WO2021228967A1

WO2021228967A1 - ACTIVE AND ORTHOGONAL HYBRID PYRROLYSYL tRNA

Info

Publication number: WO2021228967A1
Application number: PCT/EP2021/062672
Authority: WO
Inventors: Simon ELSÄSSER; Birthe MEINEKE
Original assignee: Nested Bio Ab
Priority date: 2020-05-13
Filing date: 2021-05-12
Publication date: 2021-11-18

Abstract

The present invention relates to a hybrid pyrrolysyl tRNA (PylT), which is highly orthogonal and active. The invention furthermore provides a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a pyrrolysyl-tRNA-synthetase (PylRS) (PylT/PylRS), as well as a pair of PylT/PylRS complexes, which are mutually orthogonal and active in the same system. The present invention allows for incorporation of at least one, such as two distinct, non-canonical amino acids (ncAA) into a protein.

Description

Active and orthogonal hybrid pyrrolysyl tRNA

Technical field

The present invention relates to a hybrid pyrrolysyl tRNA (PylT), which is highly orthogonal and active in combination with its corresponding pyrrolysyl-tRNA-synthetase (PylRS) with respect to endogenous PylT and PylRS, as well as other PylT/PyIRS pairs. The invention furthermore provides a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a pyrrolysyl-tRNA-synthetase (PylRS) (PylT/PyIRS), as well as a pair of PylT/PyIRS complexes, which are mutually orthogonal and active in the same system. The present invention allows for incorporation of at least one, such as two distinct, non-canonical amino acids (ncAA) into a protein.

Background

The specific nucleotide sequence of an mRNA specifies which amino acids are incorporated into a protein product of the gene from which the mRNA is transcribed.

The ribosome uses transfer RNA (tRNA) to translate the mRNA sequence into amino acid sequence of the protein. The transfer RNA (tRNA) hereby serves as a physical link between the mRNA and the amino acid sequence of the proteins. The three nucleotides of the codon, localized in the anticodon-arm of the tRNA molecule, decode the genetic code by recognizing the three nucleotides of the anticodon in the mRNA. The anticodon forms three complementary base pairs with a codon in the mRNA during protein biosynthesis. The 3’ end of the tRNA is a covalently attached to the amino acid that corresponds to the anticodon sequence. Normally, each type of tRNA molecule can be attached to only one type of amino acid, thus each organism has many types of tRNA. The covalent attachment to the 3’ end of the tRNA is catalyzed by enzymes termed aminoacyl-tRNA synthetases (aaRS).

Non-canonical amino acids (ncAA) can be incorporated into a protein by the ribosome if they are attached to the 3’ end of the tRNA analogous to natural proteinogenic amino acids, or through organic synthesis or utilizing a aaRS in vitro or in vivo.

Genetic code expansion by e.g. amber codon (TAG/UAG) suppression has been used to introduce over a hundred different ncAA into proteins in mammalian cells, however genetic code expansion by amber codon only allows for one ncAA to be incorporated into the protein. tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pairs from methanogenic archaea naturally evolved to recode amber codons for the incorporation of pyrrolysine (Pyl). The PylT/RS pairs from Methanosarcina barkerii and Methanosarcina mazei ( Mma ) have been proven to be versatile tools for genetic code expansion as they are broadly orthogonal to all tRNA/aaRS pairs in other organisms, i.e. do not cross-react with endogenous tRNAs or aaRS. In addition, unique properties of PylRS active site make them highly suitable for evolving or designing new substrate specificity.

To date, no natural orthogonal PylT/PyIRS pairs are known, which can be combined in the same system, such as mammalian cell, E.coli or yeast.

Summary

Higher organisms, such as eukaryote cells, are capable of producing a wide range of different proteins, which normally consist of standard (canonical) amino acids encoded by the standard genetic code. Bacteria and archeal species are capable of incorporating non-standard amino acids (ncAA) into proteins. The present invention allows for expanding the normal repertoire of standard amino acids, by incorporation of ncAA into proteins. Thus, the present invention allows for the incorporation of a distinct non-standard amino acid into proteins, as well as the incorporation of two distinct non standard amino acids into proteins. The invention furthermore, allows introduction of site specific incorporation of specific amino acids into proteins, hereby providing a platform of applications, such as dual colour labelling of proteins.

Interestingly the inventors have found a hybrid PylT with superior properties, having both high activity and low or no cross reactivity over any known PylT variant. Said hybrid PylT has high activity to its cognate PylRS and low cross reactivity to other PylRSs. The hybrid PylT is particular useful in a PylT/PyIRS complex as well as in a pair of PylT/PyIRS complexes comprising said hybrid PylT. The present invention allows for nonsense codon suppression, such as amber, opal and/or ochre suppression, as well as for incorporation of one or two distinct ncAA into a protein in the same system.

By using a pair of PylT/PyIRS complexes comprising a first PylT/PyIRS complex comprising said hybrid PylT and a second PylT/PyIRS complex, optionally combined with modifications to the PylT anticodons and the PylRS active sites, the two PylT/PyIRS complex can recognize different mRNA codons and pair the mRNA codon with a specific ncAA. The pair of PylT/PyRS complexes can hereby recognize two different stop codons within a single mRNA and site-specifically introduce two distinct ncAAs in a single protein of interest, thereby generate unique modified proteins. Due to the high activity of hybrid PylT there is a very high efficiency in the dual stop codon suppression/incorporation of ncAAs. Due to the low or none cross-reactivity with other PylRS, there is little or no unspecific incorporation of ncAAs.

Thus, PylT provides a platform for several genetic code expansion strategies, due to its unique properties, and its inability to be aminoacylated by another (non-cognate)

PylRS. Incorporation of multiple distinct ncAAs into a protein facilitates strategies for site-specific dual color labeling of proteins in the same systems, such as mammalian cells, E.coli and yeast, and chemically controlled site-specific tethering of protein- protein complex subunits as well as providing a foundation for synthesis of non- canonical biopolymers. The PylT is highly suitable for evolving and designing new substrate specificity.

The present invention provides hybrid PylTs based on domain swapping where the entire acceptor stem of a PylT from one species is swapped with an entire PylT acceptor stem of another species. The hybrid PylT of the present invention has a surprisingly high activity towards its cognate PylRS, while having no or very reduced activity towards its non-cognate PylRS.

The invention provides a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together forms a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence. The invention also provides a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most one nucleotide of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added.

The invention also provides a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence.

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most one nucleotide of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence.

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is native to a first species and the PylT body is native to a second species, wherein the first species is different form the second species and the acceptor stem native to the first species is different from the acceptor stem native to the second species, and the body native to the first species is different from the body native to the second species wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence.

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is native to a first species and the PylT body is native to a second species, wherein the first species is different form the second species and the acceptor stem native to the first species is different from the acceptor stem native to the second species, and the body native to the first species is different from the body native to the second species wherein at the most one nucleotide of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence.

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species.

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is native to a first species and the PylT body is native to a second species, wherein the first species is different form the second species and the acceptor stem native to the first species is different from the acceptor stem native to the second species.

In one aspect of the present invention, C consists of a nucleotide sequence having the general formula

5’-C_I-C₂-3’ wherein: a. Ci consists of a consecutive sequence of nucleotides which are complementary to A; and

C₂ consists of in the range of 0 to 5 nucleotides; and b. A consists of a consecutive sequence of nucleotides which are complementary to Ci. The invention also comprises a complex comprising a pyrrolysyl tRNA (PylT) and a pyrrolysyl-tRNA-synthetase (PylRS) (PylT/PyIRS), wherein the PylT is as defined herein above and wherein PylRS may be any PylRS which is able to amioacylate said PylT.

The present invention further provides a pair of PylT/PyIRS complexes, wherein said pair comprises a first PylT/PyIRS complex as described herein above, and a second PylT/PyIRS complex, wherein said second complex is mutually orthogonal to the first PylT/PyIRS complex.

The invention further provides a method of producing a protein using a hybrid PylT, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA- synthetase (PylRS) and/or a pair of PylT/PyIRS complexes according to the present invention.

Description of Drawings

Figure 1 shows the general structure of tRNA, divided into domain regions denoted A, B and C. A, B and C together form a consecutive sequence of nucleotides, wherein each letter (either A, B or C) represents a nucleotide individually selected from “A”, “G”, “U” or “C”. A and C (including Ci and C_å) together form a tRNA acceptor stem. Thus, by way of example a nucleotide indicated as “A” may be the same or a different nucleotide to another nucleotide indicated as “A” in Figure 1. Ci and A are generally complementary, but can contain non-Watson Crick base pairs. C_å may consist of 0 to 5 nucleotides. B forms the body of the tRNA comprising a D-arm, an anticodon region, a variable-loop and a T-arm.

Figure 2 shows that the G1 PylT/RS pair is highly active in mammalian cells (GFP reporter fluorescence). A fluorescence plate reader assay of HEK293T cell lysates harvested 24 h after transfection with PylT/GFP^150TAG reporter plasmid and Mma PylRS, G1 PylRS or Mx1201 PylRS vectors, at 9:1 ratio. GFP fluorescence is normalized to the mean of fluorescence measures with Mma PylT/RS in the same experiment. Where indicated +ncAA (darker grey), 0.2mM CpK was added to the medium at transfection. Individual datapoints, mean and standard deviation are shown from 3-4 independent experiments each including triplicate +ncAA transfections. Figure 3 shows that the G1 PylT/RS pair is highly active in mammalian cells (western blot). A western blot from HEK293T cell lysate transfected as in Figure 2 shows expression of GFP, FLAG-tagged synthetases and a b-actin loading control. Cells were cultured in the presence of 0.2 mM CpK and harvested 24 h post transfection.

Figure 4 shows cloverleaf structures of G1 wild type, Gf/Mx1201 hybrid PylT mutants and wild type Mma PylT. A) Structures of G1 PylT and mutant G1 PylT^{A41AA C55A} (G1* PylT) shown as cloverleaf. Black boxes highlight positions of the A41AA variable-loop and C55A T-arm mutations. B) Structures of Mx1201/G1 hybrid PylT (hyb PylT), Mx1201/G1 hybrid PylTA^{A41AA C55A} (hyb* PylT) and Mma PylT shown as cloverleaf. Black boxes highlight positions of the A41AA variable-loop and C55A T-arm mutations. The Mx1201 PylT derived sequences are shown in light grey, while G1 PylT derived sequences are shown in black. Light grey boxes indicate the two base pairs differing between G1 and M x1201 PylT acceptors stems.

Figure 5 shows that the engineered G1/Mx1201 hybrid PylT is orthogonal to Mma PylT) Fluorescence plate reader assay of HEK293T cell lysates harvested 24 h post transfection with PylT/GFP^150TAG reporter plasmid and a Mma or G1 PylRS vector, at 9:1 ratio. GFP fluorescence is normalized to the mean of fluorescence measures with Mma PylT/RS in the same experiment. Where indicated +ncAA (darker grey), 0.2mM CpK was added to the medium at transfection. Individual data points, mean and standard deviation are shown from minimum 2 independent experiments each including triplicate +ncAA transfections. Significance and p values were determined using two-sided Student’s T test.

B) Selectivity index of G1 PylT variants. The index is calculated for each G1 PylT variant as the ratio of cognate ( G1 PylRS) over non-cognate ( Mma PylRS) activity as determined by mean GFP fluorescence after subtracting -CpK background. Error bars indicate confidence intervals calculated using Fiellers theorem for the ratio of two means.

Figure 6 shows amino acid substrate specificity of G1 PylT/RS and G1 PylT/RS^Y125A (GFP eporter fluorescence). Fluorescence plate reader assay of HEK293T cell lysates harvested 24 h after transfection with PylT/GFP^{150 AG} reporter plasmid and A) Mma PylT/RS B) G1 PylT/RS orC) G1 PylT/RS^Y125A vector, at 4:1 ratio. After transfection, cells were grown in absence (-ncAA) or presence the indicated ncAAs (0.5 mM). GFP fluorescence is normalized to the mean fluorescence measured with CpKforthe PylT/RS pair in each experiment. Error bars show standard deviation of three replicates.

Figure 7 shows amino acid substrate specificity of G1 PylT/RS and G1 PylT/RS^Y125A (Western blot). Western blot using samples from Figure 6 A and C showing expression of GFP, FLAG-tagged PylRS variants and a b-actin loading control.

Figure 8: shows selective incorporation of two ncAAs at distinct sites in sfGFP. Intact mass determination of purified GFP containing 150TCO*K (incorporated with G1 hyb* PylT/RS^Y125A in GFP^150TAG), 150ProK (incorporated with Mma M15 PylT^UUA/RS in QPp^i50TAA) _{as we|| as} Qpp _wjth 102TCO*K and 150ProK incorporated with a combination of Mma M15 PylT^UUA/RS and G1 hyb* PylT/RS^Y125A in GFP^{102TAG 150TAA}.

Figure 9 shows production of sfGFP with two ncAAs at distinct sites. A) Live cell imaging of HEK293T cells transfected with Mma M15 PylT^UUA/RS, G1 hyb* PylT/RS^Y125A and a hyb* PylT/GFP^{102TAG 150TAA} reporter (1:1 :8 ratio) in the absence or (-) or presence (+) of 0.1 mM TCO*K and 0.25 mM ProK. Images were taken 48 h post transfection.

B) Western blot showing expression of GFP and FLAG-tagged synthetases from the same experiment as A.

Figure 10 shows incorporation of two ncAAs at distinct sites in sfGFP and their site selective biorthogonal labelling by SPIEDIAC and CuAAC. Bioorthogonal SPIEDAC and CuAAC labeling of bead captured GFP. HEK293T cells were transfected with Mma M15 PylT^UUA/RS and G1 hyb* PylT/RS^Y125A as well as a hyb* PylT expressing GFP amber suppression reporter plasmid (encoding for GFP^150TAA, GFP^150TAG _OrGFP^{102TAG 150TAA}) and grown in presence of 0.1 mM TCO*K and 0.25 mM ProK for 72 h. Cell lysates were incubated with GFP trap beads to capture produced GFP, SPIEDAC and CuAAC reactions were performed with bead bound GFP. Aliquots of the eluates were separated by SDS-PAGE and imaged for in-gel fluorescence. The gel was stained to show equal loading.

