RNA ligase variants

Abstract

The present invention provides RNA ligase variants and its use in ligation reactions with oligonucleotides.

Description

[0001] RNA Ligase variants

[0002] Oligonucleotides have become an increasingly important class of active pharmaceutical ingredients (APIs) that show promise for the treatment of several diseases, such as genetic disorders, cancer and cardiovascular diseases. Despite the great potential of this class of compounds, their synthesis remains a challenge. Oligonucleotide synthesis is commonly obtained through phosphoramidite chemistry, which requires the intensive use of solvents and expensive building blocks. Moreover, when oligonucleotides are synthesized using the phosphoramidite method, each nucleotide is added sequentially through a complex cycle of de-blocking, coupling, capping and oxidation. The thorough optimization of this synthesis yields up to 99% coupling efficiency, however when long oligonucleotide sequences are synthesized the amount of impurities due to mistakes in the coupling becomes incrementally greater. In the field, several efforts are currently underway to improve the synthesis of oligonucleotides with the purpose of obtaining these APIs in higher purity through less resource intensive techniques.

[0003] RNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between two strands of RNA or DNA at the expense of the cofactor adenosine triphosphate (ATP). These enzymes are commonly applied in molecular biology for a number of applications, namely 3 ’ end labelling of RNA and the cloning of cDNA. In particular, this work focuses on the sub-family of RNA ligases that share similarity with T4 RNA ligase 2 from the bacteriophage T4, which requires the presence of a DNA or RNA sequence overlapping the ligation site to successfully ligate two oligonucleotide strands. This auxiliary oligonucleotide is called “splint”. RNA ligases were shown to have potential for oligonucleotide synthesis by ligating together short oligonucleotide fragments to form longer oligonucleotides, therefore reducing the generation of impurities caused by the synthesis of lengthy oligonucleotide sequences.

[0004] There is a need for RNA ligases with improved ligase reaction efficiency, which can be used in the synthesis of oligonucleotides.

[0005] In a first aspect, the present invention provides an RNA ligase variant comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of Seq. Id. No. 2 and wherein the RNA ligase variant comprises at least three amino acid substitutions relative to Seq. Id. No. 2, wherein the amino acid substitutions are selected from the group consisting of S57T, S62P, N65T and N65W.

[0006] In an embodiment of the present invention, the RNA ligase variant comprises the amino acid substitutions S57T, S62P, N65W.

[0007] In an embodiment of the present invention, the RNA ligase variant comprises the amino acid substitutions S57T, S62P, N65T.

[0008] In an embodiment of the present invention, the RNA ligase variant comprises an additional amino acid substitution selected from the group consisting of F67Y, H89R, D91N, K93E, F101L, Y126H, S131G, D140A, D140N, Y145F, T187A, E189G, F195S, S204A, S204C, S230T, S233T, K237T, K237H, S238G, T257G, NT11 , E277G, E277P, E277V, T279K, T279S, A280T, A280V, K281E, K281N, D282N, G284D, G284A, K285E, K285E, K285M, V286I, M287V, G288D, L289F, V296A, H307L, L313H, K315E Q317H, G319H, V322I, I326T, I326V, R327Q, T331S, and H339N.

[0009] In an embodiment of the present invention, the RNA ligase variant comprises amino acid substitutions selected from the group consisting of:

[0010] S57T S62P N65W H89R,

[0011] S57T S62P N65W D91N,

[0012] S57T S62P N65W K93E,

[0013] S57T S62P N65W F101L,

[0014] S57T S62P N65W S131G,

[0015] S57T S62P N65W D140N,

[0016] S57T S62P N65W D140A E189G,

[0017] S57T S62P N65W F195S,

[0018] S57T S62P N65W E277V K285E,

[0019] S57T S62P N65W K285E,

[0020] S57T S62P N65W E277G G284D,

[0021] S57T S62P N65W Q317H I326T,

[0022] S57T S62P N65W V286I,

[0023] S57T S62P N65W R327Q,

[0024] S57T S62P N65W K281E,

[0025] S57T S62P N65W V272A A280V,

[0026] S57T S62P N65W M287V,

[0027] S57T S62P N65W K285E G288D H307L,

[0028] S57T S62P N65W T279S,

[0029] S57T S62P N65W L289F,

[0030] S57T S62P N65W T279K D282N M287V G288D Q319H V322I,

[0031] S57T S62P N65W T279K K281N G284A M287V G288D Q319H V322I H339N.

