C 2 -C 10 -alkynyl" denotes an alkynyl group having 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms.

The term "alkenynyl" includes unsaturated aliphatic (non-aromatic) groups having at least one C-C double bond and at least one C-C triple bond,

including straight-chain and branched-chain alkeninyl groups. The term also includes O, N, S or P alkenyl groups, i. Alkeninyl groups which have an oxygen,

Nitrogen, sulfur or phosphorus atom are bonded to a compound. The term "C ₄ _10 -alkeninyl" means an alkeninyl group having 4, 5, 6, 7, 8, 9 or 10 C atoms.

The term "cycloalkyl" includes alicyclic groups, ie, cyclic saturated aliphatic (non-aromatic) groups, eg, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl. The term also includes O, N, S or P cycloalkyl groups, ie Cycloalkyl groups linked to a compound via an oxygen, nitrogen, sulfur or phosphorus atom The terms "cycloalkenyl", "cycloalkynyl" and "cycloalkeninyl" mean, respectively, ring-shaped aliphatic (non-aromatic) alkenyls, alkynyls or alkenyls according to the above definition, wherein the double and / or triple bond (s) may be present inside or outside the ring or ring system. The term "heteroalkyl" refers to alkyl groups in which one or more

Carbon atoms of the hydrocarbon skeleton through other atoms (heteroatoms), e.g. Oxygen, nitrogen, sulfur or phosphorus atoms are replaced. The term also includes O, N, S or P heteroalkyl groups, i. Heteroalkyl groups which are bonded to a compound via an oxygen, nitrogen, sulfur or phosphorus atom. The term "heteroalkyl" also includes cycloalkyls in which one or more carbon atoms of the hydrocarbon backbone are replaced by other atoms, eg, oxygen, nitrogen, sulfur, or phosphorus, by the terms "heteroalkenyl," "heteroalkynyl," "heteroalkeninyl." are alkenyls, alkynyls and alkenyls as well

Cyclo alkenyls, cycloalkynyls and Cycloalkeninyle understood in which one or more carbon atoms of the hydrocarbon skeleton by other atoms (heteroatoms), for example, oxygen, nitrogen, sulfur or phosphorus atoms replaced. The term "C 1 -C 10 -heteroalkyl" denotes an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 C atoms and at least one heteroatom. The same applies analogously to heteroalkenyls, heteroalkynyls and heteroalkenyls.

By "azidoalkyl" herein is meant an alkyl having an azido group -N ₃ The term also includes cyclic alkyls and heteroalkyls having an azido group. "Azidoalkenyl", "azidoalkynyl" and "azidoalkeninyl" correspondingly include alkenyls, alkynyls and alkenyls, cyclic alkenyls , Alkynyls and alkenyls as well as heteroalkenyls, alkynyls and alkenyls understood. The term "substituted" means one or more substituents replacing a hydrogen atom on one or more carbon atoms of the hydrocarbon backbone, such as oxo, hydroxyl, phosphate, cyano, and amino groups, but also halogens, for example (eg F, Cl, Br, I), alkyl, cycloalkyl, aryl and heteroaryl.

By a "nucleic acid" is meant a polymer whose monomers are nucleotides A nucleotide is a compound of a sugar moiety, a nitrogen-containing heterocyclic organic base (nucleotide or nucleobase) and a phosphate group The sugar moiety is usually a pentose, in the case DNA deoxyribose, in the case of RNA ribose The nucleotides are linked via the phosphate group by means of a

Phosphodiester bridge usually between the 3'-C atom of the sugar component of a nucleoside (compound of nucleobase and sugar) and the 5'-C atom of

Sugar component of the next nucleoside. The term "nucleic acid" as used herein includes, for example, DNA, RNA and DNA / RNA mixed sequences.

A "nucleobase" is understood to mean organic bases found in RNA or DNA, which are often purines (R) and pyrimidines (Y), examples of purines are guanine (G) and adenine (A), examples for pyrimidines cytosine (C), thymine (T) and uracil (U) are phosphorylated nucleosides, for example

Nucleoside monophosphates (NMP), nucleoside diphosphates (NDP), and nucleoside triphosphates (NTP) are also referred to as nucleotides. The phosphate, diphosphate (pyrophosphate) or triphosphate group is usually linked to the 5'-C atom of the sugar component of the nucleoside, but may for example also be linked to the 3'-C atom.

A "nucleoside" is understood here as meaning organic molecules which consist of a

Sugar residue (sugar component) and an organic base (base component), e.g. a heterocyclic organic base, in particular a nitrogen-containing heterocyclic organic base, which are connected via a glycosidic bond. Of the

Sugar residue is often a pentose, eg deoxyribose or ribose, but may also be another sugar, for example a C ₃ , C ₄ or C ₆ sugar. In particular, a nucleoside is therefore a compound of the general formula (III)

where B is a nitrogen-containing heterocyclic organic base, e.g. a

Nucleobase, and R ³ and R ^{4 are} independently H or OH.

By a "nucleoside analog" is meant herein a compound that does not naturally occur in the human body, but is structurally similar to a naturally occurring nucleoside in the human body, such as, for example, from the cell and / or viral enzymes substantially corresponding to the natural nucleoside can be phosphorylated and incorporated into an RNA or DNA strand, for example, a nucleoside analog can itself be a nucleoside

for example, another compound having the above properties, for example, a compound of a heterocyclic base and an acyclic group and / or a group which is not a sugar, or a compound of a carbocyclic compound and a sugar residue. Nucleoside analogs are either structurally and / or functionally analogous nucleosides in the above sense or nucleosides. Since nucleoside analogues do not necessarily have to contain a sugar or base component in the narrower sense, here is also a component analogous to the base component (base analog) or a zur

Sugar component analog component (sugar analog) spoken. If it is spoken here of a sugar component or base component, the corresponding Analogous components of nucleoside analogues should be included, unless the context clearly states otherwise. Examples of nucleoside analogues are, for example, AZT (3'-azido-2 ', 3'-dideoxythimidine, azidothymidine), 2', 3'-dideoxyinosine (didanosine), 2 ', 3'-dideoxycytidine (zalticabin) and 2-amino-9 - ((2-hydroxyethoxy) methyl) -1H-purin-6 (9H) -one (acyclovir). Nucleoside phosphonates may also be nucleoside analogs.