Figure 11 shows dual bioorthogonal labeling of SynNotch receptor by SPIEDAC and CuAAC on the surface of live cells by SDS-PAGE. SPIEDAC and CuAAC labeling of SynNotch on the surface of live HEK293T cells. Cells were transfected with Mma M15 PylT^UUA/RS, G1 hyb* PylT/RS^Y125A and hyb* PylT/LaG17-SynNotch^{204 AA 442 AG} or hyb* PylT/LaG17-SynNotch^{204TAG 442TAA} in a 1:1:8 ratio. Transfected cells were grown in presence of 0.1 mM TCO*K and 0.25 mM ProK for 48 h. The SynNotch construct is a LaG17(aa1 -159)- Notch core (aa173-491) fusion joined by a linker with a TEV-protease cleavage site (aa160-172). Positions of amber or ochre stop codons are 204 or 442. Primary processing at the S1 site produces a 43k Da N-terminal fragments (aa1-392, including 204 stop codon) and a 11kDa C-terminal membrane spanning fragment (aa393-491 , including 442 stop codon). The unprocessed polypeptide is 54 kDa Furin-inhibiting proprotein convertase inhibitor (PPCI) was added to 50 mM at the time of transfection. To control for unspecific fluorescence, untransfected HEK293T cells were grown with 0.1 mM TCO*K and 0.25 mM ProK for 48 h (first and second lane, SynNotch -). Cells were labeled with 10 pM AF488-tetrazine and 10 pM AF647-picolyl azide in 50 pM CuS0₄ were indicated (dye +). Lysates were separated by SDS-PAGE, imaged for in-gel fluorescence and analyzed for expression of the FLAG-tagged synthetase variants and b-actin as loading control. An unfilled arrow indicates the 43 kD N-terminal fragment after S1 cleavage, filled arrows indicates unprocessed 54 kD La 17G-Syn Notch in presence of furin inhibitor. Asterisks indicate an additional SynNotch N-terminal fragment without La17G.

Figure 12 shows dual bioorthogonal labeling of SynNotch receptor by SPIEDAC and CuAAC on the surface of live cells by fluorescence live cell microscopy, and chemical- controlled crosslinking. A) Live cell imaging of SPIEDAC and CuAAC labelled HEK293T cells transfected with Mma M15 PylT^UUA/RS, G1 hyb* PylT/RS^Y125A and hyb* PylT/LaG17-SynNotch^{204 AA 442 AG} in a 1 :1 :8 ratio in the absence (-ncAA) or presence of 0.1 mM TCO*K and 0.25 mM ProK. Surface labeling by SPIEDAC with 1 pM Cy3- tetrazine and CuAAC with 5 pM sulfo-Cy5-picolyl azide was carried out 48 h post transfection. B) SPIEDAC and CuAAC mediated stapling of SynNotch on the surface of live HEK293T cells. Structure and simplified scheme of the tetrazine-Cy5-azide trifunctional linker is depicted. Cells were transfected with Mma M15 PylT^UUARS, G1 hyb* PylT/RS^Y125A and hyb* PylT/LaG17-SynNotch^{204 AG 442TAA} in a 1 :1 :8 ratio. Transfected cells were grown in presence of 0.1 mM TCO*K and 0.25 mM ProK for 48 h. Cells were labeled with 2 pM tetrazine-Cy5-azide. Initial addition leads to SPIEDAC reaction with TCO*K, subsequent CuAAC conditions allow reaction of the azide group with ProK and covalently link the SynNotch heterodimer. Figure 13 shows dual bioorthogonal labeling of G-protein coupled receptor CRFR1 by SPIEDAC and CuAAC on the surface of live cells by SDS-PAGE. SPIEDAC and CuAAC labeling of CRFRIon the surface of live HEK293T cells. Cells were transfected with Mma M15 PylT^UUA/RS, G1 hyb* PylT/RS^Y125A and hyb* PylT /CRFR1^{95TAA 263TAG} or hyb* PylT^UUA/CRFR1^{95 AG 263 AA} in a 1 :1 :8 ratio. Transfected cells were grown in presence of 0.1 mM TCO*K and 0.25 mM ProK for 48 h post transfection. Cells were labeled with 1.5 mM AF488-tetrazine and 10 pM AF647-picolyl azide in 50 pM CuS0₄. Lysates were separated by SDS-PAGE, imaged for in-gel fluorescence (at 460nm and 630nm) and analyzed by western blot for expression of HA-tagged CRFR1, FLAG-tagged synthetase variants and a b-actin loading control. Labeled CRFR1 at the cell surface corresponds to the mature complex-glycosylated receptor and runs at apparent MW of about 65-75 kDa (Coin, 2013). Additional lower molecular weight isoforms detected by anti-HA stain are high-mannose glycosylated and unglycosylated forms of CRFR1. These precursors run at an apparent MW of 50 kDa and 37 kDa, respectively.

Figure 14 shows dual bioorthogonal labeling of G-protein coupled receptor CRFR1 by SPIEDAC and CuAAC on the surface of live cells by fluorescent live cell imaging. Live cell imaging of SPIEDAC and CuAAC labelled HEK293T cells transfected with Mma M15 PylT^UUA/RS, G1 hyb* PylT/RS^Y125A and hyb* PylT/CRFR1^{95 AA 263 AG} in a 1:1:8 ratio in the absence or (-ncAA) or presence of 0.1 mM TCO*K and 0.25 mM ProK. Surface labeling by SPIEDAC with 1 pM Cy3-tetrazine and CuAAC with 5 pM sulfo-Cy5-picolyl azide was carried out 48 h post transfection.

Figure 15: Acceptor stem alignment of known archeal and bacterial PylT, showing natural diversity of the acceptor stems. The sequence of is shown in 5’ to 3’ direction with sequence A as defined in Figure 1 on the left and Ci as defined in Figure 1 on the right. Sequence B is omitted (-). The dotted lines show base pairings in the acceptor stem.

Figure 16 A) Acceptor stem alignment of Methanosarcina mazei WWM610/1-16 (Mma), Methanogenic archaeon IS04-G1 (G1), Candidatus Methanomethylophilus Alvus Mx1201 ( Mx1201 ), Methanogenic archaeon IS04H5 (H5), Candidatus Methanoplasma termitum MpT1 (MpT PylT), showing natural diversity of the acceptor stems. The sequence is shown in 5’ to 3’ direction with sequence A as defined in Figure 1 on the left and C as defined in Figure 1 on the right. Sequence B is omitted and simply represented by a dash (-). The dotted lines show base pairings in the acceptor stem. B) Schematic of G1 PylT and hyb PylT comprising an acceptor stem of Mx1201 and a body of Gf (hyb,) (described in previous figures), and two additional hybrids comprising acceptor stem of H5 and body of G1 (hyb2), and acceptor stem of MpT1 and body of G1 (hyb3). C) Fluorescence plate reader assay of HEK293T cell lysates prepared 48 h post transfection with PylT/GFP^150TAG reporter plasmid and the indicated combination of G1 PylRS and G1 PylT, or one of the additional hybrid PylT hyb2 or hyb3 constructs. 0.2mM CpK has been added at time of transfection. Relative GFP fluorescence shows that hyb2 and hyb3, like hyb1 (Figure 5) are highly active with G1 PylRS. D) Fluorescence plate reader assay of HEK293T cell lysates prepared 48 h post transfection with PylT/GFP^150TAG reporter plasmid and the indicated combination of Mma PylRS and Mma PylT, or one of the hybrid PylT constructs. 0.2mM CpK has been added at time of transfection. Relative GFP fluorescence shows that all hybrid PylT have no activity with Mma PylRS. C) and D) show that hyb, hyb 2 and hyb 3 are very activity with G1 PylRS and only have very low activity with Mma PylRS, thus the three hybrids are highly active and specific for G1 PylRS.

Figure 17 Distance Tree of selected PylT sequences from archea and bacteria.

Figure 18 Alignment of selected PylT sequences from archea and bacteria. The alignment show a high variation in the nucleotide sequences of the selected PylT.

Thus, the acceptor stem nucleotide sequence and body nucleotide sequence varies between the selected PylTs.

Detailed description Definitions

The term “amino acid” as used herein refers to any amino acid, such as any canonical and non-canonical amino acid.

The term “canonical amino acid” as used herein refers to a proteinogenic amino acid. Preferably, the proteinogenic amino acid is one of the 20 amino acids encoded by the standard genetic code. The lUPAC one and three letter codes are used to name amino acids. The term “homology” as used herein is a measure of how much one sequence resembles another sequence. Homology may e.g. be determined by determining “identity”. Herein, the term homology is preferably used interchangeably with “identity”.

The term “hybrid” as used herein refers to a hybrid PylT that is generated by mixing parts of two PylTs having different origins, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species. In other words, a hybrid PylT is a non-natural PylT having one PylT part that is native to one species and another part that is native to a different species. Thus, a hybrid PylT is a non-natural PylT, which is distinct from any wild type PylT. In particular, a hybrid PylT has an acceptor stem (A and C) native to a first species and a body (B) native to a second species, wherein the acceptor stem (A and C) native to the first species is distinct from the acceptor stem (A and C) native to the second species and wherein the body (B) native to the first species is distinct from the body (B) native to the second species. A hybrid PylT having an acceptor stem (A and C) nucleotide sequence native to a first species and a body (B) nucleotide sequence native to a second species generates a hybrid PylT having a nucleotide sequence that is distinct from a native PylT nucleotide sequence of a first species and a second species and any wild type nucleotide sequence.

The term “sequence identity” as used herein refers to the % of identical amino acids between a candidate sequence and a reference sequence following alignment. Thus, a candidate sequence sharing 80% amino acid identity with a reference sequence requires that, following alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity according to the present invention is determined by aid of computer analysis, such as, without limitations, the Clustal Omega computer alignment program for alignment of polypeptide sequences (Sievers et al. 2011 ; Li et al. 2015; McWilliam et al., 2013), and the default parameters suggested therein. The Clustal Omega software is available from EMBL-EBI at https://www.ebi.ac.uk/Tools/msa/clustalo/. Using this program with its default settings, the mature (bioactive) part of a query and a reference polypeptide are aligned. The number of fully conserved residues are counted and divided by the length of the reference polypeptide. Sequence identity as provided herein is thus calculated over the entire length of the reference sequence. The term “non-canonical amino acid” as used herein refers to a non-proteinogenic amino acid. The abbreviation ncAA refers to “non-canonical amino acid” herein.

The term “orthogonal” as used herein refers to the ability of a tRNA or a tRNA synthetase to interact only with a specific, corresponding tRNA synthetase or a specific, corresponding tRNA, respectively, without any cross-reactivity with other tRNAs or tRNA synthetases, such as endogenous tRNAs or tRNA synthetases. An orthogonal tRNA synthetase can only aminoacylate its cognate, corresponding tRNA, whereas an endogenous tRNA synthetase is not capable of aminoacylate the cognate, corresponding tRNA. A set consisting of a PylT and a PylRS is an orthogonal pair, if it does not crosstalk with an endogenous tRNA or tRNA synthetase or another introduced tRNA or tRNA synthetase, while still being functionally compatible with the ribosome and other components of translation apparatus.

The term “PylRS” as used herein refers to a pyrrolysyl-tRNA-synthetase. Wild type PylRS is normally capable of attaching pyrrolysine to its orthogonal PylT, however PylRS as used herein refers to a PylRS which is capable of attaching any amino acid to its orthogonal PylT. PylRS recognize its cognate PylT by the acceptor stem.

The term “PylT” as used herein refers to a transfer RNA molecule capable of translating an mRNA sequence into a protein. PylT consists of a consecutive sequence of polynucleotides, which in its mature form forms a cloverleaf like structure comprising an acceptor stem, a D-arm, an anticodon arm, a variable loop and a T-arm. PylT used herein as an abbreviation for “pyrrolysyl tRNA”.

The term “PylT acceptor stem” as used herein refers to the part of the mature and folded tRNA, which is recognized by a tRNA synthetase. The PylT consists of a consecutive sequence of nucleotides, which can be defined by the general formula 5’- A-B-C-3’, wherein A and C together forms the PylT acceptor stem. C consists of a nucleotide sequence, which further can be defined by the general formula 5’-C_I-C₂-3’, wherein Ci consists of a consecutive sequence of nucleotides which are complementary to A; and C_å consists of in the range of 0 to 5 nucleotides. C_å often comprises a CCA tail, whereto a tRNA synthetase can covalently link an amino acid. A consists of a consecutive sequence of nucleotides which are complementary to Ci. See Figure 1. When the PylT acceptor stem is from a first species it is meant that said species is a wild type species. Thus, the acceptor stem can be an acceptor stem of a wild type species or be an acceptor stem of a wild type species comprising at the most one nucleotide substitution or addition.

The term “PylT body” as used herein refers to the part of the mature and folded tRNA which comprises a three-nucleotide sequence anticodon. The anticodon forms the three complementary base which pairs with a codon in the mRNA during protein synthesis. The PylT consists of a consecutive sequence of nucleotides, which can be defined by the general formula 5’-A-B-C-3’, wherein B forms a PylT body. The PylT body comprises or consists of a D-arm, an anticodon arm comprising the anticodon, a variable loop and a T-arm. See Figure 1. When the PylT body is from a second species it is meant that said species is a wild type species. Thus, the PylT body can be an acceptor stem of a wild type species or be an acceptor stem of a wild type species comprising at the most six nucleotide substitutions or additions.

The term “D-arm” as used herein refers to a consecutive sequence of nucleotides, wherein the first and last nucleotides of the D-arm are capable of forming a first base pair. This first base pair is connected to other nucleotides capable of forming additional base pairs (“D-stem”) or connected to a consecutive sequence of nucleotides forming a loop, without any pairing nucleotides (“D-loop”). The first nucleotide in the D-arm is either directly connected to the 3’ terminal end of the 5’ portion of the acceptor stem or linked by one or more nucleotides to the 3’ terminal end of 5’ portion the acceptor stem A and the last nucleotide in the D-arm is either directly connected to the anticodon arm or linked by at least one, such as at least two nucleotides to the anticodon arm.