[0032] In an embodiment of the present invention, the RNA ligase variant comprises amino acid substitutions selected from the group consisting of:

[0033] S57T S62P N65W E277V K285E,

[0034] S57T S62P N65W E277P K285E,

[0035] S57T S62P N65W A280T K285E,

[0036] S57T S62P N65W K237T K285E,

[0037] S57T S62P N65W K237H K285E,

[0038] S57T S62P N65W S204A K285E,

[0039] S57T S62P N65W S204C K285E,

[0040] S57T S62P N65W S204C,

[0041] S57T S62P N65W S204A T257G K285E,

[0042] S57T S62P N65W Y145F S204A K285E,

[0043] S57T S62P N65T S204C M287V G288D Q319H V322I, S57T S62P N65W T279K D282N M287V G288D Q319H V322I,

[0044] S57T S62P N65W T279K K281N G284A M287V G288D Q319H V322I H339N.

[0045] In an embodiment of the present invention, the RNA ligase variant comprises amino acid substitutions selected from the group consisting of:

[0046] S57T S62P N65W F67Y S230T K237H S238G K285E Q317H,

[0047] S57T S62P N65W K237H S238G E277P K285E Q317H,

[0048] S57T S62P N65W K237H E277P K285E Q317H,

[0049] S57T S62P N65W F67Y S233T K237H S238G K285E Q317H I326V R327Q,

[0050] S57T S62P N65W F67Y K237H S238G E277P K285E,

[0051] S57T S62P N65W F67Y S230T K237H S238G K285E Q317H R327Q,

[0052] S57T S62P N65W K237H S238G E277P K285E Q317H R327Q,

[0053] S57T S62P N65W F67Y K237H E277P K285E Q317H,

[0054] S57T S62P N65W F67Y S233T K237H S238G E277P K285E Q317H R327Q,

[0055] S57T S62P N65W F67Y S230T K237H E277P K285E Q317H.

[0056] In an embodiment of the present invention, the RNA ligase variant comprises amino acid substitutions selected from the group consisting of:

[0057] S57T S62P N65W K285E,

[0058] S57T S62P N65W T279K K281N G284A M287V G288D Q319H V322I H339N,

[0059] S57T S62P N65W K237H K285E,

[0060] S57T S62P N65W F67Y S230T K237H S238G K285E Q317H,

[0061] S57T S62P N65W K237H S238G E277P K285E Q317H,

[0062] S57T S62P N65W K237H E277P K285E Q317H,

[0063] S57T S62P N65W F67Y S233T K237H S238G K285E Q317H I326V R327Q,

[0064] S57T S62P N65W F67Y K237H S238G E277P K285E,

[0065] S57T S62P N65W F67Y S230T K237H S238G K285E Q317H R327Q,

[0066] S57T S62P N65W K237H S238G E277P K285E Q317H R327Q,

[0067] S57T S62P N65W F67Y K237H E277P K285E Q317H,

[0068] S57T S62P N65W S230T K237H S238G K285E Q317H,

[0069] S57T S62P N65W F67Y S233T K237H E277P K285E Q317H,

[0070] S57T S62P N65W F67Y S233T K237H S238G E277P K285E Q317H R327Q,

[0071] S57T S62P N65W F67Y S230T K237H E277P K285E Q317H,

[0072] S57T S62P N65W F67Y S230T K237H S238G K285E.

[0073] In an embodiment of the present invention, the amino acid sequence of the RNA ligase variant is at least 90% identical to the amino acid sequence of Seq. Id. No. 2.

[0074] In an embodiment of the present invention, the amino acid sequence of the RNA ligase variant is at least 95% identical to the amino acid sequence of Seq. Id. No. 2.

[0075] In a second aspect, the present invention provides an RNA ligase variant comprising an amino acid sequence selected from the group consisting of Seq. Id. No. 3 - 27, 30 - 36, 39, 40 and 42 - 75. In an embodiment of the present invention, the RNA ligase variant consists of an amino acid sequence selected from the group consisting of Seq. Id. No. 3 - 27, 30 - 36, 38 - 40 and 42 - 75.

[0076] The RNA ligase variants of the present invention can be used to ligate oligonucleotide fragments to full-length oligonucleotides. In certain embodiments, the oligonucleotide is selected from the group consisting of RNA, RNA-DNA hybrids, antisense oligonucleotides, siRNAs and single stranded guide RNAs. The oligonucleotide fragments can be modified oligonucleotides comprising chemical modifications such as e.g., modified sugar moieties, modified base moieties, and modified intemucleo- side linkages.