By a "nucleotide" are phosphorylated nucleosides, for example

Nucleoside monophosphates (NMP), nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP). The phosphate, diphosphate (pyrophosphate) or triphosphate group is usually linked to the 5'-C atom of the sugar component of the nucleoside, but may for example also be linked to the 3'-C atom. By a "nucleotide analog" is accordingly understood a phosphorylated nucleoside analog.

The Total Turnover Number (TTN) is the number of moles of product formed per mole of cofactor or enzyme over the entire reaction time.

The protein according to the invention preferably catalyzes the enzymatic transfer of the

R of the compound of the following formula (I)

on the N2 of guanosine of m ⁷ GTP, m ⁷ GpppN or a compound of the following formula (II) wherein RNA is ribonucleic acid, R ^{1 is} OH or OCH ₃ , N is nucleoside, nucleotide, nucleoside or nucleotide analog, B is nucleobase, and R is selected from the group consisting of substituted or unsubstituted C ₂ _ ₁₀ alkyl, substituted or unsubstituted C ₂ _io-alkenyl, substituted or unsubstituted C ₂ _io-alkynyl,

substituted or unsubstituted C4-io-Alkeninyl, substituted or unsubstituted C ₃ i ₂ cycloalkyl, substituted or unsubstituted C ₃ _i ₂ cycloalkenyl, substituted or unsubstituted _C5-i2 cycloalkynyl, substituted or unsubstituted _C5-i2 -Cycloalkeninyl, substituted or unsubstituted Ci_io-heteroalkyl, substituted or unsubstituted C ₂ _io-Heterolkenyl, substituted or unsubstituted C ₂ _io-heteroalkynyl, substituted or unsubstituted C4-io-Heteroalkeninyl, substituted or unsubstituted Ci_io- azidoalkyl, substituted or unsubstituted C ₂ _io-Azidoalkenyl, substituted or unsubstituted C ₂ _io-azidoalkynyl, substituted or unsubstituted C ₄ _ ₁₀ -azidoalkeninyl, substituted or unsubstituted benzyl, propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butinyl CH = C-CH ₂ - CH ₂ -, penteninyl CH = C-CH = CH-CH ₂ - and azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -. In the case of benzyl, substitution in the para position is preferred, particularly preferred is substitution by an alkene, alkyne or azide.

The fact that the protein according to the invention catalyzes the transfer of the radical R of the AdoMet analog according to formula (I) does not mean that it can not also use AdoMet as a cofactor and can transfer a methyl group to the N 2 of the m ⁷ GpppN cap. Rather, it means that the reaction with the AdoMet analogue is at least also catalysed by the protein according to the invention under physiological conditions, preferably in comparison with the wild-type enzyme at a higher rate, with greater affinity for the cofactor, greater yield and / or greater total turnover number (Total Turnover Number , TTN). Preferably, the total turnover number TTN of the protein according to the invention is greater than 5, preferably>6,>7,>8,> 9 or> 10, and / or the total turnover number TTN of the protein according to the invention is at least twice the total turnover number of the wild-type enzyme, in each case based on mean values and the same AdoMet analogue, preferably AdoPropen. Further preferred in the protein according to the invention is the ratio of the total turnover numbers of AdoMet to AdoPropen, ie the ratio TTNAdoMet: TTNAdoPropen, <20, more preferably <15, more preferably <14, <13, <12, <11 or <10.

The transfer of a residue R of an S-adenosyl-L-methionine analogue catalyzed by the protein according to the invention to an mRNA is shown in the scheme reproduced below:

In the event that the protein according to the invention comprises or comprises only a partial sequence of SEQ ID NO: 2, it is particularly preferred that the protein catalyzes at least one of the above transfer reactions. It is preferred if the protein according to the invention

a. has or comprises an amino acid sequence according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, or

b. has or comprises an amino acid sequence which is homologous to the amino acid sequence according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, with the proviso that in the homologous

Amino acid sequence at the position corresponding to the position 34 according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, is not valine, or

c. has or comprises an amino acid sequence which is homologous to the amino acid sequence according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, wherein the homologous amino acid sequence to the amino acid sequence according to SEQ ID NO: 3, 4, 5 , 6, 7, 8, 9 or 10 is more than 85%, preferably at least 90%, more preferably at least 95% identical, with the proviso that in the amino acid sequence at the position corresponding to position 34 according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, not valine, or

d. a contiguous partial sequence of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 80, 90 or at least 100 amino acids, preferably at least 110, 120, 130, 140,

150, 160, 170, 180, 190 or at least 200 amino acids, more preferably at least 210, 220, 230, 240 or at least 250 amino acids of the amino acid sequence according to a, b or c, or comprises, with the proviso that the partial sequence the amino acid Position 34 according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10 or the corresponding homologous

Includes amino acid, and

e. does not have the amino acid sequence of SEQ ID NO: l 1.

Preferably, at position 34 of the amino acid sequence of SEQ ID NO: 2 is not isoleucine or threonine. Particularly preferred is at position 34 of the amino acid sequence according to SEQ ID NO: 2 alanine, glycine or methionine, preferably alanine. At position 76 of the amino acid sequence according to SEQ ID NO: 2 is preferably aspartic acid, threonine, leucine or valine, particularly preferably aspartic acid, and / or at position 92 of

Amino acid sequence according to SEQ ID NO: 2 is preferably arginine or alanine. At position 34 according to SEQ ID NO: 2 alanine is particularly preferred, at position 76 according to SEQ ID NO: 2 aspartic acid and at position 92 of the amino acid sequence according to SEQ ID NO: 2 arginine or alanine. The radical R is preferably propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butinyl CH ^ C-CH ₂ -CH ₂ -, penteninyl CH = C-CH = CH-CH ₂ -, benzyl Ph-CH ₂ - or azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -, more preferably propenyl CH ₂ = CH-CH ₂ -, benzyl Ph-CH ₂ - or penteninyl CH = C-CH = CH-CH ₂ -.