The term “anticodon-arm” as used herein refers to a consecutive sequence of nucleotides, wherein the first and last nucleotides of the anticodon-arm are capable of forming a first base pair. This first base pair is connected to other nucleotides capable of forming additional base pairs (“anticodon-stem”) or connected to a consecutive sequence of nucleotides forming a loop, without any pairing nucleotides (“anticodon- loop”). The anticodon-loop comprises a three-nucleotide sequence anticodon. The first nucleotide in the anticodon-arm is either directly connected to the D-arm or linked by at least one, such as at least two nucleotides to the D-arm. The last nucleotide in the anticodon-arm is directly connected to the variable-loop. The term “variable-loop” (equivalent with the term “variable-arm”) as used herein refers to a consecutive sequence of nucleotides connecting the anticodon-arm and the T-arm. The variable-loop of PylT species does not contain any paired bases.

The term “T-arm” as used herein refers to a consecutive sequence of nucleotides, wherein the first and last nucleotides of the T-arm are capable of forming a first base pair. This first base pair is connected to other nucleotides capable of forming additional base pairs (“T-stem”) or connected to a consecutive sequence of nucleotides forming a loop, without any pairing nucleotides (“T-loop”). The first nucleotide in the T-arm is directly connected to the variable loop. The last nucleotide in the anticodon-arm is either directly connected to the 3’ terminal end of the acceptor stem C₂ or linked by at least one, such as at least two nucleotides to the 3’ terminal end of the acceptor stem C₂.

The term “complementary” as used herein refers to a consecutive sequence of nucleotides which are capable of base pairing with another consecutive sequence of nucleotides. The base pairs may be selected from:

Canonical base pairs/Watson-Crick base pairs:

A-U and C-G, and

Noncanonical base pair/non-Watson-Crick base pairs:

A-G (Hoogsten base pair)

G-U (Wobble base pair).

See Figure 15 for examples of such base pairs.

The term “from a species” as used herein refers to a wild type species. Thus, if a specific part of a PylT is from a specific species, said part corresponds to the same part of a wild type species. In other words, said specific part is “native to” said specific species. The term “from a species” is thus used interchangeably with “native to”. Thus, “native to” a species and “from a species” as used herein refers to a nucleotide sequence or amino acid sequence as would be found endogenously in a host organism. Meaning that a PylT native to a specific species is found endogenously in that specific species and a PylT from a specific species is found endogenously in that specific species. The term “wild type” as used herein refers to a naturally occurring sequence of a nucleic acid at a genetic locus in the genome of an organism. A wild type PylT is a natural PylT endogenous to an organism, which has not been engineered in any way. Opposite, a hybrid PylT is a non-natural PylT that is not endogenous to an organism, this type of PylT have been engineered by e.g. mutations or domain swapping.

The term “non-sense codon” or “stop codon” are used interchangeably herein. The terms refer to a group of codons selected from the groups of amber (UAG), ochre (UAA) or opal (UAG) codons. These codons normally signals a termination of translation into proteins, however these codons are used in the present invention as coding codons capable of coding ncAAs.

The term “anticodon” as used herein refers to nucleotide triplets contained within a tRNA body, which determines the codon specificity of said tRNA. The hybrid PylT according the present invention typically contains anticodons selected from the group consisting of CUA, UUA and UCA. B of PylT may comprise an anticodon selected from the group of CUA, UUA and UCA.

The term “substitution” as used herein refers to a change in a nucleotide within a sequence of nucleotides, wherein a specific nucleotide is replaced with a different nucleotide, which is not the same nucleotide. The total length of the sequence of nucleotides is not changed.

The term “addition” as used herein refers to the addition of a nucleotide within a sequence of nucleotides. The added nucleotide may be any nucleotide. If one nucleotide is added to a sequence of nucleotides, the total length of the sequence is increased by one.

Hybrid pyrrolysyl tRNA (PylT)

The inventors have explored a new family of PylTs and their cognate PylRS enzymes from a seventh order of methanogenic archaea useful in genetic code expansion. The inventors have engineered a new highly active and orthogonal hybrid PylT. Said hybrid PylT is useful in any system, such as mammalian cells, E. coli and yeast. In some embodiments, the hybrid PylT is especially useful in mammalian cells. The present invention relates to a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

The present invention also relates to a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

In other words, the present invention relates to a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

Thus, the present invention also relates to a hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

Thus, A and C together forms a PylT acceptor stem. The PylT acceptor stem can further be defined by: a. C consists of a nucleotide sequence having the general formula

5’-C_I-C₂-3’ wherein Ci consists of a consecutive sequence of nucleotides which are complementary to A; and

C₂ consists of in the range of 0 to 5 nucleotides; and b. A consists of a consecutive sequence of nucleotides which are complementary to Ci.

In one embodiment, A comprises or consists of seven 5’-terminal nucleotides and/or C comprises or consists of eleven 3’-terminal nucleotides.

In one embodiment, A comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO: 12. In one embodiment, C comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

In a preferred embodiment, A comprises or consists of a sequence of nucleotides of SEQ ID NO:5, SEQ ID: 6, SEQ ID: 7 or SEQ ID NO:13. In a preferred embodiment, C comprises or consists of a sequence of nucleotides of SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:19.

In one embodiment, B comprises or consists of a sequence of nucleotides from position 6 to 60 of SEQ ID NO:1 or comprises or consists of a sequence of nucleotides from position 6 to 61 of SEQ ID NO:2.

Thus, in one embodiment, the hybrid PylT comprises or consists of a sequence of nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:28 and SEQ ID NO:29.

The different regions of the hybrid PylT of SEQ ID NO:1 are indicated herein below:

5’-GGGGGACG^*G^*U^*C^*C^*G^*G^*C^*G^*A^*G^*C^*AAACGGGUCUCUAAAACCUGUA_vA_vG_v

CGGGGL/L/CGACCCCCCGGUCUCUCGCCA-3’

Bold indicates the acceptor stem, i.e. nucleotides 1 to 7 and 61 to 74 of SEQ ID NO:1 ^'indicates the D-arm, i.e. nucleotides 8 to 19 of SEQ ID NO:1.

Underlined indicates the anticodon-arm, i.e. nucleotides 22 to 40 of SEQ ID NO:1, wherein nucleotides 30 to 32 represent the anticodon region vindicates the variable-loop, i.e. nucleotides 41 to 43 of SEQ ID NO:1.

Italic indicates the T-arm, i.e. nucleotides 44 to 60 of SEQ ID NO:1.

A hybrid PylT having one or more substitutions or additions The hybrid PylT according to the present invention, comprises a PylT acceptor stem, A and C, and a PylT body, B, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most two, such as at the most one nucleotide of said hybrid PylT acceptor stem and/or at the most six, such as at the most five, such as at the most four, such as at the most three, such as at the most two, such as at the most one nucleotides of said hybrid PylT body have been substituted and/or added.

In one embodiment of the present invention, the hybrid PylT acceptor stem, A and C, may comprise at the most two, such as at the most one nucleotide substitution(s).

Thus, the hybrid PylT acceptor stem may comprise one or two nucleotide substitutions in A, Ci or C2. The substitution(s) may be any substitution(s) with the proviso that the substitution(s) do not generate a PylT acceptor stem which is identical to the acceptor stem of the second species. The nucleotide substitution is preferably in A and/or C2. If the mutation is present in Ci it is preferred that the three most 3’ terminal nucleotides of Ci are conserved, and the fourth nucleotide, i.e. the most 5’ terminal nucleotide of Ci is substituted.

In one embodiment, A comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12. In another embodiment of the present invention, A comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11 and SEQ ID NO:12, wherein one nucleotide is substituted. In another embodiment of the present invention, A comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11 and SEQ ID NO:12, wherein two nucleotides are substituted.

In one embodiment, C comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19. In another embodiment of the present invention, C comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, wherein one nucleotide is substituted. In another embodiment of the present invention, C comprises or consists of a sequence of nucleotides selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO: 19, wherein two nucleotides are substituted.

In a preferred embodiment, A comprises or consists of a sequence of nucleotides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:13. In another preferred embodiment of the present invention, A comprises or consists of a sequence of nucleotides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:13, wherein one nucleotide is substituted. In another preferred embodiment of the present invention, A comprises or consists of a sequence of nucleotides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:13, wherein two nucleotides are substituted.

In one preferred embodiment, C comprises or consists of a sequence of nucleotides of SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:19. In another preferred embodiment of the present invention, C comprises or consists of a sequence of nucleotides of SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:19, wherein one nucleotide is substituted. In another preferred embodiment of the present invention, C comprises or consists of a sequence of nucleotides of SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:19, wherein two nucleotides are substituted.

In one embodiment of the present invention, the hybrid PylT body, B, may comprise at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2, such as at the most 1 nucleotide substitutions or nucleotide additions. The substitution(s) may by any substitution(s) of nucleotide(s). The nucleotide addition(s) may be an addition(s) of any nucleotide(s). With the proviso that the substitution(s) do not generate a PylT body which is identical to the body of the first species.

In one embodiment, B comprises or consists of a sequence of nucleotides from position 6 to 60 of SEQ ID NO:1 or comprises or consists of a sequence of nucleotides from position 6 to 61 of SEQ ID NO:2. In another embodiment, B comprises or consists of a sequence of nucleotides from position 6 to 60 of SEQ ID NO:1 or comprises or consists of a sequence of nucleotides from position 6 to 61 of SEQ ID NO:2, wherein at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2, such as at the most 1 nucleotides are substituted or added.

In one embodiment, the hybrid PylT comprises or consists of a sequence of nucleotides of SEQ ID NO:1 or SEQ ID NO:2. In another embodiment, the hybrid PylT comprises or consists of a sequence of nucleotides of SEQ ID NO:1 or SEQ ID NO:2, wherein at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2, such as at the most 1 nucleotides are substituted or added.

In one embodiment, the nucleotide substitution(s) and/or nucleotide addition(s) are in the T-arm and/or the variable loop and/or the anticodon-armof B.

In one embodiment, the nucleotide substitution(s) may be any substitution(s) of nucleotide(s) in the T-arm, wherein the T-arm comprises or consists of a sequence of nucleotides 44 to 60 of SEQ ID NO:1. In one embodiment, said hybrid PylT comprises a substitution of C to A in the T-arm, wherein the T-arm comprises or consists of a sequence of nucleotides 44 to 60 of SEQ ID NO:1. In one embodiment, said hybrid PylT comprises a substitution of C to A at position 55 of SEQ ID NO:1.

In one embodiment, the nucleotide addition(s) may be any addition(s) of a nucleotide(s) in the variable loop, wherein the variable loop comprises or consists of a sequence of nucleotides 41 to 43 of SEQ ID NO:1. In one embodiment, the hybrid PylT comprises an addition of A to AA in the variable loop, wherein the variable loop may comprises or consists of a sequence of nucleotides 41 to 43 of SEQ ID NO:1. In one embodiment, the hybrid PylT comprises an addition of A to AA at position 41 of SEQ ID NO:1.

In one embodiment, the hybrid PylT comprises or consists of a sequence nucleotides of SEQ ID NO:2.

A hybrid PylT of the present invention may be modified to recognize any codon present in the mRNA, however, preferably it recognizes a stop codon such as amber, opal and ochre codons. The codon may be any three letter codon present in the mRNA. The codon may be any quadruplicates, i.e. four letter code present in the mRNA. Thus, a PylT according to the present invention may comprise one or more substitutions in the variable loop. In particular, the one or more substitutions may be within the anticodon of the hybrid PylT allowing the PylT to recognize any codon.

In one embodiment, the nucleotide substitutions may be any substitution or addition of a nucleotide in the anticodon-arm, wherein the anticodon-arm may comprises or consists of nucleotides 22 to 44 of SEQ ID NO:1 or SEQ ID NO:2. In particular, the nucleotide substitutions may be any substitution of the nucleotides in the anticodon region, wherein the anticodon region comprises or consists of nucleotides 30 to 32 of SEQ ID NO:1 or SEQ ID NO:2, and/or an addition of a nucleotide to the anticodon region of comprising or consisting of comprises or consists of nucleotides 30 to 32 of SEQ ID NO:1 or SEQ ID NO:2.

In a preferred embodiment the hybrid PylT is capable of recognizing a non-sense codon. In some embodiments of the invention the hybrid PylT is capable of recognizing an amber codon (UAG), ochre codon (UAA) or opal (UAG) codon.

In one embodiment of the present invention, B of hybrid PylT comprises an anticodon selected from the group of CUA, UCA and UUA. In one embodiment of the present invention, B of hybrid PylT comprises a CUA anticodon.

The hybrid PylT may be altered to record a different stop codon, in particular by substituting the nucleotides 30 to 32 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:28 or SEQ ID NO:29 or adding at least one nucleotides to the nucleotides 30 to 32 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:28 or SEQ ID NO:29 hereby generating a four nucleotides anticodon. Useful methods of modifying the tRNA anticodon are for example described in Dumas et al. 2015 or Nui et al. 2013. Nui et al., describes generation of four nucleotides anticodons. The PylT anticodon may also be modified as described in example 4.

A hybrid PylT is a PylT that is generated by mixing parts of two PylT having different origins, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species. As can be seen from figure 18 the PylT nucleotide sequence alignment of selected PylT show a high nucleotide variation. Hence, the acceptor stem nucleotide sequence and body nucleotide sequence differ greatly between different species. Importantly, the acceptor stem is the most conserved functional domain within the PylT. Thus, if the acceptor stem nucleotide sequences of two PylTs from two different species differ, then the two PylT body nucleotide sequences will also differ.

In other words, if an acceptor stem of a PylT from a first species is different to an acceptor stem of a PylT from a second species, then the body of said PylT from the first species is also different from the body of said PylT from the second species.

A PylT/PyIRS complex

The present invention also relates to a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a pyrrolysyl-tRNA-synthetase (PylRS) (PylT/PyIRS), wherein the hybrid PylT is as defined in section “Pyrrolysyl tRNA (PylT)” and wherein PylRS may be any PylRS which is active to said PylT. The PylT/PyIRS complex comprising the hybrid PylT may also be termed the “first PylT/PyIRS” complex” herein. Furthermore, the PylRS of the complex comprising the hybrid PylT may also be termed the “first PylRS” herein.

A pyrrolysyl-tRNA-synthetase (PylRS) is an enzyme capable of attaching an appropriate amino acid to its orthogonal tRNA. PylRSs recognize their cognate PylT by the acceptor stem, A and C. The PylRS covalently links an amino acid to Ci of the 3’ terminal end of the tRNA.