[0077] DEFINITIONS

[0078] The term “ligate” (or “ligating”, “ligation”, etc.) refers to the formation of phosphodiester bonds between the ribose and / or deoxyribose moieties of oligonucleotides, and particularly, the 3'-OH and 5'-P groups of these oligonucleotides.

[0079] The term “ligase” refers to an enzyme that catalyzes the formation of phosphodiester bonds between the ribose and / or deoxyribose moieties of oligonucleotides.

[0080] The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide may comprise one or more modified nucleosides such as 2’ sugar modified nucleosides. The oligonucleotide may comprise one or more modified internucleoside linkages, such as one or more phosphorothioate intemucleoside linkages.

[0081] The term “antisense oligonucleotide” as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. Antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. Preferably, the antisense oligonucleotides are single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than 50% across of the full length of the oligonucleotide. The antisense oligonucleotide can comprise one or more modified nucleosides or nucleotides, such as 2’ sugar modified nucleosides.

[0082] The term “contiguous nucleotide sequence” refers to the region of the antisense oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term “contiguous nucleobase sequence” and the term “oligonucleotide motif sequence”. In some embodiments, all the nucleosides of the oligonucleotide constitute the contiguous nucleotide sequence. In some embodiments, the oligonucleotide comprises the contiguous nucleotide sequence, such as an F-G-F’ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group (e.g., a conjugate group) to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the nucleobase sequence of the antisense oligonucleotide is the contiguous nucleotide sequence.

[0083] The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. The RNA strands may have the same or a different number of nucleotides.

[0084] The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus.

[0085] The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0086] As used herein, the term "siRNA" is defined as a double-stranded RNA comprising a first and second strand and having a central complementary portion between the first and second strands and terminal portions that are optionally complementary between the first and second strands or with a target nucleic acid. Each strand in the complex may have a length or from about 12 to about 24 nucleosides and may further comprise a central complementary portion having one of these defined lengths. Each strand may further comprise a terminal unhybridized portion having from 1 to about 6 nucleo- bases in length. The siRNAs may also have no terminal portions (overhangs) which is referred to as being blunt ended. The two strands of a siRNA can be linked internally leaving free 3’ or 5’ termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5’ or 3’ terminus producing an extension of single-stranded character.

[0087] As used herein, the term “guide RNA” or “gRNA” refers to a site-specific targeting RNA that can bind an RNA-guided endonuclease to form a complex, and direct the activities of the bound RNA- guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid. The gRNA is a duplex RNA made up of two parts: crisprRNA (crRNA), a nucleotide sequence complementary to the target DNA, and a tracrRNA, which serves as a binding scaffold for the Cas nuclease, wherein the crRNA and tracrRNA hybridize to each other to form a duplex.

[0088] As used herein, a single guide RNA (sgRNA) refers to a guide RNA which comprises the CRISPR RNA and the tracer RNA in one molecule.

[0089] As used herein, the term “Cas endonuclease” or “Cas nuclease” refers to an RNA-guided DNA endonuclease associated with the CRISPR adaptive immunity system. Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides and nucleosides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.

[0090] The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. Advantageously, one or more of the modified nucleosides of the antisense oligonucleotide comprise a modified sugar moiety. The term “modified nucleoside” may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson- Crick base pairing.

[0091] The term “modified intemucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. The oligonucleotides may therefore comprise one or more modified internucleoside linkages such as a one or more phosphorothioate intemucleoside linkages, or one or more phosphorodithioate internucleoside linkages.

[0092] In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80% or such as at least 90% of the intemucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments, all of the intemucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.

[0093] In some advantageous embodiments, all the internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate, or all the intemucleoside linkages of the oligonucleotide are phosphorothioate linkages.

[0094] It is recognized that, as disclosed in EP 2 742 135, antisense oligonucleotides may comprise other intemucleoside linkages (other than phosphodiester, phosphorothioate and phosphorodithioate), for example alkyl phosphonate / methyl phosphonate intemucleoside, which according to EP 2 742 135 may for example be tolerated in an otherwise DNA phosphorothioate gap region.

[0095] The term “nucleobase” includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al. (2012) and Bergstrom (2009).

[0096] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5- propynyl-cytosine, 5 -propynyl -uracil, 5-bromouracil 5-thiazolo-uracil, 2 -thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-amino- purine.

[0097] The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g., A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine.