In a further aspect, the present invention also relates to a nucleic acid which codes for a protein according to the invention.

In yet another aspect, the present invention relates to a method for

Modification of the m ⁷ GpppN cap of an RNA molecule, particularly an mRNA molecule, comprising the step of contacting an m ⁷ GpppN-capped RNA molecule with a protein according to the first aspect of the invention in the presence of an AdoMet analog according to the following formula (I)

wherein R is selected from the group consisting of substituted or unsubstituted C ₂ _ io alkyl, substituted or unsubstituted C ₂ _io-alkenyl, substituted or

unsubstituted C ₂ _io alkynyl, substituted or unsubstituted C4-io-Alkeninyl, substituted or unsubstituted C ₃ _i ₂ cycloalkyl, substituted or unsubstituted C ₃ i ₂ cycloalkenyl, substituted or unsubstituted _C5-i2 cycloalkynyl, substituted or unsubstituted C5 -i ₂ -cycloalkeninyl, substituted or unsubstituted Ci_io-heteroalkyl, substituted or unsubstituted C ₂ _io-heterolkenyl, substituted or unsubstituted C ₂ _io-heteroalkynyl, substituted or unsubstituted C ₄ -o-heteroalkeninyl, substituted or unsubstituted Ci_io-azido-alkyl, substituted or unsubstituted C ₂ _io azidoalkenyl, substituted or unsubstituted C ₂ _io-azidoalkynyl, substituted or unsubstituted C4-io-Azidoalkeninyl, substituted or unsubstituted benzyl, Ph-CH ₂ -, propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butynyl CH = C-CH ₂ -CH ₂ -, CH Penteninyl ^ C-CH = CH-CH ₂ - and azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -,

under conditions where there is a transfer of the R residue to the N2 guanosine of the m ⁷ GpppN cap.

The radical R is preferably propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butinyl CH ^ C-CH ₂ -CH ₂ -, penteninyl CH = C-CH = CH-CH ₂ -, benzyl Ph-CH ₂ - or azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -, more preferably propenyl CH ₂ = CH-CH ₂ -, benzyl Ph-CH ₂ - or penteninyl CH ^ C-CH = CH-CH ₂ -.

It is particularly preferred if in this method a protein according to the invention is used which has the amino acid sequence according to SEQ ID NO: 2 and in which at position 34 of the amino acid sequence according to SEQ ID NO: 2 alanine, glycine or methionine, preferably alanine Position 76 of the amino acid sequence according to SEQ ID NO: 2

Aspartic acid, threonine, leucine or valine, preferably aspartic acid, and at position 92 of the amino acid sequence of SEQ ID NO: 2 is arginine or alanine, and AdoPropen, AdoEnYn or AdoBenzyl is used as the AdoMet analog. Further preferred is a protein in which position 34 according to SEQ ID NO: 2 is alanine, at position 76 according to SEQ ID NO: 2 is aspartic acid and at position 92 of the amino acid sequence according to SEQ ID NO: 2 arginine or alanine, in the presence of AdoPropen, AdoEnYn or AdoBenzyl under appropriate conditions with the RNA, preferably mRNA, brought into contact to run the enzymatic reaction. Following enzymatic modification of the m ⁷ GpppN cap, the residue R transferred to the N2 of guanosine may be further modified, for example, by an enzymatic or non-enzymatic chemical route.

A preferred possibility is to carry out a chemical modification of the radical R by means of a bioorthogonal chemical reaction, preferably a bioorthogonal click reaction. Such reactions are known in the art and include, for example, photoclick methods, thiol-ene-click methods, and Cycloaddition reactions, eg, the Cu (I) -catalyzed Huisgencycloaddition (see, for example, HC Kolb, MG Finn, KB Sharpless, 2001, Angew Chem 97, 11, 2056-2075; HC Kolb, MG Finn, KB Sharpless, 2001, Angew Chem Int. Ed., 40, 11, 2004-2021; CN Bowman and CE Hoyle, Angew Chem. Int. Ed., 2010, 49, 1540-1573; SS van Berkel, et al, Angew Chem., 2011, 123, 89-8989; E. Lallana et al, Angew Chem 2011, 123, 8956-8966; AH El-Sagheerab, T. Brown, Chem. Soc. Rev. 2010, 39, 1388-1405; CS McKay, et al , Chem.

Commun. 2010, 46, 931-933; W. Song, et al, J. Am. Chem. Soc. 2008, 130, 9654-9655; P.M.E. Grhma, et al, Angew. Chem. Int. Ed. 2008, 47, 8350-8358; C.R. Becer, et al, Angew. Chem. Int. Ed. 2009, 48, 4900-4908; T.R. Chan, et al, Org. Lett. 2004, 6, 2853-2855; C.

Uttamapinant, et al, Angew. Chem. Int. Ed. 2012, 51, 5852-5856; Y. Wang, et al, Angew. Chem. Int. Ed. 2009, 48, 5330-5333).

For example, a "photoclick reaction" is understood as meaning the 1,3-dipolar cycloaddition of an alkene with a nitrile imine to form a pyrazoline cycloadduct, and the basic prerequisite for the formation of the cycloadduct is similar

Frontier orbital energies of the reactants used were assumed. Thus, for the Photoclick reaction it could be shown that the reactivity is influenced by changing the energy of the highest occupied orbital in the nitrile-imine and thus can be counted as cycloadditions of type I (see Y. Wang, W. Song, WJ Hu, Q. Lin, Angew Chem. Int. Ed. Engl. 2009, 48, 5330-3). The Photoclick reaction is not only characterized by bioorthogonality, but also by the possibility of forming fluorescent products from non-fluorescent starting materials, which is particularly advantageous for the suppression of background signals in live cell applications. Clues as to whether a photoclick-based functionalization of the mRNA cap is possible, the expert can for example by means of Kohn-Sham density functional theory calculations (KS-DFT calculations, see W. Kohn, LJ Sham, Phys., Rev. 1965, 140, AI 133-1138).