A hybrid PylT according to the present invention retains a high activity only to its orthogonal PylRS, and a low reactivity to other PylRS (Example 1 and 2).

In one embodiment, the first PylRS is of the same species as the hybrid PylT body, B.

In other words, PylRS is from a second species and the hybrid PylT body is from the same second species.

In one embodiment of the present invention, said first PylRS comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22 or functional homologues thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% homology thereto. In one embodiment of the present invention, said first PylRS comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:20-22 or functional homologues thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto.

In a preferred embodiment, the first PylRS comprises or consists of an amino acid sequence of SEQ ID NO:22 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% homology thereto.

In a preferred embodiment, the first PylRS comprises or consists of an amino acid sequence of SEQ ID NO:22 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto.

The hybrid PylT/PyIRS complex is capable of co-translational incorporation of an ncAA into proteins via genetic code expansion. Preferably, the hybrid PylT/PyIRS complex does not recognize the 20 canonical amino acids. Identification of an ncAA which exclusively is incorporated by the first PylRS can be performed as described in Example 4. Optionally, the active site of the first PylRS may be altered to accommodate a specific ncAA.

The activity and orthogonality of a PylT/PyIRS complex can be tested by any standard methods. It can, for example, be tested as described in Examples 1 and 2 herein below.

In some embodiments of the present invention, the first PylRS has one or more amino acid substitutions and/or amino acids additions. In one embodiment the first PylRS may comprise one or more amino acid substitutions, such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more amino acid substitutions.

In one embodiment the first PylRS is a Methanogenic archaeon IS04- G1 PylRS, wherein said Methanogenic archaeon IS04- G1 PylRS comprises one or more amino acid substitutions, such as 2 or more, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more amino acid substitutions.

The amino acid substitution may be any amino acid substitution at amino acid position 125, 128, 167 and/or 204 of SEQ ID NO:24. In one embodiment, the amino acid substitution(s) is selected from the group consisting of alanine (A), glycine (G), serine (S), Threonine (T), and cysteine (C). In a preferred embodiment, the PylRS is a Methanogenic archaeon IS04- G1 having an Y125A mutation.

In one embodiment of the present invention, said first PylRS comprises or consists of an amino acid of SEQ ID NO:26 or functional homologues thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto.

A pair of PylT/PyIRS complexes

Despite a progress of identifying new PylRS enzymes and their cognate PylTs progress, combinations of orthogonal synthetase pairs in mammalian cells have been inefficient in dual stop codon suppression (Meineke et al. , 2018; Serfling et al., 2018a; Zheng et al., 2017).

Successful suppression of two stop codons depends on

1) availability of mutually orthogonal tRNA/aaRS pairs that are also orthogonal to the host cell,

2) non-canonical amino acids selectively aminoacylated by only one of the tRNA/aaRS pairs,

3) suppression efficiency of the stop codons used.

These limitations explain why, to date, dual fluorescent labeling studies in mammalian cells have relied on workarounds using a single suppressor with two compatible non- canonical amino acids or a single non-canonical amino acids with two compatible dyes to stochastically create molecules with dual labels (Das et al., 2018; Gust et al., 2018; Lu et al., 2019; Nikic et al., 2014).

Importantly, the present invention provides a pair of PylT/PyIRS complexes, wherein said pair comprises a first PylT/PyIRS complex as described in section “A PylT/PyIRS pair” herein above, comprising a hybrid PylT and a first PylRS, and a second PylT/PyIRS complex, wherein said second complex is mutually orthogonal to the first PylT/PyIRS complex in the same system. The PylT and PylRS of said second complex may also be termed second PylT and a second PylRS. Said second PylT/PyIRS complex may comprise any PylT. Thus, is may comprise a hybrid PylT or a wild type PylT. It is preferred that said second PylT/PyIRS complex comprises a wild type PylT.

The invention provides a pair of PylT/PyIRS complexes, which are both active and orthogonal to the tRNA synthetases and tRNAs used by the host organism for natural translation and mutually orthogonal with respect to other orthogonal tRNA synthetases and tRNAs. It is preferred that said first and said second PylT/PyIRS complexes selectively incorporate distinct ncAAs.

In some embodiments of the present invention, said first PylRS from the first PylT/PyIRS complex recognize a first ncAA and said second PylRS from the second PylT/PyIRS complex recognize a first ncAA, wherein the first ncAA is different from the second ncAA.

A pair of PylT/PyIRS complexes allows for incorporation of at least one, such as two distinct non-canonical amino acids (ncAA) into a protein. Incorporation of multiple distinct ncAAs into a protein facilitates site-specific dual color labeling of proteins in the same system, and chemically controlled site-specific tethering of protein-protein complex subunits as well as providing a foundation for the encoded synthesis of non- canonical biopolymers.

In one embodiment of the present invention, B of the second PylT comprises an anticodon selected from the group of CUA, UCA and UUA. It is preferred that the second PylT comprises an anticodon which is different from the anticodon of the hybrid PylT of said first PylT/PyIRS complex. In one embodiment, B of the second PylT comprises an UUA anticodon.

In some embodiments of the present invention, said B of the first PylT comprises a CUA anticodon and said B of the second PylT an UUA anticodon. By combining the first PylT/PyIRS complex comprising an optimized amber suppressor (UAG) with a second PylT/PyIRS complex comprising an optimized ochre suppressor (UAA) PylT/RS, the inventors can efficiently recode amber and ochre codons in the same mRNA in the same cell, such as mammalian cells, and introduce two site specific fluorophores by using compatible bioorthogonal reactions (see Example 6).

Method of producing a protein

The present invention also concerns a method of producing a protein using a hybrid PylT, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA- synthetase (PylRS) and/or a pair of PylT/PyIRS complexes according to the present invention. The method of producing the protein may include any method suitable for producing a protein using a hybrid PylT, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA-synthetase (PylRS) and/or a pair of PylT/PyIRS complexes according to the present invention.

In one embodiment, the method of producing a protein comprises the steps of providing a host cell expressing a hybrid PylT, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA-synthetase (PylRS) and/or a pair of PylT/PyIRS complexes according to the present invention; culturing the host cell under conditions allowing the host cell to produce the protein; and optionally lysing the cell and recover and/or isolate the protein.

Lysing the cell can be performed by any suitable method known to a person skilled in the art. The protein may be isolated using any suitable method.

In another embodiment, the method of producing a protein comprises the steps of providing a cell-free expression system; contacting said cell-free expression system with a hybrid PylT, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA- synthetase (PylRS) and/or a pair of PylT/PyIRS complexes according to the present invention; and optionally recover and/or isolate the protein. In some embodiments the cell-free expression system is a lysate, such as an E. coli lysate and/or a rabbit reticulocyte lysate. In another embodiment, the cell-free expression system is a wheat germ extract.

In some embodiments of the present invention, the protein comprises a non-canonical amino acid (ncAA). The protein may comprise at least one ncAA, such as at least two ncAA. Said at least two ncAA may preferably be two distinct ncAA.

The ncAA may be any of the ncAAs described in the section “Non-canonical amino acids”.

It is preferred that the protein produced by the method according to the invention comprises an ncAA, wherein the ncAA is linked to a label, a marker, active compound, drug and/or prodrug. In one embodiment the ncAA is subsequently linked to a label, a marker, active compound, drug and/or prodrug through a chemical reaction, preferably a biorthogonal reaction.

In a preferred embodiment, the protein is an antibody or antibody fragment. In particular the protein is an antibody or antibody fragment comprising at least one ncAA linked to a marker, active compound, drug and/or prodrug. In another embodiment, the protein is an antibody or antibody fragment comprising at least two ncAA, wherein the at least two ncAA are linked to a marker, active compound, drug and/or prodrug. The at least two ncAA may be linked to the same or a different marker, active compound, drug and/or prodrug.

In one embodiment the protein is a green fluorescent protein (GFP). This is demonstrated in Example 4, Figures 8 and 10.

Host cell

The present invention also concerns a host cell expressing a hybrid PylT, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA-synthetase (PylRS) and/or a pair of PylT/PyIRS complexes according to the present invention. In some embodiments of the present invention, the host cell encodes a hybrid PylT, a PylT/PyIRS complex and/or a pair of PylT/PyIRS complexes according to the present invention.

The host cell may be any host cell, such as human or mammalian cell lines, or human or mammalian primary cells. In particular, HEK293T or CHO. It is preferred that said host cell is a eukaryote cell, such as any mammalian cell.

A host cell according to the present invention may be comprised within a host organism, such as an animal, optionally a mice, C. elegans or D. melanogaster.

The inventors also show that this strategy allows live-cell site-specific dual color labeling of proteins on mammalian cells, as well as chemically controlled site-specific tethering of protein-protein complex subunits (See Examples 5 and 6).

Species

Cellular life forms can be divided into archaes, bacteria and eukaryote domains. Archaea and bacteria are classified as prokaryote life forms. Several sets of endogenous tRNA synthetases and tRNAs exist within these cellular life forms. A tRNA and tRNA synthetase pair that are orthogonal in one life form are commonly not orthogonal in another life form. Thus, is difficult to identify a PylT, PylT/PyIRS and a pair of PylT/PyIRS complexes which is/are functional in different systems and is/are highly active, mutually orthogonal and do not interact with endogenous PylT and/or PylT/PyIRS complexes.

Hybrid PylT

The present invention is directed to a hybrid PylT, wherein the hybrid PylT acceptor stem, A and C, is form one species and the hybrid PylT body, B, is from a second species. In other words, the present invention is directed to a hybrid PylT, wherein the hybrid PylT acceptor stem, A and C, is native to a first species and the hybrid PylT body, B, is native to second species, wherein the hybrid PylT is different from a wild type PylT. The first species and the second species may be selected from any bacteria or archea. In a preferred embodiment, the first and second species are archea. In one embodiment, the first species is of the genus Candidatus Methanomethylophilus. In another embodiment, the first species is of the genus Candidatus Methanoplasma.

For example, the first species is selected from the group of Candidatus Methanomethylophilus alvus Mx1201, Candidatus Methanomethylophilus sp.1R26 and Candidatus Methanomethylophilus sp. UBA 18, Candiatus Methanomethylophilaceae, Methanomassiliicoccales, Methanogenic archaeon IS04-H5 and Candidatus Methanoplasma termitum MpT1,.

In a preferred embodiment, the first species is Candidatus Methanomethylophilus alvus Mx1201, Methanogenic archaeon ISO-H5 or Candidatus Methanoplasma termitum MpT1.

In a preferred embodiment, the second species is Methanogenic archaeon IS04-G1.

A PylT/PylRS complex

The present invention is also directed to a complex comprising a hybrid PylT/PylRS, said complex is also referred to as a first PylT/PylRS complex.

In other words, in one embodiment the PylRS is from a second species and the PylT body is from a second species, wherein the second species is the same species.

In one embodiment of the present invention, the first PylRS is archea. In one embodiment, said first PylRS is selected from Methanomethylophilus alvus Mx1201 or Methanogenic archaeon IS04-G1. It is preferred, that said PylRS is Methanogenic archaeon IS04-G1.

A pair of PylT/PylRS complexes

The invention is furthermore directed to a pair of PylT/PylRS complexes, wherein said pair comprises a first PylT/PylRS complex as described herein in section “A PylT/PylRS complex”, and a second PylT/PylRS complex, wherein said second complex is mutually orthogonal to the first PylT/PylRS complex. In one embodiment, said second PylT/PyIRS complex is archea. In one embodiment, said second PylT/PyIRS complex is a Methanosarcinaceae. In one embodiment, said second PylT/PyIRS complex is a Methanosarcina. In a preferred embodiment, said second PylT/PyIRS complex is Methanosarcina mazei.

In one embodiment of the present invention, said PylT of said second PylT/PyIRS is archea. In one embodiment, said second PylT is archea. In one embodiment, said second PylT is a Methanosarcinaceae. In one embodiment, said second PylT is a Methanosarcina. In a preferred embodiment, said second PylT is Methanosarcina mazei.

In one embodiment of the present invention, said PylRS of said second PylT/PyIRS is archea. In one embodiment, said second PylRS is archea. In one embodiment, said second PylRS is a Methanosarcinaceae. In one embodiment, said second PylRS is a Methanosarcina. In a preferred embodiment, said second PylRS is Methanosarcina mazei.

Said second PylT and said second PylRS may be from the same species or from different species. It is preferred that said second PylT and said second PylRS are from the same species. In some embodiments of the invention, said second PylT and said second PylRS are both Methanosarcina mazei (Mma).

Non-canonical amino acids (ncAA)

The present invention provides a PylT, a PylT/PyIRS complex and a pair of PylT/PyIRS complexes as defined in the following sections “A pyrrolysyl PylT”, “A PylT/PyIRS complex” and “A pair of PylT/PyIRS complexes” described herein above, they are capable of incorporating one or more ncAAs in to a protein.

Many different ncAAs exist, allowing for a larger repertoire of new protein functions when incorporated into proteins.

In one embodiment, the ncAA can be selected from the group consisting of cyclopropene-L-lysine (CpK), exo-Bicyclo [6.1.0] nonyne-L-lysine (BCNK), axial trans- cyclooct-2-ene-L-lysine (TCO*K),, N-Propargyl-L-lysine (ProK) and N-e-((2- Azidoethoxy)carbonyl)-L-lysine (AzeoK), 4-fluorotryptophane (4FW); 4- methyltryptophane (4MW); 5-fluorotryptophane (5FW); 5-hydroxytryptophane (50HW); 5-methyltryptophane (5MW); 6-fluorotryptophane (6FW), 6-methyltryptophane (6MW); 7-azatryptophane (7azaW). azidohomoalanine (Aha); 2-naphthylalanine (2NpA); 2- anthrylalanine (AntA), acetyl-lysine (AcK); Dansyl-lysine (DaK), homophenylalanine (hF); para-nitrophenylalanine (pNF); para-fluorophenylalanine (pFF); para- trifluoromethylphenylalanine (tF F); para-iodophenylalanine (loF); para- azidophenylalanine (AzF); para-azidomethylphenylalanine (AzMeF); para- acetylphenylalanine (AcF); para-benzoylphenylalanine (Bpa); para- bipyridylphenylalanine (Bpy), 2-methyltyrosine (2MeY); 3-iodotyrosine (3IY); 3- chlorotyrosine (3CIY); 3-azidotyrosine (3AzY); propargyloxyphenylalanine (Ppa), allylglycine (AIG); homopropargylglycine (HpG), selenomethionine (SeM); biocytin (Bet); 1-3,4-dihydroxyphenylalanine (l-DOPA), dabcyl-diaminopropionic (Dbc); b- anthraniloyl-l-a^-diaminopropionic acid (Adp), (7-hydroxy-coumarin-4-yl)ethylglycine (Hco); bodipy-fl-aminophenylalanine (BODIPY-FL-Phe); Bodipy558- aminophenylalanine (BODIPY558-Phe); and 4-[(6-[tetramethylrhodamine-5-(and-6)- carboxamido]hexanoyl)amino]phenylalanine (TAMRA-X-Phe).