[0098] The term “modified oligonucleotide” describes an oligonucleotide comprising one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term “chimeric” oligonucleotide is a term that has been used in the literature to describe oligonucleotides comprising sugar modified nucleosides and DNA nucleosides. The antisense oligonucleotide may be chimeric oligonucleotide.

[0099] The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleo- sides / nucleotides. Watson-Crick base pairs are guanine (G) - cytosine (C) and adenine (A) - thymine (T) / uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al. (2012) and Bergstrom (2009)).

[0100] The term “% complementary” as used herein, refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., oligonucleotide) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g., a target sequence or sequence motif). The percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson-Crick base pair) between the two sequences (when aligned with the target sequence 5 ’-3’ and the oligonucleotide sequence from 3’- 5 ’), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase / nucleotide which does not align (form a base pair) is termed a mismatch. Insertions and deletions are not allowed in the calculation of percentage complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g., 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

[0101] The term “fully complementary”, refers to 100% complementarity.

[0102] The term “identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g., a sequence motif). The percentage of identity is thus calculated by counting the number of aligned nucleobases that are identical (a Match) between two sequences (in the contiguous nucleotide sequence of the compound and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. Therefore, Percentage of Identity = (Matches x 100) / Length of aligned region (e.g., the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g., 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

[0103] The antisense oligonucleotide may comprise one or more nucleosides which have a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

[0104] Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0105] Such modifications include those where the ribose ring structure is modified, e.g., by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

[0106] Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’ -OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.

[0107] A 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’ - 4’ biradical bridged) nucleosides.

[0108] Indeed, much focus has been spent on developing 2’ sugar substituted nucleosides, and numerous 2’ substituted nucleosides have been found to have beneficial properties when incorporated into antisense oligonucleotides. For example, the 2’ modified sugar may provide enhanced binding affinity and / or increased nuclease resistance to the antisense oligonucleotide. Examples of 2’ substituted modified nucleosides are 2’-O-alkyl-RNA, 2’-O-methyl-RNA, 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside. For further examples, please see Freier et al. (1997), Uhlmann (2000) and Deleavey et al. (2012). Below are illustrations of some 2’ substituted modified nucleosides.

[0109] EXAMPLES

[0110] The present invention relates to the enzyme engineering of an RNA ligase from the T4 RNA ligase 2 sub-family for the synthesis of oligonucleotides. This enzyme (rLI2_28) was demonstrated to have a 10-fold higher reaction rate compared to T4 RNA ligase 2. Moreover, the catalytic features of this enzyme were further enhanced through several rounds of enzyme engineering, yielding enzyme variants with great potential for oligonucleotide API synthesis.

[0111] Ligases sequences

[0112] In the initial phase of the project, orthologs were recruited based on the sequence corresponding to RNA ligase 2 from the T4 phage (Ho et al. (2002), Nandakumar et al. (2004), Nandakumar et al. (2005) and Nandakumar et al. (2006)). The sequences of T4 RNA ligase 2 (Seq. Id. No. 1) and the best ortholog (rLI2_28 = Seq. Id. No. 2) are reported below.

[0113] T4 RNA Ligase 2 = Seq. Id. No. 1

[0114] MFKKYSSLENHYNSKFIEKLYSLGLTGGEWVAREKIHGTNFSLIIERDKVTCAKRT- GPILPAEDFFGYEIILKNYADSIKAVQDIMETSAVVSYQVFGEFAGPGIQKNVDYCDK DFYVF DIIVTTESGDVTYVDDYMMESFCNTFKFKMAPLLGRGKFEELIKLPNDLDSWQ DYNFT VDHAGLV DANKCVWNAEAKGEVFTAEGYVLKPCYPSWLRNGNRVA IKCKNSKFSEK KKSDKPIKAKVELSEADNKLVGILACYVTLNRVNNVISKIGEIGPKDFGKVMGLTVQD ILEETSRE GITLTQADNP SLIKKELVKMVQD VLRP AWIEL VS rLI2_28 = Seq. Id. No. 2

[0115] MEFNSYSSLENHHNGKFISKIRELALDSGEWVAREKIHGTNFSVLITPDLIQP CKRSGVILSAENFFGYEIIMKRYKESFEGLRASLSHLDLKSIQVFGEFAG GGIQKEV DYGEK EFYVFDMLMNYGGEVSYAEDKVVEDLANHYGFKMAPLLGRGKFEDLIKIPNDFQSR VNDF NALASGGNTVETNETVWEVFADEEGPNVSEGYVLKPNKPAYLPNGSRVAIKCKNSKFSEKK KSDKLIQPPKELSEIDADVLTKFSEYATWNRVSNWSKLGEVTAKDFGKVMGLTLQDIFVE AEREGLEISHAEDPALVKKQLQKLVQEVIRERWTELLTD