With the aid of this preferred embodiment of the method according to the invention, the cap structure can be attached to the 5 'end of RNAs which have such a cap structure or can be provided with such a cap structure in a two-stage or two-part RNA

if appropriate, multistage processes can also be specifically modified by a chemoenzymatic route. In the first step, the cap structure with the help of a according to the invention enzymatically modified by instead of a methyl radical, a radical R is transferred to the N2 of the m ⁷ GpppN cap. In a second, chemical step, the RNA modified with this radical can then be reacted with suitable molecules, for example with a suitable biomarker (eg biotin), for example by means of known methods of click chemistry, and further modified. These molecules also include columnar materials that allow immobilization of the RNAs through the cap structure. This immobilization may be, for example, directly or indirectly via non-covalent

Interactions take place with a corresponding matrix. But it can also be via a covalent bond, whereby the interaction of e.g. becomes more stable with the matrix, allowing, for example, more stringent washing steps that allow others

Separate components more efficiently. Thus, e.g. mRNA be isolated specifically from complex cell lysates.

In yet another aspect, the present invention relates to a test kit comprising a protein according to the first aspect of the invention and an AdoMet analog according to the following formula

unsubstituted C ₂ _io alkynyl, substituted or unsubstituted C4-io-Alkeninyl, substituted or unsubstituted C ₃ -i ₂ cycloalkyl, substituted or unsubstituted C ₃ i ₂ cycloalkenyl, substituted or unsubstituted _C5-i2 cycloalkynyl, substituted or unsubstituted _C5-i2 -Cycloalkeninyl, substituted or unsubstituted Ci_io-heteroalkyl, substituted or unsubstituted C ₂ _io-Heterolkenyl, substituted or unsubstituted C ₂ _io-heteroalkynyl, substituted or unsubstituted C4-io-Heteroalkeninyl, substituted or unsubstituted Ci_io-azidoalkyl, substituted or unsubstituted C ₂ _ ₁₀ - azidoalkenyl, substituted or unsubstituted substituted or unsubstituted C4-io-Azidoalkeninyl, substituted or unsubstituted benzyl, Ph-CH ₂ - propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butynyl CH = C-CH ₂ -CH ₂ -, Penteninyl CH ^ C-CH = CH-CH ₂ - and azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -.

In a particularly preferred embodiment of the test kit according to the invention, the AdoMet analog AdoPropen, AdoEnYn or AdoBenzyl, the protein has a

Amino acid sequence according to SEQ ID NO: 2 and has at position 34 according to SEQ ID NO: 2 alanine, at position 76 according to SEQ ID NO: 2 aspartic acid and at position 92 of

Amino acid sequence according to SEQ ID NO: 2 arginine or alanine.

In the following, the invention will be based solely on exemplary embodiments

Illustrative purposes explained in more detail.

1. Synthesis of S-adenosyl-L-methionine analogs

1.1 Synthesis of 5 '- [(S) - [(3S) -3-amino-3-carboxypropyl] prop-2-enylsulfonio] -5'-deoxyadenosine (AdoPropen)

The AdoMet analog AdoPropen was prepared by a method of Dalhoff et al. (Dalhoff, C., et al, Nat. Chem. Biol. 2006, 2, 31-32). For the synthesis of AdoPropen 20 mg of S-adenosyl-L-homocysteine (52 μιηοΐ) were dissolved with stirring in 3 mL of formic acid: acetic acid 1: 1. The solution was cooled by stirring for 10 minutes in an ice bath, before 264 μΙ _^ (3Α2 mmol) of 3-bromopropene was added. Subsequently, the reaction solution was stirred for 4 days at room temperature and stopped by adding 30 ml of cold, didemineralisertem water. The aqueous phase was extracted 3 times with 5 mL diethyl ether each time and

then the water is removed under reduced pressure. The resulting solid was dissolved in 5 mL of deionized water + 0.01% TFA and purified by RP-HPLC.

1.2 Synthesis of 5 '- [(S) - [(3S) -3-amino-3-carboxypropyl] pent-2-en-4-ynylsulfonio] -5'-deoxyadenosine (AdoEnYn) The AdoMet analog AdoEnYn was prepared according to Peters et al. (Peters, S. Willnow, M. Duisken,

H. Kleine, T. Macherey, K.E. Duncan, D.W. Litchfield, B. Luscher, E. Weinhold, Angew Chem Int Ed Engl 2010, 49, 5170).

For the synthesis of the AdoMet analogue AdoEnYn, pent-2-en-4-yn-1-ol was converted to the methanesulfonic acid ester in a first step. 240 mg (6.00 mmol) were added

Sodium hydroxide resuspended in 6 mL dichloromethane, 426 μί (5.50 mmol)

Methanesulfonylchlorid added and the suspension cooled in an ice bath. Subsequently, a mixture of (E) - and (Z) -pent-2-en-4-yn-1-ol (472 μΕ, 6.02 mmol) was added and the reaction mixture was stirred for 16 hours at room temperature. It was extracted with 50 mL of saturated sodium bicarbonate solution. The solvent was removed in vacuo and the activated alcohol directly in 1 mL of a solution of methanoic acid and

Ethanoic acid (1: 1) dissolved. This solution was added to 7.2 mg (19 μιηοΐ) of S-adenosyl-L-homocysteine and stirred at room temperature for 14 hours. This was then added to 30 mL ddH ₂ 0 and extracted three times with 50 mL diethyl ether. The aqueous phase was frozen and lyophilized. The residue was taken up in 2.5 mL ddH ₂ O with 0.01% TFA and analyzed by HR-ESI-MS. This was followed by purification by preparative HPLC.