The ncAA can be incorporated alone or in combination with one or more additional ncAA.

It is preferred that the ncAA does not comprise any of the 20 canonical amino acids.

Non-limiting examples of uses of ncAA are listed below:

Biorthogonal chemistry

Chemical or photo-decaging

Photoactivation, Photoinhibition, Photoswitching

Photocrosslinking

Spectroscopic probe, Imaging

Mimicking posttranslational modifications

Examples

General method and materials DNA constructs The sfGFP^150TAG reporter construct with four tandem h7SK -Mma PylT repeats, as well as expression plasmids for Mma PylRS, Mx1201 PylRS and Mx1201 PylT mutants have been described previously (Meineke et al. 2018). All Mma PylT carry the U25C mutation previously described (Schmied et. al., 2014; Meineke et al. 2018). Analogous constructs for G1 PylRS and G1 PylT mutants, as well as M15 PylT single and quadruple tandem repeat plasmids were generated for this study based on PB510B-1 (System Biosciences) and pUC-derived vectors. Amber suppression plasmids for the CRFR1 and SynNotch receptors were cloned by replacing GFP in the reporter constructs. All DNA constructs were verified by Sanger sequencing.

Cell Culture and Transfection

HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, GlutaMAX™, Thermo) supplemented with 10 % (v/v) FBS at 37 °C and 5 % CO2 atmosphere. For transfection (1.5-2.0) x 10⁵ cells/ml were seeded 24 h before transient transfection with 1 pg DNA per ml culture using TranslT-LT1 (Mirus) according to the manufacturer’s instructions. ncAAs were added at the time of transfection and cells were harvested after 24 or 48 hours, as indicated. A modified protocol was used for large scale GFP expression for bead purification, increasing the amount of total DNA to 6 pg per ml culture and transfecting (5.0-8.0) x 10⁵ cells/ml with 2 pg polyethylenimine (PEI) per pg DNA. Cells were harvested after 72 hours.

Noncanonical amino acids ncAAs used in this study were prepared from 100 mM stock solutions. Unless noted otherwise final concentrations of 0.2 mM CpK, 0.1 mM TCO*K and 0.25 mM ProK were used.

Quantification of GFP expression

Transfected HEK293T cells were lysed in RIPA buffer supplemented with 1x complete protease inhibitor (Roche) 24 h or 48 h post transfection. The insoluble fraction was removed by centrifugation. GFP bottom fluorescence of aliquots was measured in a Tecan Infinite M200 pro plate reader (excitation 485 nm, emission 518 nm). Fluorescence measurements were normalized to total protein content of each sample as determined by Pierce BCA assay kit (Fisher Scientific) on the same samples. Summary statistics were calculated and visualized in R using ggpubr and mratios packages. T test was performed using ggpubr compare_means function. Fiellers confidence intervals of ratios were calculated using mratios ttestratio function.

Intact Mass Spectrometry

HEK293T cells were transfected, cultured in the presence of ncAA for 72 hours and lysed in RIPA buffer supplemented with 1x complete protease inhibitor (Roche) 24 h or 48 h post transfection. The insoluble fraction was removed by centrifugation.

Expressed GFP was captured on GFP-Trap_MA magnetic beads (ChromoTEK), washed and eluted in 1 % (v/v) acetic acid. Purified GFP samples were desalted and rebuffered into 100 mM ammonium acetate, pH 7.5, using ZebaSpin columns with a 7 kDa cut-off (Thermo). Samples were directly infused into an Orbitrap Fusion Tribrid mass spectrometer equipped with an offline nanospray source using borosilicate capillaries (Thermo). The capillary voltage was 1.5 kV and the pressure in the ion routing multipole was maintained at 0.11 torr. Spectra were acquired in the Orbitrap mass analyzer operated in high mass mode at a resolution of 60.000 between 800- 3000 m/z. Data were analyzed using Excalibur (Thermo).

Bioorthogonal labeling on beads

HEK293T cells were transfected, cultured in the presence of ncAA for 72 hours and lysed in RIPA buffer with 1x complete protease inhibitor (Roche). The insoluble fraction was removed by centrifugation. Expressed GFP was captured on GFP-Trap_MA magnetic beads (ChromoTEK) and washed with PBS. CuAAC was carried out on aliquots in 1 mM CuS0₄, 1 mM TCEP, 100 mM THPTA and 1 mM AF488-picolyl azide for 1 hour at 24 °C, 450 rpm. For subsequent SPIEDAC 1 mM silicon rhodamine-tetrazine (SiR-Tetrazine, Spirochrome, SC-008) was added and incubated at 4 °C overnight. Excess dye was washed off and proteins were eluted in 1 % (v/v) acetic acid. Equal amounts of GFP from different samples were separated on 4-20% Tris-glycine gels (BioRad) and exposed for in-gel fluorescence at 460 nm and 630 nm in a GE AI600 imager. The gel was stained with InstantBlue (Expedeon) to visualize protein bands.

Live cell Imaging for GFP expression

GFP expressing HEK293T cells were visualized in a ZOE Fluorescent Cell Imager (BioRad).

Labeling of surface receptor proteins on live cells Transfected HEK293T cells grown in the presence of 0.1 mM TCO*K and 0.25 mM ProK for 48h were washed with PBS and labeled with 1.5-10 mM tetrazine dye (AFDye 488 Tetrazine, Click Chemistry Tools) in DM EM + 10 % FBS for 15 min at 37°C. Cells were again washed with PBS and labeled with 10 pM picolyl azide dye (AFDye 647-Picolyl- Azide, Jena Bioscience), in 50 pM CuSCU, 250 pM THPTA in 2.5 mM ascorbic acid for 5 min at 4°C (Hong et al. , 2010). For labeling with the trifunctional linker, cells were washed and incubated with 2 pM tet-Cy5-azide in DM EM + 10 % FBS for 30 min at 37°C. After washing, CuAAC was carried out under the same conditions as above. Cells were collected in cold PBS, spun down and lysed in PBS with 0.1 % (v/v) triton X-100 and 1x complete protease inhibitor (Roche). Aliquots were separated on 4-20% Tris-glycine gels (BioRad) and exposed for in-gel fluorescence at 460 nm and 630 nm in a GE AI600 imager and further analyzed by Western blot.

Synthesis of tet-Cy5-azide trifuntional linker

N-(azide-PEG3)-N'-(PEG4-NHS ester)-Cy5 (N-Cy5-NHS, BroadPharm) was coupled with tetrazine amine (SiChem) at 2:1 molar ratio in PBS at 30 °C for 42 h. A 200 pM stock solutions were prepared in 100 mM Tris-HCI pH 8.0.

SDS-PAGE and Western blot

Aliquots of cell lysate were separated on 4-20 % Tris-glycine gels (BioRad) and transferred to nitrocellulose membranes. Expression of GFP reporter, CRFR1-HA and FLAG-PyIRS was confirmed by immunoblotting with antibodies against GFP (Santa Cruz, sc-9996), HA-HRP (Roche, 12013819001), FLAG-HRP (Sigma, A8592), b-actin (cell signaling, 4970), and corresponding secondary HRP-conjugated antibodies when needed (BioRad).

Live Cell Fluorescence Microscopy

Transfected HEK293T cells were cultured in 96 well plates in the presence of 0.1 mM TCO*K and 0.25 mM ProK for 48 hours. Cells were washed with PBS and cultured in fresh medium for two hours before SPIEDAC labeling with 1 pM Cy3 tetrazine (Click Chemistry Tools, 1204-1) in DMEM + 10 % (v/v) FBS for 30 minutes at 37 °C. After washing, CuAAC was carried out on the cells for 5 minutes at 4 °C or RT, in 50 pM CuS0₄, 250 pM THPTA, 2.5 mM ascorbic acid and 5 pM sulfo-Cy5-picolyl azide (Jena Bioscience, CLK-1177-1) in PBS (Hong et al., 2010). Subsequently, the cells were washed and counterstained with 5 pM Hoechst33342 (Life Technologies) in DMEM + 10 % (v/v) FBS for 30 minutes at 37 °C. Imaging was performed on a Nikon Eclipse Ti2 inverted widefield microscope, using a 20x (0.75 NA) objective and filter sets for DAPI, GFP, Cy3 and Cy5 fluorescence.

Example 1 - G1 PylT/RS activity in mammalian cells

Alignment of Mma PylRS C-terminal, Mx1201 PylRS and G1 PylRS were performed and the performance of each G1 PylRS/PylT pair, Mxl201 PylRS/PylT pair and Mwa PylT/RS pair were tested.

Material and method

Alignment was performed using CLUSTAL OMEGA.

Plasmids expressing Mma, G1 or Mx1201 PylRS were cotransfected in HEK293T cells with a reporter plasmid expressing four copies of PylT variant and the sfGFP^150TAG reporter. Amber suppression in presence of cyclopropene-L-lysine (CpK) was assessed by measuring GFP fluorescence in cell lysates.

Results

The inventors found that G1 PylRS encodes for a 273 amino acid protein with strong homology to the PylRS encoded by Mx1201, and homology to the C-terminal domain of Mma PylRS. PylT encoded by G1 and Mx1201 are also very similar (82.6% sequence identity). G1 PylT and Mx1201 PylT share the small D-arm which sets them apart from other PylTs. Notably, unlike Mx1201 PylT, G1 PylT does not have a “broken” anticodon stem.

The G1 PylRS/PylT pair performed better than the Mx1201 PylRS/PylT pair in mammalian cells and displayed higher efficiency, comparable to the Mma PylT/RS pair. The G1 PylRS enzyme also is highly efficient with the Mx1201 PylT showing that G1 and Mx1201 PylRS/PylT pairs are not orthogonal to each other and G1 PylRS appears to be a more active enzyme than Mx1201 PylRS when combined with the same Mx1201 PylT. (Figure 2, 3).

Mma PylRS/ G1 PylT produces 5% GFP fluorescence compared to Mma PylRS/PylT, it was found that G1 PylT has little amber suppression activity with Mma PylRS in mammalian cells. However, with 5% activity, the G1 PylT is not orthogonal to Mma PylRS. (Figure 2, 3). In summary, the G1 and Mma PylRS/PylT pairs are each efficient in amber suppression, but their respective PylT-PyIRS interaction are different enough to drastically reduce non-cognate interactions. These properties of G1 PylRS/PylT provide a promising starting point for generating a new pair orthogonal to Mma PylRS/PylT.

Example 2 - Generation of a hybrid PylT and a mutant hybrid PylT

In order to generate a new orthogonal G1 PylRS/PylT pair, the inventors needed to abrogate the low but measurable activity (~5%) of G1 PylT with Mma PylRS. The inventors tested the activity and orthogonality of wild type G1 PylT, PylT, a hybrid PylT, a G1 PylT with an extended variable-loop carrying two mutations: A41AA, and C55A mutations (hereafter referred to as G1* PylT, Figure 4A), as well as a hybrid PylT with an extended variable-loop carrying two mutations: A41AA, and C55A mutations (hereafter referred to as hyb* PylT, Figure 4B). Hybrid PylT were obtained by transplantation of acceptor stem between archeal species.

Material and method:

Plasmids expressing Mma or G1 PylRS were cotransfected in HEK293T cells with a reporter plasmid expressing four copies of PylT variant and the sfGFP^150TAG reporter. Amber suppression in presence of cyclopropene-L-lysine (CpK) was assessed by measuring GFP fluorescence in cell lysates.

Results:

The activity of the different PylRS/PylT pairs is shown relative to th eMma PylRS/PylT pair (Figure 5A). As measure for orthogonality the ratio of cognate over non-cognate activity was calculated (Figure 5B).

Extending the variable-loop of G1 PylT did not significantly reduce non-cognate activity with Mma PylT (4.2% versus 5%) (Figure 5A and B).

An additional area for orthogonalization could be the acceptor stem of PylT, which interacts with the PylRS aminoacylation active site. To increase orthogonality, interaction with the non-cognate PylRS should be minimized. The acceptor stem of G1 PylT shares three identical base pairs with Mma PylT, while the more divergent Mxl 201 PylT shares only two (Figure 4B). Thus the inventors transplanted th eMxl201 acceptor stem onto G1 PylT, creating a hybrid PylT (hyb PylT) see Figure 4B. The hybrid maintained high activity with G1 PylRS, but had almost no activity with Mma PylRS, leading to a fourfold increase in specificity (Figure 5A and B).

Maximal orthogonality, a 10-fold increase in specificity, was achieved by combining the acceptor stem hybrid with the variable- loop/C55A double mutation (hyb* PylT, Figure 5A and B). Thus, the mutated hybrid PylT (hyb*PylT) is selectively active with G1 PylRS. Consequently, G1 PylRS/hyb*PylT and Mma PylRS/PylT are mutually orthogonal pairs.

Example 3 - Exclusive recognition of ncAA by the G1 PylRS

Example 2 showed that the engineered G1 hyb*PylT/RS pair, orthogonal to Mma PylT/RS and the host cell with regard to tRNA-aaRS interaction, provided a good starting point for efficient and selective dual ncAA incorporation. The next challenge represented the identification of a pair of ncAAs that would each be exclusively recognized by one of the PylRS enzymes only, and encode orthogonal chemical reactivity for site-specific dual labeling.

The inventors sought to explore the potential combination of combining one strain- promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reactivity with one Cu(l)-catalyzed azide-alkyne cycloaddition (CuAAC) reactivity, which do not interfere with each other.