[0116] Cloning and transformation of wild-type T4 RNA ligase 2 and rLI2_28

[0117] The genes coding for the two wild-type ligases were synthesized and cloned into pET29b(+) by TWIST Bioscience (South San Francisco, USA). E. coli NEBlO-beta cells and E. coli BL21(DE3) (New England Biolabs, USA) were employed as cloning and expression strain, respectively. LB agar plates containing 50 mg / L kanamycin were used for transformation of chemically competent cells. The sequences were verified by Sanger sequencing (Microsynth AG, Balgach).

[0118] Expression of wild-type T4 RNA ligase 2 and rLI2_28

[0119] Cultivation in 96-deepwell plates

[0120] Bacterial cells were cultivated in a 96-deepwell plate (DWP) format with V-bottom shape and conical base aids. Precultures were started by inoculation of fresh single transformants into LB medium containing 50 mg / L kanamycin, followed by incubation at 30 °C with shaking at 300 rpm for 18 hours (Duetz system, Kuhner shaker). Main cultures in DWP were started by inoculation of 8 pL preculture into 500 pl ZYM-5052 autoinduction medium without trace elements containing 50 mg / L kanamycin. The cultures were incubated at 20 °C, 300 rpm for 20 hours or at 16 °C, 300 rpm for 30 hours without humidity (Duetz system, Kuhner shaker). Prior to cell harvesting, optical cell densities at 600 nm were measured in a Tecan Infinite spectrophotometer. Cells were pelleted by centrifugation at 4 °C, 3,300 g for 20 minutes and supernatants were discarded by inverting the plates. Cell pellets were lysed by adding 0.2 mL 25 mM Tris-HCl buffer pH 7.5 containing 1 mg / mL lysozyme from chicken egg white and 0.75 mg / mL polymyxin B. Cell suspensions were incubated at 30 °C with shaking at 300 rpm for 1 hour (Duetz system, Kuehner shaker), followed by centrifugation at 4 °C, 3,300 g for 30 minutes. Freshly extracted or frozen supernatants were used for ligation reactions.

[0121] Ligation reaction preparation for enzyme screening

[0122] Biocatalytic reactions were carried out in PCR tubes or in a 96-well PCR plate format. The reactions contained 50 mM Tris-HCl, 1.5 mM MgCL, 1 mM DTT, 0.4 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were: 0.1 mM 3’0H-49-mer RNA substrate, 0.11 mM 5’PO4-51-mer RNA substrate, and 0.12 mM of 28-mer DNA adapter. Reactions were performed with 0.1% to 1% (v / v) fresh clarified lysate in a final reaction volume of 40 pL.

[0123] Reactions were quenched by adding 40 pL phenol / chloroform / isoamyl alcohol (PCI) and diluted with 60 pL water. Samples were mixed and centrifuged at 3,300 g for 2 minutes for phase separation. Afterwards, 50 pL of the upper aqueous phase were collected, further diluted with 50 pL water, and analyzed by HPLC-UV.

[0124] In Table 1 the sequences of the oligonucleotides tested are reported. Table 1. List of oligonucleotides used in the ligation reactions.

[0125] N = A, C, G, T / U; mN: 2’-0-methylated nucleotide; fN: 2 ’-fluorinated nucleotide; rNo: ribonucleotide; dNo: deoxyribonucleotide; No: phosphodiester bond; Ns: phosphorothioate bond; 08: lipocap.

[0126] HPLC analysis sgRNA - Long method

[0127] Standards and quenched samples were measured by HPLC-UV at 260 nm on an Agilent 1290 Infinity II HPLC instrument coupled to DAD and equipped with a Waters Acquity UPLC BEH Amide column (1.7 pm, 2.1 x 100 mm). The column was heated to 80 °C and the injection volume was 1 pL. Mobile phase A consisted of 70% (v / v) acetonitrile + 70 mM ammonium acetate (pH 5.5), while mobile phase B consisted of 30% (v / v) acetonitrile + 70 mM ammonium acetate (pH 5.5). The following gradient method was used at a flow rate of 0.6 mL / min (max. 900 bar): 0 - 0.1 min, 60% B; 0. 1 - 2.5 min, 60 - 80% B; 2.5 - 3.3 min, 80% B, 3.3 - 3.4 min, 100 %B.