I.3 Synthesis of 5 '- [(S) - [(3S) -3-amino-3-carboxypropyl] benzyl] -5'-deoxyadenosine (AdoBenzyl)

The AdoMet analog AdoBenzyl was analogously to the method of Dalhoff et al. (Dalhoff, C., et al, Nat. Chem. Biol. 2006, 2, 31-32). For the synthesis of AdoBenzyl 9.2 mg S-adenosyl-L-homocysteine (24 μιηοΐ) with stirring in 1.38 mL

Formic acid: acetic acid 1: 1 dissolved. The solution was cooled by stirring for 10 minutes in an ice bath before 171.2 μΐ _^ (1:44 mmol) of benzyl bromide was added. Subsequently, the reaction solution was stirred for 4 days at room temperature and stopped by adding 15 mL of cold, didemineralisertem water. The aqueous phase was washed 3 times with 2.5 mL each

Extracted diethyl ether and then the water removed under reduced pressure. The resulting solid was dissolved in 2.5 mL of deionized water + 0.01% TFA and purified by RP-HPLC. 2. HPLC-coupled activity assay

With the aid of an HPLC-coupled activity assay, the enzymatically catalysed transfer of the residues carried by synthetically produced AdoMet analogues by the WT-GlaTgs2 (SEQ ID NO: 1) and the proteins according to the invention according to SEQ ID NO: 4-10 propenyl (AdoPropen), Penteninyl (AdoEnYn) and benzyl (AdoBenzyl) on the N2 atom of the

Guanosins of m ⁷ GpppA (A = adenine) examined. A typical approach of volume 8 is summarized in Table 1.

Table 1: Components used for the activity assay using m ⁷ GpppA.

Component volume final concentration

GlaTgs2 or GlaTgs2 variant 5.6 μΕ 15-50 μΜ

MTAN / LuxS (l: l) 0.16 μΕ 4.1 μM or 3.0 μΜ

m ⁷ GpppA [10 mM] 0.22 μΕ 275 μΜ

AdoMet analogue 0.33 - 0.6 μΕ 365 - 740 μΜ

Reaction Buffer 2 Ad 8 μΕ

Reaction Buffer 2: 50 mM Tris; 10 mM MgCl ₂ ; 100 mM NH ₄ OAc; pH 8.4

MTAN stands for 5'-methylthioadenosine / S-adenosylhomocysteine nucleosidase. LuxS stands for S-ribosylhomocysteine lyase. For larger runs of 10 or 20 μΐ _^ , a proportionate adjustment was made. The amount of AdoMet analog used was adjusted so that the area of the im

Chromatogram occurring peaks of a diastereomer of the AdoMet analogue of the area of m ⁷ GpppA caused signal corresponded. The reaction was usually stopped immediately after preparation of the reaction (tO) and after three hours at 37 ° C (tl80) and analyzed by analytical HPLC and MALDI-TOF.

The following Table 2 shows the activity of GlaTgs2-WT in comparison with examined GlaTgs2 variants, whereby AdoPropen was used as AdoMet analogue. Table 2: Activity of GlaTgs2-WT and GlaTgs2 variants on AdoPropen

Enzyme SEQ ID NO: Activity TTN

GlaTgs2-WT 1 + 3 ± 2

GlaTgs2-V34A 4 +++ 10 ± 2

GlaTgs2-V34G 5 ++ 5

GlaTgs2-V34M 6 ++ 6

GlaTgs2-V34A, D76L 7 + 1

GlaTgs2-V34A, D76T 8 + 2

GlaTgs2-V34A, D76V 9 + 1

GlaTgs2-V34A, R92A 10 +++ 8

TTN = Total Turnover Number. Amino acid exchanges are indicated in the manner known to those skilled in the art (original amino acid position - new amino acid) using the one letter code for amino acids. For example, V34A means that the original amino acid valine at position 34 has been replaced by alanine.

It turns out that the proteins according to the invention had at least one activity comparable or higher with the wild-type enzyme.

For the same amount of enzyme, 95% of the m ⁷ GpppA was converted, for example, by the variant GlaTgs2-V34A in the case of AdoPropen, 10% in the case of AdoEnYn. For example, with the variant GlaTgs2-V34A also a transfer of benzyl was found.

Enzymatic parameters for the GlaTgs2 variant according to SEQ ID NO: 4 with respect to

AdoPropen are shown in Table 3:

Table 3: Enzymatic parameters for GlaTgs2-WT and GlaTgs2-VV34A with respect to AdoPropen Protein K -cat TTN T ₅₀ (15min)

GlaTgs2-WT 151 ± 19 μΜ 0.09 ± 0.08 min 3 39.9 ± 0.2 ° C

GlaTgs2-V34A 57 ± 29 μΜ 8 ± 0.08 min 10 40.4 ± 0.2 ° C

The GlaTgs2 variant GlaTgs2-V34A (SEQ ID NO: 4) has a higher affinity for AdoPropen and a higher activity with AdoPropen compared to the wild-type enzyme GlaTgs2-WT. The thermal stability corresponds to that of the wild-type enzyme. AdoMet kinetic parameters were similar to those of the wild-type enzyme (S. Hausmann, S. Shuman, J Biol Chem 2005, 280, 32101-32106.). 3. Thermostability

The stability of a protein characterizes its ability to tolerate denaturing influences, such as an increase in ambient temperature, within certain limits and to maintain the native conformation. As a measure of the thermal stability of the Tso value can be used. is the temperature at which an enzyme loses 50% of its activity after 15 minutes of incubation. To determine T50, the proteins to be analyzed were heated for 15 minutes at different temperatures in the thermocycler while a sample of the protein was incubated on ice. In the following activity tests were carried out with the previously heated proteins. The sample incubated on ice was used as a reference, which had 100% activity. After normalization of the values it was possible to determine using a Boltzmann fit with the software Origin.