The inventors tested substrate specificity of wildtype Mma PylRS, wildtype G1 PylRS and mutant G1 PylRS^Y125A.

Material and methods:

HEK293T cells were transiently transfected with either of the three PylT/RS pairs: Mma PylT/RS, G1 PylT/RS and G1 PylT/RS^Y125A together with a GFP^150TAG reporter plasmid carrying additional four tandem repeats of the respective PylT and tested for incorporation of different ncAAs. Hereby testing the incorporation of CpK, BCNK, TCO*K, N-Propargyl-L-lysine (ProK) and N-£-((2-Azidoethoxy)carbonyl)-L-lysine (AzeoK) (Figure 6A-C). Results:

Mma PylT incorporated AzeoK, CpK, ProK with high efficiency, but neither BCNK or TCO*K (Figure 6A). G1 PylRS incorporated CpK with high efficiency, AzeoK, ProK with low efficiency, and neither BCNK or TCO*K (Figure 6B). G1 PylRS^Y125A efficiently incorporated CpK, BCNK, TCO*K. While it also incorporated AzeoK, it was very inefficient with ProK (Figure 6C).

The selectivity of Mma PylT/RS for ProK and G1 PylRS^Y125A for TCO*K was further confirmed by western blot (Figure 7).

These experiments show that TCO*K and ProK can be incorporated as orthogonal ncAA with G1 PylT/RS^Y125A and Mma PylT/RS, respectively, thus allowing dual stop suppression in mammalian cells.

Example 4 - Recoding of PylT stop codon recognition

To allow for site-specific incorporation of the two ncAAs TCO*K and ProK, the inventors next altered one of the PylTs to recode a different stop codon.

Ochre codon suppression proved less efficient than opal suppression, but ochre codons make up a quarter of stop codons in the human genome, while half of the stop codons are opal. Thus, least interference with endogenous codons is expected with an amber/ochre combination.

As the efficiency of recoding of two distinct stop codons in a target protein will be rate- limited by the less efficient suppressor, the efficiency of ochre suppression with Mma PylT was sought to be improved.

Material and method:

The inventors introduced 6 mutations into PylT, generating the variant M 15 PylT which have a high steady state level in the cell (Serfling et. al. , 2018, doi 10.1093/nar/ gkx2018).

The inventors first generated plasmids with a single copy of amber or ochre suppressor M15 PylT with h7SK promoter (M15^CUA and M15^UUA, respectively). In combination with Mma PylRS and the GFP^150TAG reporter the inventors tested their nonsense suppression efficiency.

The PylT/RS pair Mma M15PylT^UUA/RS was combined with the PylT/RS pair G1 hyb* PylT/RS^Y125A along with a GFP reporter in a three-plasmid system carrying 4xPylT arrays for both suppressors. The GFP gene contained a TAG codon at position 102 and TAA at position 150. To establish dual ncAA incorporation and orthogonal fluorescent labeling, GFP was first produces containing either TCO*K or ProK in position 102 or 150, respectively, see Figure 8. Both suppressors were combined to incorporate TCO*K and ProK into GFP^102TAGi50TAA (Figure 8, 9 and 10).

Results

First, near-optimal suppression by wildtype PylT^CUA could not be further enhanced with M15^CUA. M15^UUA was ~ 2-fold more active than its wildtype PylT^UUA counterpart, confirming the activity enhancement conferred by the M15 variant for Mma PylT.

Single incorporation of ProK by Mma M15PylT^UUA /RS and TCO*K by G1 hyb* PylT/RS^Y125A and dual incorporation by intact mass spectrometry was confirmed Figure 8. Further analysis of proteolytic fragments by MS/MS confirmed the respective sites of TCO*K and ProK to be 102 and 150. Each stop codon was only suppressed by the respective cognate PylT and full-length GFP was only produced in the presence of both ncAAs Figure 9. ProK and TCO*K could be selective labeled on purified GFP with 488- picolyl azide by CuAAC and SiR-tetrazine via SPIEDAC, respectively. Figure 10.

Example 5 - Dual labelling on live cells

The efficient dual labeling strategy was next applied on live cells. Dual color fluorescent labeling and imaging using two organic dyes provides potential for a variety of exciting applications including single molecule tracking and FRET to study the trafficking, processing, conformational heterogeneity of surface receptors.

CuAAC and SPIEDAC can be performed on the surface of live cells (Hong et al. , 2010; Nikic et al., 2014). While fluorescent labeling schemes using a combination of ncAAs and other covalent or non-covalent fluorescent tags have been achieved on purified transmembrane receptor fragments, dual ncAA incorporation and fluorescent labeling has not been achieved on live cells. The inventors combined dual ncAA incorporation with CuAAC/SPIEDAC dual fluorescent labeling for two transmembrane proteins, Notch receptor and a G-protein coupled receptor (GPCR).

Notch receptor is a cell surface receptor with well-documented functions in the developing embryo and the nervous system. Notch receptor undergoes a constitutive proteolytic processing step after folding in the ER by a furin-like protease (S1 site), separating a large N-terminal extracellular receptor fragment from the C-terminal transmembrane domain Figure 6A. Resulting fragments remain non-covalently linked and are resistant to further proteolysis because a signal-sensitive cleavage site, S2, is maintained in an autoinhibited conformation until the Notch receptor is activated by a ligand (such as Delta, Serrate or Lag2). The negative regulatory region (NRR) with the buried S2 site consists of a tight fold of three Lin-12/Notch Repeats (LNR) and the juxtamembrane heterodimerization domain (HD). Receptor activation is thought to involve a mechanic force-dependent dissociation of the LNRs from the HD, exposing S2. S2 cleavage triggers subsequent proteolysis of an intermembrane site, releasing the intracellular signaling domain.

Material and methods

For this example SynNotch was chosen, it is a synthetic minimal Notch receptor, where the EGF repeats of human Notchl are replaced with a nanobody. Amber and ochre nonsense codons replaced Leu1465 and Asn1703 (numbered according to original Notchl gene; 204 and 442 in the SynNotch construct). The residues are placed in the first LNR and juxtamembrane HD fragment.

Mma M15PylT^UUA/RS and G1 hyb* PylT/RS^Y125Awere expressed together with SynNotch^{204TAA 442TAG} or SynNotch^{204TAG 442TAA}to express SynNotch on the cell surface with ProK and TCO*K in position 204 and 442, or vice versa. Subsequently, SPIEDAC and CuAAC were sequentially performed with two orthogonal fluorescent dyes on live cells. As a control, the reactions were also performed on untransfected cells.

Results

Analysis of the live-cell labeled receptor in an SDS-PAGE showed that SynNotch receptor was correctly processed into a 43 kDa N-terminal and 11kDa C-terminal fragment at the constitutive S1 cleavage site, Figure 11. For visualization on an SDS- PAGE, ProK was labeled with A647-picolylazide and TCO*K with A488-tetrazine. The label at position 204 tracked with the 43kDa fragment while the label at 442 was on the 11 kDa fragmen, Figure 11. An additional long fragment of ca 30kDa was observed corresponding to a N-terminal truncated primary product either by proteolytic cleavage or secondary translation initiation within the nanobody, Figure 11. We used a Furin/Proprotein Convertase inhibitor to partially inhibit S1 cleavage, as evident from a full-length 54kDa SynNoth fragment now labeled with both dyes, Figure 11. Dual labeling was equally efficient with both possible configurations of stop codon, ncAA and fluorescent dye, Figure 11.

Live cell imaging of SynNotch was performed after dual fluorescent labeling using Cy5- picolylacide and Cy3-tetrazine, Figure 12A. Mma M15PylT^UUA/RS and G1 hyb* PylT/RS^Y125Awere expressed together with SynNotch^{204TAA 442TAG}to produce SynNotch with ProK and TCO*K in position 204 and 442. Subsequently, SPIEDAC and CuAAC were sequentially performed with two orthogonal fluorescent dyes on live cells.

Dual labeling was only observed after SynNotch expression in the presence of both ncAAs, Figure 12A. Selectivity and orthogonality of labeling reactions were also confirmed on fixed cells.

Dual ncAA incorporation not only provides ability of dual color fluorescent labeling, but other exciting uses of orthogonal chemistries on the living cell. Here, we exploit the two ncAAs and selective control of SPIEDAC and CuAAC reactions to introduce a trifunctional linker molecule for fluorescent labeling and chemically-controlled crosslinking of the SynNotch fragments. We synthesized a trifunctional terazine-Cy5- azide and reacted it with SynNotch bearing ProK and TCO*K in either configuration, Figure 12B. In the absence of Cu(l) this lead to a fluorescent labeling through SPIEDAC of either only the large or only the small SynNotch S1 cleavage product, depending on the position of TCO*K. Because we have introduced the ncAA in close spatial proximity (<15A) to each other, we could subsequently use the ProK reactivity in a Cu(l) dependent manner to generate a precise intermolecular crosslink between the SynNotch fragments Figure 12B. In summary, the inventors demonstrate that orthogonal chemistries on two site- specifically incorporated ncAA can be exploited for fluorescent labeling and chemically controlled crosslinking.

Example 6 - Dual labeling of CRF1R

To further evaluate the present invention, the inventors sought to introduce dual ncAA into a second transmembrane receptor. The class B GPCR corticotropin releasing factor type 1 receptor (CRF1 R). CRFI R has an extracellular domain serving as a receptor for the peptide hormone CRF1 and a 7-transmembrane domain involved in signal transduction to the intracellular side. CRF1 R has been studied using a variety of genetic code expansion techniques.

The present invention could be applied to allow efficient dual fluorescent labeling of CRF1 R.

Material and methods

Positions 95 and 263 were used for ncAA and are located in the extracellular domain and extracellular loop 2, respectively.

CRF1 R ^{95TAA 263TAG} _was combined with a C-terminal HA tag into a plasmid with four copies of Mma M15PylT^UUA and four copies G1 hyb* PylT. HEK293T cells were cotransfected with Mma M15PylT^UUA /RS and G1 hyb* PylT/RS^Y125A expression plasmids in presence of TCO*K and ProK, Figure 13 and 14. For analysis on an SDS- PAGE, we labeled the receptor on live cells using either A488-tetrazine and A647- azide or both, Figure 13.

Results

Labeling was always highly specific for CRF1R, and dual fluorescent labeling was as efficient as the individual reactions, Figure 13. We also varied the order of stop codons for dual labeling with the same result, Figure 13. Efficient labeling for live cell microscopy was showed by SDS-PAGE, Figure 13. CRFR1 ^{95TAA 263TAG} _Was expressed in the presence of either or both ProK and TCO*K, and subsequently labeled using Cy5-picolylazide and Cy3-tetrazine, Figure 14. Fluorescent labeling was only observed when full-length CRF1R was expressed in the presence of both ProK and TCO*K, Figure 14. Example 7 - Additional PylT hybrid

The inventors sought to explore additional hybrid PylTs generating hybrid PylT 2 (H5- G1, hyb2) and hybrid PylT 3 (MpT1-G1, hyb3) by transplanting the acceptor stem of Methanogenic archeon IS04H5 or Candidatus Methanoplasma termitum MpT1, respectively, onto the body of G1 PylT. The relevant acceptor stem sequences are given in Figure 16A and a scheme of the hybrid PylTs are given in Figure 16B. Activity of hybrid 2 and hybrid 3 were tested with Mma PylRS and G1 PylRS.

Material and method:

The inventors first generated plasmids with a single copy of amber or ochre suppressor hyb2 or hyb3 PylT with h7SK promoter (hyb2 and hyb3, respectively). The inventors tested cognate activity of hyb2 and hyb3 relative to the original G1 PylT in combination with G1 PylRS and the GFP^150TAG. (Figure 16C, D) The inventors further tested non cognate activity of hyb2 and hyb3 relative to Mma PylT in combination with Mma PylRS and the GFP^150TAG .(Figure 16 C, E). To this end, HEK293T cells were cotransfected with hybrid single copy plasmids, a sfGFP150TAG plasmid and either a Mma or G1 PylRS expression plasmid in absence of presence of 0.2 mM CpK. 48h after transfection, cells were imaged with a fluorescent microscope for GFP fluorescence (Figure 16C). Cells were lysed and GFP fluorescence in lysate was determined in a plate reader assay (Figure 16D, E).

Results

Hybrid 2 and hybrid 3 showed similarly high activity with G1 PylRS and the same reduced activity with Mma PylRS observed for the Mx1201/G1 hybrid in Example 2, Figure 16. Thus, transplanting the acceptor stem of Candidatus Methanomethylophilus alvus Mx1201, Methanogenic archeon IS04H5 or Candidatus Methanoplasma termitum MpT 1 onto a PylT body of Methanogenic archaeon IS04-G1 generated three hybrids retaining activity with G1 PylRS while exhibiting no-cross reactivity with Mma PylRS.