[0128] HPLC analysis sgRNA - Short method

[0129] Standards and quenched samples were measured by HPLC-UV at 260 nm on an Agilent 1290 Infinity II HPLC instrument coupled to DAD and equipped with a Waters Acquity Premier BEH Amide VanGuard FIT (1.7 pm, 2.1 x 50 mm). The column was heated to 80 °C and the injection volume was 1 pL. Mobile phase A consisted of 70% (v / v) acetonitrile + 70 mM ammonium acetate (pH 5.5), while mobile phase B consisted of 15% (v / v) acetonitrile + 70 mM ammonium acetate (pH 5.5). The following gradient method was used at a flow rate of 0.9 mL / min (max. 1200 bar): 0 - 0.1 min, 55% B; 0.1 - 0.9 min, 55 - 77% B; 0.9 - 0.91 min, 77 - 90% B, 0.91 - 1.1 min, 90% B. This method was used solely for the rapid RNA ligase mutants screening. Confirmation experiments to determine conversion were performed using the “Long method”.

[0130] HPLC analysis siRNA

[0131] Standards and quenched samples were measured by HPLC-UV at 260 nm on an Agilent 1290 Infinity II HPLC instrument coupled to DAD and LC / MSD, and equipped with a Waters Acquity UPLC BEH C18 column (1.7 pm, 2.1 x 50 mm). The column was heated to 80 °C and the injection volume was 1 pL. Mobile phase A consisted of 2.5% (v / v) methanol in 200 mM HFIP and 1.65 mM TEA water solution, while mobile phase B consisted of 80% (v / v) methanol in 200 mM HFIP and 1.65 mM TEA water solution. The following gradient method was used at a flow rate of 0.4 mL / min (max. 1000 bar): 0 min - 0.6 min, 5 - 10% B; 0.6 - 9.6 min, 10 - 31% B, 9.6 min - 9.7 min, 31 - 100% B.

[0132] Comparison of T4 RNA ligase 2 and rLI2_28

[0133] To define the best protein scaffold for subsequent enzyme engineering, we evaluated the performance of rLI2_28 and T4 RNA ligase 2 over a time course using low biocatalyst loads, namely 1% and 0.5% (v / v) lysate (Table 2).

[0134] At 1% (v / v) lysate rLI2_28 reached conversions of 92.8% after 2 h, clearly outcompeting T4 RNA ligase 2 (44.1% conversion) under the same conditions.

[0135] Moreover, at 0.5% (v / v) lysate, rLI2_28 yielded conversions of 89.5%, respectively, while T4 RNA ligase 2 resulted in only 20.8% conversion. Under these conditions, candidate rLI2_28 also displayed the highest reaction rate, yielding conversions above 50% after only 30 minutes, while T4 RNA ligase 2 only showed 5.1% conversion after 30 minutes in the same conditions (Table 2).

[0136] Table 2. rLI2_28 and T4 RNA ligase 2 tested at several time points and different biocatalyst load. Reactions were incubated at 23 °C.

[0137] Screening of RNA ligase rLI2_28 variants obtained through enzyme engineering

[0138] Molecular modelling

[0139] Two conformers of the enzyme rLI2_28, the AMP cofactor complex (“compact”) and the nucleic acid duplex complex (“loose”), were modelled using Swiss-Model (Waterhouse et al. (2018)) and the crystal structures of T4 RNA ligase 2 as templates (PDB ID: 2HVQ and 2HVS, respectively; Nandakumar et al. (2006)). Additionally, a nicked 20-mer RNA:DNA hybrid model was modeled using PyMOL (version 2.5.5). Investigation into conformational dynamics was carried out, and all models were optimized via Molecular Dynamics (MD) simulations employing OpenMM (version 8.1.0) (Eastman et al. (2010); Eastman et al. (2024)).

[0140] To predict potential protein-ligand interactions, the refined RNA:DNA hybrid was docked into “compact” and “loose” conformer of rLI2_28 employing the standalone protein-protein and protein- DNA docking tool HDOCKlite (Y an et al. (2020)). The resulting complexes were manually investigated, and putatively productive complexes were utilized for further MD simulations to derive amino acids with high contact frequency toward the RNA:DNA hybrid.