As can be seen from Table 4, the GlaTgs2 variants GlaTgs2-V34A (SEQ ID NO: 4), GlaTgs2-V34G (SEQ ID NO: 5) and GlaTgs2-V34M (SEQ ID NO: 6) had a similar

Thermostability or even showed greater thermal stability, ie greater stability to higher temperatures. Table 4: Thermostability of the GlaTgs2 variants GlaTgs2-V34A, -V34M and -V34G compared to the wild-type. Variant SEQ ID NO: thermostability, T

WT 1 39.9 ± 0.2

Val34Ala 4 40.4 ± 0.2

Val34Gly 5 44.8

Val34Met 6 49.0

4. Chemical Modification of Enzymatically Modified RNA Caps by Click Chemistry

4.1 Biotinylation by Thiol-Ene-Click (TEC) 800 μΜ m 1 GpppA were alkenylated enzymatically to propenyl 2 m 1 GpppA 1 (a 2 m 1 GpppA, propenyl at N 2 of m ⁷ GpppA) and the reaction was terminated by addition of 1 / 10 volumes of 1 M perchloric acid or incubated for 5 min at 68 ° C.

The batches were centrifuged and used for the reaction with biotin-thiol 2. For this they were degassed with argon for about 30 seconds and with 1 mM exclusion of air

Radical starter VA-044 and biotin-thiol 2 (about 50-fold, molar excess) were added. The batches were incubated for 8 h at 44 ° C and analyzed by HPLC and MALDI-TOF-MS.

VA044 = 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride 4.2 Fluorescence labeling by Cu-Click reaction

The Cu-Click reaction was according to Tomkuviene et al. (Tomkuviene, B. Clouet-d'Orval, I. Cemiauskas, E. Weinhold, S. Klimasauskas, Programmable sequence-specific click-labeling of RNA using archaeal box C / D RNP methyltransferases, Nucleic Acids Research, 2012, 40 , 14, 6765). For this purpose, initially about 100 μΜ pent-2-en-4-ynyl ² m ⁷ GpppA

(p ² m7GpppA or EnYn ² m ⁷ GpppA) 4 was prepared enzymatically and the reaction stopped by adding 1/10 volume 1 M perchloric acid.

For the Cu-click reaction, a 300 mM CuBr solution (in DMSO / tBuOH 3: 1) was freshly prepared and diluted 1:10 with 111 mM TBTA solution (in DMSO / tBuOH 3: 1). To 10 of the enzyme-catalyzed reaction were then 8 DMSO / tBuOH, 3 μΐ _{^ of} the 30 mM CuBr solution (in TBTA) and 2.5 μΐ, Eterneon-azide 5 (Eterneon-Azid 480/635, Jena Bioscience GmbH, cat CLK-FA15-1) (2.5mM in DMSO / tBuOH). The reaction was incubated for one hour at 37 ° C with occasional vortexing and analyzed by gel electrophoresis. TBTA = tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine), DMSO =

Dimethy sulfo xid

4.3 Fluorescence labeling by means of cross metathesis

Cross-metathesis was used to fluorescently label allyl-modified mRNA caps using flurorescein-o-acrylate 8 or another fluorescently-labeled acrylate and a second-generation Hoveyda Grubbs catalyst 10, according to the general scheme outlined below.

For this example, the allyl-modified mRNA cap 7 with two equivalents of fluorescein o-acrylate 8, which was dissolved in DMSO, and 2 mol% of Hoveyda Grubbs-

Catalyst 10, which was taken up in 30% tBuOH in water, also incubated in 30% tBuOH in water for five hours at room temperature or 37 ° C with exclusion of light. The analysis was carried out by means of 20% polyacrylamide gel electrophoresis and in-gel

Fluorescence detection.

4.4 Functionalization of Cap Structures by Photoclick Reaction In the following, a fluorescence labeling of an alkenylated and an alkynylated mRNA cap by means of a photoclick reaction is described by way of example. For this purpose, the nitrile-Imine I Ia and 12a of tetrazoles 11 and 12 were reacted with the cap analog m ⁷ GpppA.

First m GpppA ⁷ in the presence of AdoPropen and GlaTgs2-V34A to a ² m were 1 mM reacted GpppA ⁷ and the resulting mixture was then heated to 68 ° C, and dialyzed to remove unreacted AdoPropen. The removal of unreacted AdoPropene served to prevent it from also reacting with the nitrile imine in the subsequent Photoclick reaction. After these steps, the aqueous solution of a ² m ⁷ GpppA with acetonitrile and tetrazole 11 (617 μΜ) was added and irradiated after careful mixing in a black microtiter plate for five minutes at 254 nm. It was found that the irradiation with minimal separation of reaction and

Light source was essential for the successful implementation and the formation of the reactive nitrile imine apparently disappeared or was minimized by increasing the distance. The components contained in the sample were gel-electrophoretically separated after an incubation of up to 20 hours at 4 ° C. and analyzed for the appearance of fluorescent products. For this purpose, the gel was illuminated at a wavelength of 365 nm and photographed. For the reaction, which was carried out in the presence of the alkenylated cap a ² m ⁷ GpppA, a turquoise-fluorescent product could be detected. A parallel analysis of the gel by means of UV shadowing showed that the detected fluorescent product had a lower electro-mobility and thus presumably a higher molecular weight than the cap. This is consistent with the possibility that the corresponding band will produce the expected Photoclick product (P ¹ adenosine (5 ') -P ³ - [N ² -ethyl-2- (4- (4-methoxyphenyl) -2-phenylpyrazoline ), 7-methylguanosine (5 ')] triphosphate, synonym: N ² -methoxypyrazolinethyl-m ⁷ GpppA) 14, since this would have a higher molecular weight than the cap analog a ² m ⁷ GpppA 13 with the same charge. In

Control approaches could not be detected a corresponding fluorescent signal. Controls included performing bioconversion without enzyme, without

AdoPropen, without m7GpppA and in the presence of denatured enzyme. In all these controls, therefore, no alkenylated cap, which should be the substrate for the photoclick reaction, could be demonstrably formed. Since, in the absence of α ² m ⁷ GpppA, no fluorescent product could be observed after the photoclick reaction, it follows that fluorescence labeling of the alkenylated cap by means of tetrazole 11 was successful and specific. This was also verified by mass spectrometric analysis by HPLC-ESI-TOF-MS. Here, the mass of the expected photoclick product 14 could be detected in the reaction mixture (calc. [M] ⁺ = 1051.23 m / z; det. [M] ⁺ = 1051.23 m / z). To further characterize the photoclick reaction of tetrazole 11 and a ² m ⁷ GpppA 13, also with respect to potential applications in cells, a first kinetic analysis was performed. For this purpose, the reaction was carried out as described above, but in each case part of the sample was analyzed after incubation for 5, 30, 120 and 240 minutes by detecting fluorescent products. It was shown that the Photoclick product 14 was already detected after 5 minutes, based on the detected