Items

1. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most one nucleotide of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most one nucleotide of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is native to a first species and the PylT body is native to a second species, wherein the first species is different form the second species and the acceptor stem native to the first species is different from the acceptor stem native to the second species, and the body native to the first species is different from the body native to the second species wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is native to a first species and the PylT body is native to a second species, wherein the first species is different form the second species and the acceptor stem native to the first species is different from the acceptor stem native to the second species, and the body native to the first species is different from the body native to the second species wherein at the most one nucleotide of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species. A hybrid pyrrolysyl tRNA (PylT), wherein PylT consists of a nucleotide sequence having the general formula

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together form a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is native to a first species and the PylT body is native to a second species, wherein the first species is different form the second species and the acceptor stem native to the first species is different from the acceptor stem native to the second species. The hybrid PylT according to any one of the preceding items, wherein a. C consists of a nucleotide sequence having the general formula

C₂ consists of in the range of 0 to 5 nucleotides; and b. A consists of a consecutive sequence of nucleotides which are complementary to Ci. The hybrid PylT according to any one of the preceding items, wherein the A comprises or consists of seven 5’-terminal nucleotides and/or C comprises or consists of eleven 3’-terminal nucleotides. The hybrid PylT according to any one of the preceding items, wherein the first and second species are species of bacteria or archea. The hybrid PylT according to any one of the preceding items, wherein the first and second species are archea. The hybrid PylT according to any one of the preceding items, wherein the first species is selected from the group consisting of Candidatus Methanomethylophilus alvus Mx1201, Candidatus Methanomethylophilus sp.1R26, Candidatus Methanomethylophilus sp. UBA78, Candiatus Methanomethylophilaceae and Methanomassiliicoccales. The hybrid PylT according to any one of the preceding items, wherein the first species is selected from the group consisting of Candidatus Methanomethylophilus alvus Mx1201, Candidatus Methanomethylophilus sp.1R26, Candidatus Methanomethylophilus sp. UBA78, Candiatus Methanomethylophilaceae, Methanomassiliicoccales, Methanogenic archaeon ISO-H5 and Candidatus Methanoplasma termitum MpT1. The hybrid PylT according to any one of the preceding items, wherein the first species is Candidatus Methanomethylophilus alvus Mx1201. The hybrid PylT according to any one of the preceding items, wherein the first species is Methanogenic archaeon ISO-H5. The hybrid PylT according to any one of the preceding items, wherein the first species is Candidatus Methanoplasma termitum MpT1. The hybrid PylT according to any one of the preceding items, wherein the second species is of the genus Methanomicrobiales. The hybrid PylT according to any one of the preceding items, wherein the second species is a Methanogenic archaeon IS04-G1. The hybrid PylT according to any one of the preceding items, wherein A comprises or consists of a sequence of nucleotides of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. The hybrid PylT according to any one of the preceding items, wherein C comprises or consists of a sequence of nucleotides of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19. The hybrid PylT according to any one of the preceding items, wherein A comprises or consists of a sequence of nucleotides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:13. The hybrid PylT according to any one of the preceding items, wherein C comprises or consists of a sequence of nucleotides of SEQ ID NO: 14, SEQ ID NO:15 or SEQ ID NO:19. The hybrid PylT according to any one of the preceding items, wherein B comprises an anticodon selected from the group of CUA, UCA and UUA. The hybrid PylT according to any one of the preceding items, wherein B comprises a CUA anticodon. The hybrid PylT according to any one of the preceding items, wherein B comprises or consists of a sequence of nucleotides of from position 6 to 60 of SEQ ID NO:1 or comprise or consists of a sequence of nucleotides from position 6 to 61 of SEQ ID NO:2. The hybrid PylT according to any one of the preceding items, wherein the hybrid PylT comprises or consists of a sequence of nucleotides of SEQ ID NO:1 or SEQ ID NO:2. The hybrid PylT according to any one of the preceding items, wherein the hybrid PylT comprises a substitution of C to A at position 55 of SEQ ID NO:1. The hybrid PylT according to any one of the preceding items, wherein the hybrid PylT comprises an addition of A to AA at position 41 of SEQ ID NO:1. A complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl- tRNA-synthetase (PylRS) (PylT/PyIRS), wherein the hybrid PylT is as defined in any one of the preceding items and wherein the first PylRS may be any PylRS which is active to said hybrid PylT. The complex according to item 30, wherein said first PylRS is of the same species as the hybrid PylT body, B. The complex according to any one of items 30-31, wherein said first PylRS is archea. The complex according to any one of items 30-32, wherein said first PylRS is selected from Methanomethylophilus alvus Mx1201 or Methanogenic archaeon IS04-G1. The complex according to any one of items 30-33, wherein said first PylRS is Methanogenic archaeon IS04-G1. The complex according to any one of items 30-34, wherein said first PylRS comprises or consists of an amino acid sequence of SEQ ID NO:22 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% homology thereto. The complex according to any one of items 30-35, wherein said first PylRS comprises or consists of an amino acid sequence of SEQ ID NO:22 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto. The complex according to any one of items 30-36, wherein said first PylRS comprises or consists of an amino acid sequence of SEQ ID NO:26 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto. A pair of PylT/PyIRS complexes, wherein said pair comprises a first hybrid PylT/PyIRS complex according to any one of items 30 to 35, and a second PylT/PyIRS complex, wherein said second complex is mutually orthogonal to the first PylT/PyIRS complex. The pair according to item 38, wherein said second PylT/PyIRS complex is archea. The pair according to any one of items 38-39, wherein said second PylT/PyIRS complex is Methanosarcina mazei. The pair according to any one of items 38-40, wherein said second PylT comprises or consists of a nucleotide sequence of SEQ ID NO: 30. The pair according to any one of items 38-41 , wherein said second PylRS comprises or consists of an amino acid sequence of SEQ ID NO:24 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% homology thereto. The pair according to any one of items 38-42, wherein said second PylRS comprises or consists of an amino acid sequence of SEQ ID NO:24 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto. The pair according to any one of items 38-43, wherein B of said second PylT comprises an UUA anticodon. The pair according to any one of items 38-44, wherein said first PylRS recognizes a first non-canonical amino acid and said second PylRS recognizes a second non-canonical amino acid, wherein the first non-canonical amino acid is different from the second non-canonical amino acid. A host cell expressing a hybrid PylT according to any one of items 1 to 29, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA- synthetase (PylRS) according to any one of items 30 to 37, and/or a pair of PylT/PyIRS complexes according to any one of items 38 to 45. A method of producing a protein using a hybrid PylT according to any one of items 1 to 29, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA-synthetase (PylRS) according to any one of items 30 to 37 and/or a pair of PylT/PyIRS complexes according to any one of items 38 to 45. The method according to item 47, wherein the method comprises the steps of providing a host cell expressing a hybrid PylT according to any one of items 1 to 29, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl- tRNA-synthetase (PylRS) according to any one of items 30 to 37 and/or a pair of PylT/PyIRS complexes according to any one of items 38 to 45; culturing the host cell under conditions allowing the host cell to produce the protein; and optionally lysing the cell and recover and/or isolate the protein. The method according to item 47, wherein the method comprises the steps of providing a cell-free expression system; contacting said cell-free expression system with a hybrid PylT according to any one of items 1 to 29, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA-synthetase (PylRS) according to any one of items 30 to 37 and/or a pair of PylT/PyIRS complexes according to any one of items 38 to 45; and optionally recover and/or isolate the protein The method according to item 49, wherein the cell-free expression system is a lysate and/or wheat germ extract. The method according to item 50, wherein the lysate is an E. coli lysate and/or a rabbit reticulocyte lysate. Sequences

SEQ ID NO: 1 Hybrid PylT G1-Mx1201

5’-GGGGGACGGUCCGGCGAGCAAACGGGUCUCUAAAACCUGUAAGCGGGGUU

CGACCCCCCGGUCUCUCGCCA-3’

SEQ ID NO: 2

Mutated hybrid PylT G1-Mx1201

5’- GGGGGACGGUCCGGCGAGCAAACGGGUCUCUAAAACCUGUAAAGCGGGGU UCGACACCCCGGUCUCUCGCCA-3’

The underlined nucleotides correspond to the nucleotides which are mutated according to the present invention.

SEQ ID NO: 3

Acceptor stem A of Methanosarcina horonobensis HB1; Methanosalsum zhilinae DSM 4017; Methanohalophilus halophilus Z7982; Methanosarcina vacuolata Z761; Methanosarcina barkeri 3; Methanosarcina mazei WWM610; Methanosarcina siciliae T4; Methanosarcina thermophila TM1; Methanohalobium evestigatum Z7303; Methanohalophilus portucalensis FDF1T; Methanomethylovorans hollandica DSM 15978; Methanohalophilus mahii DSM 5219 5’-GGAAACC-3’

SEQ ID NO:4

Acceptor stem A of Methanosarcina barkeri MS; Methanosarcina barkeri CM1 5’-GGGAACC-3’

SEQ ID NO:5

Acceptor stem A of Candidatus Methanomethylophilus alvus Mx1201;

Thermoplasmatales archaeon BRNA 1

5’-GGGGGAC-3’

SEQ ID NO:6 Acceptor stem A of Methanogenic archaeon IS04H5 chromosome 5’-GGGGGGC-3’

SEQ ID NO:7

Acceptor stem A of Candidatus Methanoplasma termitum MpT1 5’- GGGAGAC-3’

SEQ ID NO:8

Acceptor stem A of Methanosarcina lacustris Z7289; Methanolobus psychrophilus R15 5’- GGAAAUC -3’

SEQ ID NO:9

Acceptor stem A of Methanococcoides methylutens MM1; Methanococcoides burtonii

DSM 6242

5’- GGAGACU -3’

SEQ ID NO:10

Acceptor stem A of Desulfitobacterium dehalogenans; Desulfosporosinus orientis DSM 765; Thermacetogenium phaeum; Desulfosporosinus meridiei; Desulfitobacterium hafniense; Dehalobacterium formicoaceticum 5’- GGGGGGU-3’

SEQ ID NO: 11

Acceptor stem A of Eubacterium limosum 5’- GGAGGGU-3’

SEQ ID NO 12:

Acceptor stem A of Methanogenic archaeon IS04-G1 5’- GGAGGGC-3’

SEQ ID NO:13

Acceptor stem C of Methanosarcina horonobensis HB1; Methanosalsum zhilinae DSM 4017; Methanosarcina barkeri MS; Methanohalophilus halophilus Z7982; Methanosarcina vacuolata Z761; Methanosarcina barkeri 3; Methanosarcina mazei WWM610; Methanosarcina siciliae T4; Methanosarcina thermophila TM1; Methanosarcina lacustris Z7289; Methanosarcina barkeri CM1; Methanohalobium evestigatum Z7303; Methanohalophilus portucalensis FDF1T; Methanomethylovorans hollandica DSM 15978; Methanohalophilus mahii DSM 5219; Methanolobus psychrophilus R15 5’-GGUUUCCGCCA-3’

SEQ ID N0:14

Acceptor stem C of Candidatus Methanomethylophilus alvus Mx1201; Thermoplasmatales archaeon BRNA1; Candidatus Methanoplasma termitum MpT1 5’-GUCUCUCGCCA-3’

SEQ ID NO:15

Acceptor stem C of Methanogenic archaeon IS04H5 chromosome 5’-GCCUCUCGCCA -3’

SEQ ID NO:16

Acceptor stem C of Methanococcoides methylutens MM1; Methanococcoides burtonii DSM 6242

5’-AGUUUCCGCCA-3’

SEQ ID NO:17

Acceptor stem C of Desulfitobacterium dehalogenans; Desulfosporosinus orientis DSM 765; Desulfosporosinus meridiei; Thermacetogenium phaeum; Desulfitobacterium hafniense; Dehalobacterium formicoaceticum 5’-ACUCCCCGCCA-3’

SEQ ID NO:18

Acceptor stem C of Eubacterium limosum 5’-ACUCUCCACCA-3’

SEQ ID NO:19

Acceptor stem C of Methanogenic archaeon IS04-G1 5’- GCCUUUCGCCA-3’

SEQ ID NO:20 Amino acid sequence of pyrrolysyl-tRNA-synthetase of Candidatus Methanomethylophilus alvus Mx1201

MTVKYTDAQIQRLREYGNGTYEQKVFEDLASRDAAFSKEMSVASTDNEKKIKGMI ANPSRHGLTQLMNDIADALVAEGFIEVRTPIFISKDALARMTITEDKPLFKQVFWIDEKR ALRPMLAPNLYSVMRDLRDHTDGPVKIFEMGSCFRKESHSGMHLEEFTMLNLVDMG PRGDATEVLKNYISVVM KAAGLPDYDLVQEESDVYKETIDVEINGQEVCSAAVGPHYL DAAHDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKIN

SEQ ID NO:21

Polynucleotide sequence of pyrrolysyl-tRNA-synthetase of Candidatus Methanomethylophilus alvus Mx1201

AT GACGGT AAAGT AT ACGGATGCACAGAT ACAGCGCCTCAGGGAAT ACGGCAAC

GGGACCTATGAGCAGAAGGTCTTCGAGGACCTCGCATCGAGGGATGCTGCCTTC

TCCAAGGAGATGTCCGTCGCCTCTACCGACAACGAGAAGAAGATCAAGGGGATG

ATCGCCAATCCGTCCCGTCATGGATTGACCCAGCTGATGAACGACATCGCCGACG

CATTGGTCGCCGAGGGTTTCATCGAGGTCCGTACGCCCATATTCATATCGAAGGA

TGCGCTGGCACGTATGACCATCACCGAGGACAAGCCCCTTTTCAAGCAGGTCTTC

TGGATCGACGAGAAAAGGGCGCTCAGGCCT AT GCT GGCACCT AACCTTT ATTCCG

TCAT GAGGGACCT GAGGGACCAT ACGGACGGTCCGGT GAAGATCTTCGAGAT GG

GTTCCTGCTTCAGGAAGGAGTCCCACAGCGGGATGCATCTGGAGGAGTTCACCA

TGCTGAACCTCGTGGACATGGGTCCCCGCGGAGACGCCACGGAGGTCCTGAAGA

ACTACATATCGGTCGTGATGAAGGCGGCCGGTCTCCCGGACTACGACCTCGTAC

AGGAGGAGTCCGACGTATACAAGGAGACCATAGACGTGGAGATCAACGGTCAGG

AGGTCTGTTCCGCAGCCGTCGGTCCACACTATCTCGATGCGGCCCACGATGTCC

ACGAGCCTTGGTCCGGAGCGGGATTCGGTCTCGAACGCCTGCTGACCATCAGGG

AGAAGT ACAGCACCGT GAAGAAGGGAGGAGCCAGCATCAGCT ACCTCAACGGT G

CG AAG AT CAACT G A

SEQ ID NO:22

Amino acid sequence of pyrrolysyl-tRNA-synthetase of Methanogenic archaeon IS04- G1

MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVI

ANPRNDLTDLENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKR ALRPMHAMNLYKVMRELRDHTKGPVKIFEIGSCFRKESKSSTHLEEFTMLNLVEMGP