[0141] Preparation of mutant libraries

[0142] Libraries were constructed by Overlap Extension PCR (OE-PCR) using Q5 DNA polymerase, mutagenic primers, and the general In-Fusion flanking primers. Upon each OE-PCR, only one part of the gene was amplified - depending on the library either the region from A / ?oI to Hindlll or from Hindlll to Xho\. The resulting fragment was inserted by In-Fusion cloning into linearized pET22b(+) containing the other unmodified gene region. This strategy was applied to enable only one sequencing event per construct. Hits were sent to Sanger sequencing (Microsynth, Balgach) using standard sequencing primer T7probis or T7term, accordingly.

[0143] E. coll NEBlO-beta cells and E. coll BL21(DE3) (New England Biolabs, USA) were employed as cloning and expression strain, respectively. LB agar plates containing 200 mg / L ampicillin were used for transformation of chemically competent cells.

[0144] Robotic cultivation in 96-well microtiter plates

[0145] Bacterial cells were picked and cultivated on a Tecan Fluent robotic platform in flat-bottom 96-well microtiter plates (Thermo Fisher Scientific) with 180 pL LB medium containing 200 mg / L ampicillin, followed by incubation at 30 °C with shaking at 800 rpm for 20 hours. Main cultures were prepared by inoculating 10 pL preculture in 160 pL ZYM-5052 autoinduction medium without trace elements (10 g / L peptone, 5 g / L yeast extract, 5 g / L glycerol, 0.55 g / L glucose monohydrate, 2.1 g / L lactose monohydrate, 10.6 g / L sodium phosphate dibasic salt, 3.4 g / L potassium phosphate monobasic salt, 2.15 g / L ammonium chloride, 0.59 g / L sodium chloride, 0.663 g / L ammonium sulfate, 2 mM magnesium sulfate) and supplemented with 200 mg / L ampicillin. Cultures were incubated at 20 °C with shaking at 850 rpm for 20 hours. Cells were pelleted by centrifugation at 4 °C, 4060 rpm for 20 minutes and supernatants were discarded by inverting the plates. Cell lysis proceeded by addition of 160 pL lysis buffer (25 mM Tris-HCl buffer pH 7.5 containing 1 mg / mL lysozyme for chicken egg white, 0.75 mg / mL polymyxin B and 0.1 % Triton-X), followed by incubation at 30 °C with shaking at 850 rpm for 1 hour. After centrifugation at 4 °C, 4,060 rpm for 30 minutes, the supernatants were transferred to new microtiter plates. Freshly extracted supernatants were used for screening. All plates contained lysates from strains harboring the negative control plasmid, the parental enzyme, or other positive controls.

[0146] Expression of ligases in shake flasks

[0147] The expression of selected ligases was performed in 2 L flasks. 50 pl of E. coli BL21(DE3) Singles Competent Cells were transformed with 3 pL of the desired plasmid solution. After addition of the plasmid, the cells were kept on ice for 20 minutes, heat shocked at 42 °C for 45 seconds, and kept on ice for another 3 minutes. After adding 250 pL of prewarmed SOC medium, the cells were incubated for 50 minutes at 37 °C and 800 rpm. After quickly spinning down the tube, 20 pL of the cell suspension from the bottom of the tube were plated on an LB Agar plate containing the corresponding antibiotic. The plates were then incubated overnight at 37 °C. The next day, an overnight culture was started by inoculating 15 mL of LB medium containing the corresponding antibiotic with one colony from the LB Agar plate. The cultures were incubated overnight at 37 °C and 180 rpm in a rotary incubator.

[0148] Alternatively, if a glycerol stock was available, the overnight culture was inoculated with 20 pL of the glycerol stock. The overnight culture was then incubated overnight at 37 °C and 180 rpm in a rotary incubator.

[0149] The next day, 5 mL of the overnight culture was transferred into 500 mL of TB medium containing the corresponding antibiotic. After approximately 4 hours, the expression was induced by adding IPTG (1 mM) to the cells and the shake flasks were incubated at 20 °C overnight. The next day, the cells were harvested and frozen on dry-ice in a minigrip bag, before storing the cells at -80 °C.

[0150] Cell lysis and ligase purification

[0151] Following extensive optimization, enzyme lysis and purification was performed by resuspending approximately 2 g of pellet in 10 mL of binding buffer. Sonication was performed for 3 min (1 min on, 1 min off, amplitude 12). The obtained crude extract was centrifuged for 45 minutes at 30,000 ref using a Beckman Avanti J-20XP centrifuge equipped with a JA-18 rotor.