Fluorescence intensity at later times no further reaction was observed. It shows that the reaction is also suitable for the visualization of mRNA in living cells due to their kinetics, since a visible signal can be obtained shortly after induction. The investigation of PC3 cells after a five-minute irradiation with UV light (λ = 254 nm) showed, moreover, that although an effect on the

Morphology of the cells, but not on their vitality was observed, so that an application is made possible without the cells by the UV irradiation in the

Dying off investigation. In addition to the formation of a fluorescent product under

Using a ² m ⁷ GpppA as starting material and the high reaction rate, the duration of the irradiation to activate the tetrazole is thus compatible with applications in living cells.

The Photoclick reactions described above were also successfully performed for the combination nitrile-imine 12a and a ² m ⁷ GpppA 13, and the corresponding Photoclick product (P ¹ adenosine (5 ') -P ³ - [N ² -ethyl -2- (4- (4-methylbenzoate) -2-phenylpyrazoline), 7-methylguanosine (5 ')] triphosphate, synonym: N ² -benzoatepyrazolinethyl-m ⁷ GpppA) 16.

It was observed that the photoclick products of tetrazoles 11 and 12 with a ² m ⁷ GpppA appear to differ in their emission maxima. The Photoclick reactions may therefore also be used to generate in vivo at any wavelength emitting fluorophores.

4.4.2 Fluorescence Labeling of an Alkynylated mRNA Cap A Photoclick reaction was also performed for the combination nitrile-imine I 1a and p ² m ⁷ GpppA 15.

For this purpose, the above-described Photoclick reaction for fluorescence labeling of a ² m ⁷ GpppA using tetrazole 11 was used to modify the alkynylated cap analogue p ² m ⁷ GpppA 15. The bioconversion of m7 GpppA with AdoEnYn was carried out with GlaTgs2-V34A and as a control in the presence of the denatured enzyme. This ensured that the same components were present in both batches, but the formation of the photoclick starting material p ² m ⁷ GpppA did not occur in the control. After initiation of the photoclick reaction by illumination with a wavelength of 254 nm, the reaction and control mixture were incubated for 20 hours at 4 ° C and after gel electrophoretic separation of the constituents contained in the formation of a fluorescent cycloadduct characterized. In the case of illumination of the gel with a wavelength of 365 nm, a fluorescent band could be detected in the reaction mixture which did not appear in the control and thus could be added to the corresponding pyrazoline (P ¹ - adenosine (5 ') - P ³ - [N ² - but-2-en-4- (4- (4-methoxyphenyl) -2-phenylpyrazolin) yl, 7-methylguanosine (5 ')] triphosphate, synonym: N ² -methoxypyrazolinebutenyl-m ⁷ GpppA) 17 could be assigned.

OMe

Claims

1. Isolated or synthetic protein that

a. has or comprises an amino acid sequence according to SEQ ID NO: 2, or

b. has or comprises an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 2, provided that valine is not present in the homologous amino acid sequence at the position corresponding to position 34 of SEQ ID NO: 2, or c , an amino acid sequence which is homologous to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequence to the amino acid sequence according to SEQ ID NO: 2 to more than 85%, preferably at least 90%, particularly preferably at least 95% identical with the proviso that in the homologous amino acid sequence at the position corresponding to position 34 of SEQ ID NO: 2 is not valine, or d. a contiguous partial sequence of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 80, 90 or at least 100 amino acids, preferably at least 110, 120, 130, 140, 150, 160 , 170, 180, 190 or at least 200 amino acids, more preferably at least 210, 220, 230, 240 or at least 250 amino acids of the amino acid sequence according to a, b or c, or with the proviso that the partial sequence comprises the amino acid at position 34 SEQ ID NO: 2 or the corresponding homologous amino acid, and

e. does not have the amino acid sequence of SEQ ID NO: l 1.

2. Protein according to claim 1, wherein the protein is the transfer of the radical R of

Compound of the general formula (I)

on the N2 of guanosine of m ⁷ GTP, m ⁷ GpppN or a compound according to the following formula (II)

enzymatically catalyzed, and wherein RNA ribonucleic acid, R ¹ OH or OCH _3, n nucleoside, nucleotide, nucleoside or nucleotide analog means B is nucleobase, and R is selected from the group consisting of substituted or unsubstituted C ₂ _ ₁₀ Alkyl, substituted or unsubstituted C ₂ _io-alkenyl,

substituted or unsubstituted C ₂ _io alkynyl, substituted or unsubstituted C ₄ _ ₁₀ - Alkeninyl, substituted or unsubstituted C ₃ -i ₂ cycloalkyl, substituted or unsubstituted C ₃ _i ₂ cycloalkenyl, substituted or unsubstituted _C5-i2 cycloalkynyl, substituted or unsubstituted _C5-i2 -Cycloalkeninyl, substituted or unsubstituted Ci_io-heteroalkyl, substituted or unsubstituted C ₂ _io-Heterolkenyl, substituted or unsubstituted C ₂ _io-heteroalkynyl, substituted or unsubstituted C ₄ _ ₁₀ - Heteroalkeninyl, substituted or unsubstituted Ci_io- Azidoalkyl, substituted or unsubstituted C ₂ _io-azidoalkenyl, substituted or unsubstituted C ₂ _io-azidoalkynyl, substituted or unsubstituted C ₄ -ozo-azido-alkenynyl, substituted or unsubstituted benzyl, propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butinyl CH = C-CH ₂ -CH ₂ -,

Penteninyl CH = C-CH = CH-CH ₂ - and azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -.