DGDPMEHLKMYIGDIMDAVGVEYTTSREESDVYVETLDVEINGTEVASGAVGPHKLD

PAHDVHEPWAGIGFGLERLLMLKNGKSNARKTGKSITYLNGYKLD

SEQ ID NO:23

DNA sequence of pyrrolysyl-tRNA-synthetase of Methanogenic archaeon IS04-G1

AT GGTT GTCAAGTTCACCGATTCCCAGATCCAGCACCT AATGGAGT ATGGGGACA

ATGATTGGTCGGAGGCAGAGTTCGAGGATGCCGCCGCCAGGGATAAGGAATTCT

CAT CAC AGTT CTCCAAACT G AAATCCGCC AACG AT AAGGGCCTCAAGGAT GTCAT

CGCCAACCCGCGCAACGACCTCACCGACCTGGAGAACAAGATCAGGGAGAAGCT

TGCAGCCAGGGGTTTCATTGAAGTTCACACGCCCATATTCGTTTCAAAGAGCGCC

CTCGCCAAGATGACGATCACCGAGGACCACCCGCTGTTCAAGCAGGTGTTCTGG

ATCGACGACAAGCGTGCGCTCAGGCCCAT GCACGCAAT GAACCT GT ACAAGGTC

ATGAGGGAGCTCAGGGACCACACGAAGGGTCCGGTCAAGATCTTCGAGATCGGC

TCCTGCTTCAGGAAGGAGTCCAAGAGCTCCACCCACCTCGAGGAGTTCACGATG

CTGAACCTGGTGGAGATGGGCCCCGACGGGGATCCGATGGAGCATCTGAAGATG

TACATCGGGGACATCATGGACGCCGTCGGCGTCGAATATACCACATCCCGCGAG

GAGTCCGACGTGTACGTCGAGACCCTCGACGTGGAGATCAACGGTACGGAGGTC

GCTTCCGGAGCGGTCGGACCTCACAAGCTGGATCCGGCACACGACGTCCACGAG

CCGTGGGCAGGGATCGGATTCGGACTGGAGCGCCTGCTCATGCTGAAGAACGGC

AAAAGCAACGCCAGGAAGACAGGCAAGAGCATCACGTATCTGAACGGATACAAAC

TGGACTGA

SEQ ID NO:24

Amino acid sequence of pyrrolysyl-tRNA-synthetase of Methanosarcina mazei

MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARA

LRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAP

KPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNP

ITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQI

YAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNF

CLRPMLAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSG

CTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWG

IDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL

SEQ ID NO:25 DNA sequence of pyrrolysyl-tRNA-synthetase of Methanosarcina mazei AT GG AT AAAAAACCACT AAAC ACT CT GAT AT CTGCAACCGGGCT CTGG AT GTCCA GGACCGG AACAATT CAT AAAAT AAAACACCACGAAGT CT CTCGAAGCAAAATCT AT ATT GAAATGGCATGCGGAGACCACCTT GTT GT AAACAACTCCAGGAGCAGCAGGA CTGCAAGAGCGCTCAGGCACCACAAATACAGGAAGACCTGCAAACGCTGCAGGG TTTCGG AT GAGG AT CT C AAT AAGTTCCT CAC AAAGGCAAACGAAGACCAGACAAG CGTAAAAGTCAAGGTCGTTTCTGCCCCTACCAGAACGAAAAAGGCAATGCCAAAA TCCGTTGCGAGAGCCCCGAAACCTCTTGAGAATACAGAAGCGGCACAGGCTCAA CCTT CTGG AT CT AAATTTT CACCTGCG AT ACCGGTTTCCACCCAAG AGTCAGTTT C T GTCCCGGCAT CT GTTT CAACAT CAAT AT C AAGCATTT CT ACAGG AGCAACT GCAT CCGCACT GGT AAAAGGGAAT ACGAACCCCATT ACATCCAT GTCTGCCCCT GTTCA GGCAAGTGCCCCCGCACTT ACGAAGAGCCAGACT GACAGGCTT GAAGTCCT GTT AAACCCAAAAGATGAGATTTCCCTGAATTCCGGCAAGCCTTTCAGGGAGCTTGAG TCCGAATTGCTCTCTCGCAGAAAAAAAGACCTGCAGCAGATCTACGCGGAAGAAA GGGAGAATT ATCTGGGGAAACTCGAGCGT GAAATT ACCAGGTTCTTT GT GGACAG GGGTTTT CTGG AAAT AAAATCCCCG ATCCT GATCCCT CTT G AGT AT ATCG AAAGGA TGGGCATT GAT AAT GAT ACCG AACTTT C AAAACAGAT CTT CAGGGTT GACAAG AAC TTCTGCCT GAGACCCATGCTTGCTCCAAACCTTT ACAACT ACCTGCGCAAGCTT GA CAGGGCCCTGCCT GATCCAAT AAAAATTTTT GAAAT AGGCCCATGCT ACAGAAAAG AGTCCGACGGCAAAGAACACCTCGAAGAGTTTACCATGCTGAACTTCTGCCAGAT GGGATCGGGATGCACACGGGAAAATCTT GAAAGCAT AATT ACAGACTTCCT GAAC CACCTGGGAATT GATTTCAAGATCGT AGGCGATTCCTGCATGGTCT ATGGGGAT A CCCTT GAT GT AATGCACGGAGACCTGGAACTTTCCTCTGCAGT AGTCGGACCCAT ACCGCTTGACCGGGAATGGGGTATTGATAAACCCTGGATAGGGGCAGGTTTCGG GCTCG AACGCCTT CT CAAGGTT AAACACG ACTTT AAAAAT AT CAAG AGAGCT GC AA GGTCCGAGTCTT ACT AT AACGGGATTTCT ACCAACCT GT AA

SEQ ID NO:26

Amino acid sequence of pyrrolysyl-tRNA-synthetase of Methanogenic archaeon IS04- G1, Y125A mutant

MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIAN

PRNDLTDLENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALR

PMHAMNLAKVMRELRDHTKGPVKIFEIGSCFRKESKSSTHLEEFTMLNLVEMGPDGD

PMEHLKMYIGDIMDAVGVEYTTSREESDVYVETLDVEINGTEVASGAVGPHKLDPAHD

VHEPWAGIGFGLERLLMLKNGKSNARKTGKSITYLNGYKLD SEQ ID NO: 27

DNA sequence of pyrrolysyl-tRNA-synthetase of Methanosarcina mazei, Y125A mutant

AT GGTT GTCAAGTTCACCGATTCCCAGATCCAGCACCT AATGGAGT ATGGGGACA

ATGATTGGTCGGAGGCAGAGTTCGAGGATGCCGCCGCCAGGGATAAGGAATTCT

CAT CACAGTT CTCCAAACT GAAATCCGCCAACG AT AAGGGCCT CAAGG AT GT CAT

CGCCAACCCGCGCAACGACCTCACCGACCTGGAGAACAAGATCAGGGAGAAGCT

TGCAGCCAGGGGTTTCATTGAAGTTCACACGCCCATATTCGTTTCAAAGAGCGCC

CTCGCCAAGATGACGATCACCGAGGACCACCCGCTGTTCAAGCAGGTGTTCTGG

ATCGACGACAAGCGTGCGCTCAGGCCCATGCACGCAATGAACCTGgccAAGGTCA

TGAGGGAGCTCAGGGACCACACGAAGGGTCCGGTCAAGATCTTCGAGATCGGCT

CCTGCTTCAGGAAGGAGTCCAAGAGCTCCACCCACCTCGAGGAGTTCACGATGC

TGAACCTGGTGGAGATGGGCCCCGACGGGGATCCGATGGAGCATCTGAAGATGT

ACATCGGGGACATCATGGACGCCGTCGGCGTCGAATATACCACATCCCGCGAGG

AGTCCGACGTGTACGTCGAGACCCTCGACGTGGAGATCAACGGTACGGAGGTCG

CTTCCGGAGCGGTCGGACCTCACAAGCTGGATCCGGCACACGACGTCCACGAGC

CGTGGGCAGGGATCGGATTCGGACTGGAGCGCCTGCTCATGCTGAAGAACGGCA

AAAGCAACGCCAGGAAGACAGGCAAGAGCATCACGTATCTGAACGGATACAAACT

GGACTGA

SEC ID NO: 28 Hybrid PylT G1-ISO-H5

5’- GGGGGGCGCUCCGGCGAGCAAACGGGUCUCUAAAACCUGUAAGCGGGG UUCGACCCCCCGGCCUCUCGCCA -3’

SEC ID NO: 29 Hybrid PylT G1-MpT1

5’- GGGAGACGCUCCGGCGAGCAAACGGGUCUCUAAAACCUGUAAGCGGGGUUC GACCCCCCGGUCUCUCGCCA-3’

SEQ ID NO: 30 Mma PylT

5’- GGAAACCUGAUCAUGUAGAUCGAACGGACUCUAAAUCCGUUCAGCCGGGU UAGAUUCCCGGGGUUUCCGCCA-3’ References

Das, D.K., Govindan, R., Nikic-Spiegel, I., Krammer, F., Lemke, E.A., and Munro, J.B. (2018). Direct visualization of the conformational dynamics of single influenza hemagglutinin trimers. Cell 174 , 926-937. e12.

Dumas, A., Lercher, L, Spicer C. D., and Davis, B. G. (2015). “Designing logical codon reassignment - Expanding the chemistry in biology“,doi:10.1039/C4SC01534G

Gust , A., Jakob, L., Zeitler, D.M., Bruckmann, A., Kramm, K., Willkomm, S., Tinnefeld, P., Meister, G., and Grohmann, D. (2018). Site-Specific Labelling of Native Mammalian Proteins for Single-Molecule FRET Measurements. Chembiochem 19 , 780-783.

Lu, M., Ma, X., Castillo-Menendez, L.R., Gorman, J., Alsahafi, N., Ermel, U., Terry,

D.S., Chambers, M., Peng, D., Zhang, B., et al. (2019). Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET. Nature 568 , 415—419

Meineke, B., Heimgartner, J., Lafranchi, L., and Elsasser, S.J. (2018). Methanomethylophilus alvus Mx1201 Provides Basis for Mutual Orthogonal Pyrrolysyl tRNA/Aminoacyl-tRNA Synthetase Pairs in Mammalian Cells. ACS Chem. Biol. 13 , 3087-3096.

Nikic, I., Plass, T., Schraidt, O., Szymahski, J., Briggs, J.A.G., Schultz, C., and Lemke,

E.A. (2014). Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy. Angew Chem Int Ed Engl 53 , 2245-2249.

Niu et. al., 2013. “An expanded genetic code in mammalian cells with a functional quadruplet codon." DOI: 10.1021/cb4001662

Serfling, R., Seidel, L., Bottke, T., and Coin, I. (2018a). Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells. J. Vis. Exp. Serfling, R., Lorenz, C., Etzel, M., Schicht, G., Bottke, T., Mori, M., and Coin, I.

(2018b). Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells. Nucleic Acids Res. 46 , 1-10.

Zheng, Y., Addy, P.S., Mukherjee, R., and Chatterjee, A. (2017). Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells. Chem. Sci. 8 , 7211-7217.

Li et al. (2015 April 06) Nucleic Acids Research 43 (W1) :W580-4 PMID: 25845596.

McWilliam et al., (2013 May 13) Nucleic Acids Research 41 (Web Server issue) :W597- 600 PMID: 23671338

Sievers et al. (2011 October 11) Molecular Systems Biology 7 :539, PMID: 21988835

Claims

5’-A-B-C-3’ wherein each of A, B and C consists of a consecutive sequence of nucleotides, wherein A and C together forms a PylT acceptor stem; and B forms a PylT body, wherein the PylT acceptor stem is from a first species and the PylT body is from a second species, wherein the first species is different form the second species and the acceptor stem from the first species is different from the acceptor stem of the second species, wherein at the most two nucleotides of said PylT acceptor stem and/or at the most six nucleotides of said PylT body have been substituted and/or added.

2. The hybrid PylT according to claim 1, with the proviso that the hybrid PylT nucleotide sequence is different from a PylT nucleotide sequence native to the first species and second species and the hybrid PylT nucleotide sequence is different from a wild type PylT nucleotide sequence.

3. The hybrid PylT according to claim 1, wherein a. C consists of a nucleotide sequence having the general formula

5’-C_I-C₂-3’ wherein Ci consists of a consecutive sequence of nucleotides which are at complementary to A; and

4. The hybrid PylT according to any one of the preceding claims, wherein the first and second species are species of bacteria or archea.

5. The hybrid PylT according to any one of the preceding claims, wherein the first species is Candidatus Methanomethylophilus alvus Mx1201.

6. The hybrid PylT according to any one of the preceding claims, wherein the second species is a Methanogenic archaeon IS04-G1 .

7. The hybrid PylT according to any one of the preceding claims, wherein A comprises or consists of a sequence of nucleotides of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

8. The hybrid PylT according to any one of the preceding claims, wherein C comprises or consists of a sequence of nucleotides of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

9. The hybrid PylT according to any one of the preceding claims, wherein B comprises or consists of a sequence of nucleotides of from position 6 to 60 of SEQ ID NO:1 or comprise or consists of a sequence of nucleotides from position 6 to 61 of SEQ ID NO:2.

10. The hybrid PylT according to any one of the preceding claims, wherein the hybrid PylT comprises or consists of a sequence of nucleotides of SEQ ID NO:1 or SEQ ID NO:2.

11. A complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl- tRNA-synthetase (PylRS) (PylT/PyIRS), wherein the hybrid PylT is as defined in any one of the preceding claims and wherein the first PylRS may be any PylRS which is active to said hybrid PylT.

12. The complex according to claim 11 , wherein said first PylRS is of the same species as the hybrid PylT body, B.

13. The complex according to any one of claims 11 to 12, wherein said first PylRS comprises or consists of an amino acid sequence of SEQ ID NO:22 or SEQ ID NO:26 or a functional homologue thereof, having at least 70%, such as at least 80%, such as at least 90%, such as at least 95% sequence identity thereto.

14. A pair of PylT/PyIRS complexes, wherein said pair comprises a first hybrid PylT/PyIRS complex according to any one of claims 11 to 13, and a second PylT/PyIRS complex, wherein said second complex is mutually orthogonal to the first PylT/PyIRS complex.

15. The pair according to claim 14, wherein said second PylT/PyIRS complex is Methanosarcina mazei.

16. The pair according to any one of claims 14 to 16, wherein said first PylRS recognizes a first non-canonical amino acid and said second PylRS recognizes a second non-canonical amino acid, wherein the first non-canonical amino acid is different from the second non-canonical amino acid.

17. A method of producing a protein using a hybrid PylT according to any one of claims 1 to 10, a complex comprising a hybrid pyrrolysyl tRNA (PylT) and a first pyrrolysyl-tRNA-synthetase (PylRS) according to any one of claims 11 to 13 and/or a pair of PylT/PyIRS complexes according to any one of claims 14 to 16.