3. Protein according to claim 1 or 2, wherein the protein

b. has or comprises an amino acid sequence which is homologous to the amino acid sequence according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, with the proviso that in the homologous amino acid sequence at the position corresponding to the position 34 according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, is not valine, or

c. has or comprises an amino acid sequence which is homologous to the amino acid sequence according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, wherein the amino acid sequence to the Amino acid sequence according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10 to more than 85%, preferably at least 90%, particularly preferably at least 95% identical, with the proviso that in the homologous Amino acid sequence at the position corresponding to the position 34 according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10, is not valine, or

d. a contiguous partial sequence of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 80, 90 or at least 100 amino acids, preferably at least 110, 120, 130, 140, 150, 160 , 170, 180, 190 or at least 200 amino acids, more preferably at least 210, 220, 230, 240 or at least 250 amino acids of the amino acid sequence according to a, b or c, or with the proviso that the partial sequence comprises the amino acid at position 34 SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or 10 or the corresponding homologous amino acid, and

e. does not have the amino acid sequence of SEQ ID NO: l 1.

4. Protein according to one of the preceding claims, wherein at position 34 of the

Amino acid sequence according to SEQ ID NO: 2 alanine, glycine or methionine, preferably alanine.

5. Protein according to one of the preceding claims, wherein at position 76 of the

Amino acid sequence according to SEQ ID NO: 2 aspartic acid, threonine, leucine or valine, preferably aspartic acid, and / or at position 92 of the amino acid sequence according to SEQ ID NO: 2 arginine or alanine.

A protein according to any one of the preceding claims wherein R is propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butinyl CH = C-CH ₂ -CH ₂ -, penteninyl CH = C-CH = CH -CH ₂ -, benzyl, Ph-CH ₂ - or azidobutenyl, N ₃ -CH ₂ -CH = CH-CH ₂ -, preferably propenyl CH ₂ = CH-CH ₂ -, benzyl, Ph-CH ₂ - or penteninyl CH = C- CH = CH-CH ₂ -.

7. nucleic acid which codes for a protein according to any one of claims 1 to 6.

8. A method for modifying the m ⁷ GpppN cap of an RNA molecule, in particular an mRNA molecule, comprising the step of contacting one with an m ⁷ GpppN Capped RNA molecule with a protein according to any one of claims 1 to 6 in the presence of an AdoMet analog according to the following formula (I)

unsubstituted C ₂ _io alkynyl, substituted or unsubstituted C4-io-Alkeninyl, substituted or unsubstituted C ₃ _i ₂ cycloalkyl, substituted or unsubstituted C ₃ i ₂ cycloalkenyl, substituted or unsubstituted _C5-i2 cycloalkynyl, substituted or unsubstituted C5 -i ₂ -cycloalkeninyl, substituted or unsubstituted Ci_io-heteroalkyl, substituted or unsubstituted C ₂ _io-heterolkenyl, substituted or unsubstituted C ₂ _io-heteroalkynyl, substituted or unsubstituted C ₄ -o-heteroalkeninyl, substituted or unsubstituted Ci_io-azido-alkyl, substituted or unsubstituted C ₂ _io-azidoalkenyl, substituted or unsubstituted C ₂ _io-azidoalkynyl, substituted or unsubstituted C ₄ -ozo-azido-alkenynyl, substituted or unsubstituted benzyl, propenyl CH ₂ = CH-CH ₂ -, propynyl CH = C-CH ₂ -, butynyl CH = C-CH ₂ -CH ₂ -, penteninyl CH 4 C-CH = CH-CH ₂ - and azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -,

9. The method of claim 8, wherein the RNA in the presence of AdoPropen or

AdoEnYn is brought into contact with a protein which has the amino acid sequence according to SEQ ID NO: 2 and in which at position 34 according to SEQ ID NO: 2 is alanine, at position 76 according to SEQ ID NO: 2 is aspartic acid and at position 92 of Amino acid sequence according to SEQ ID NO: 2 arginine or alanine.

10. The method according to any one of claims 8 or 9, comprising the further step of the subsequent chemical modification of the transferred to the N2 of the guanosine of the m ⁷ GpppN cap residue R.

1 1. The method of claim 10, wherein the chemical modification of the radical R takes place by means of a Photoclick reaction.

A test kit comprising a protein according to any one of claims 1 to 6 and an AdoMet analog according to the following formula (I)

unsubstituted C ₂ _io alkynyl, substituted or unsubstituted C4-io-Alkeninyl, substituted or unsubstituted C ₃ _i ₂ cycloalkyl, substituted or unsubstituted C ₃ i ₂ cycloalkenyl, substituted or unsubstituted C ₅ -i ₂ cycloalkynyl, substituted or unsubstituted C ₅ -i ₂ -cycloalkeninyl, substituted or unsubstituted Ci_io-heteroalkyl, substituted or unsubstituted C ₂ _io-Heterolkenyl, substituted or unsubstituted C ₂ _io-heteroalkynyl, substituted or unsubstituted C ₄ -o-heteroalkeninyl, substituted or unsubstituted Ci_io-azido-alkyl, substituted or unsubstituted C ₂ _io-azidoalkenyl, substituted or unsubstituted C ₂ _io-azidoalkynyl, substituted or unsubstituted C ₄ -10-azido-alkenynyl, substituted or unsubstituted benzyl, propenyl CH ₂ CHCH-CH ₂ -, propynyl CH = C-CH ₂ -, Butinyl CH = C-CH ₂ -CH ₂ -, penteninyl CH ₃ C-CH = CH-CH ₂ - and azidobutenyl N ₃ -CH ₂ -CH = CH-CH ₂ -.

13. The test kit according to claim 12, wherein the AdoMet analog is AdoPropen, AdoEnYn or AdoBenzyl, the protein has an amino acid sequence according to SEQ ID NO: 2 and the protein at position 34 according to SEQ ID NO: 2 has alanine, at position 76 according to SEQ ID NO: 2 has aspartic acid and has arginine or alanine at position 92 of the amino acid sequence according to SEQ ID NO: 2.