[0152] In the meantime, the His GraviTrap columns (1 ml Ni-NTA Superflow gravity columns 1.5 ml, Qiagen) were equilibrated with 10 mL water and 10 mL binding buffer (25 mM Tris-HCl, 0.5 M NaCl, 25 mM imidazole, pH 7.5). After the equilibration, the supernatant of the centrifuged crude extract was loaded completely onto the columns. Subsequently, the bound protein was washed twice with 10 mL binding buffer, before it was eluted with 2.7 mL of elution buffer (25 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 7.5).

[0153] Buffer exchange was performed after the elution by two passes in a PD10 Desalting Columns containing 8.3 mL of Sephadex™ G-25 resin (Cytiva). The column was equilibrated 5 times with ligase storage buffer (50 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol, pH 8.0). Next, 2.5 mL of the purified protein were loaded onto the first column, before eluting the protein with 3.5 mL of ligase storage buffer. 2.5 mL of this eluate were then loaded onto the second PD10 column, and again the ligase was eluted with 3.5 mL of ligase storage buffer. Ligation reaction preparation for enzyme screening with purified enzymes

[0154] Biocatalytic reactions were carried out in PCR tubes or in a 96-well PCR plate format. The reactions contained 50 mM Tris-HCl, 15 mM MgCT. 1 mM DTT, 2 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were 0.1 mM 3’0H-49-mer RNA substrate, 0.11 mM 5’PC>4- 51-mer RNA substrate, and 0. 12 mM of 28-mer DNA adapter. Reactions were performed with 0.01% to 0.025% (v / v) purified enzyme in a final reaction volume of 40 pL.

[0155] Reactions were sampled by withdrawing 10 pL from the reaction solution and mixing them with 1 pL EDTA 0.5 M and 10 pL water. After 10 minutes incubation, the solution was diluted with 180 pL water and the solution was filtered over Ami con Ultra Centrifugal Filter (3 kDa MWCO, 500 pL) by centrifuging the filter for 3 minutes at 13,000 rpm. The filter washed twice with 100 pL water and finally the oligonucleotides were eluted by inverting the filter and centrifuging again at 1000 ref for 3 minutes. The sample was injected directly on the LC-MS for quantification of the reaction outcome.

[0156] Summary of the results of enzyme engineering

[0157] Different amino acids were selected during the enzyme engineering campaign and hereby are reported the combinations for which an improvement over the parent, rLI2_28, was observed.

[0158] Improvement over the parent rLI2_28, defined as FIOP (Fold Improvement Over Parent) were calculated according to the following formula:

[0159] FIOP = 100 * (Conversion of the mutant enzyme / Conversion of the parent enzyme)

[0160] The variants identified during the first round of engineering, which focused on an N-terminal domain loop, the interdomain loop and a C-terminal a-helix region, are consolidated in Table 3. The Seq. Id. No. in the tables below disclosing the different combinations of mutations in the ligases of the invention refer to ligase variants which comprise the specific combination of mutations.

[0161] Table 3. Summary of the FIOP of the best enzymes from the 1stround of enzyme engineering. Reactions contained 0.25% (v / v) lysate and were incubated for 30 minutes at 23 °C.

[0162] Building on the findings of the 1stround of enzyme engineering, in the 2ndround it was decided to combine the most promising sites identified in the 1stround in a combinatorial fashion, namely Serine 57, Serine 62 and Asparagine 65. A summary of the most active combination is reported in Table 4.

[0163] Table 4. Summary of the FIOP of the best enzymes from the 2ndround of enzyme engineering. Reactions contained 0.25% (v / v) lysate and were incubated for 30 minutes at 23 °C.

[0164] In a subsequent experiment, the activity of the wild-type enzyme rLI2-28 was compared with the triple mutant S57T S62P N65T, which was highlighted in later mutation rounds but that was not identified in the initial phases of the enzyme engineering.

[0165] In this specific instance, the enzymes were purified using His-tag chromatography and gel filtration, as detailed above.

[0166] The mutant S75T S62P N65T was compared to the wild-type enzyme rLI2_28 in a reaction, carried out as described in the paragraphs above and the results are reported in Table 5. Table 5. FIOP of the enzyme containing the mutations S57T S62P N65T.

[0167] For the following 3rdround of enzyme engineering, the focus shifted to residues that are within 5 A from the AMP cofactor, according to the enzyme model that was built. The residues that showed an improvement over the wild type rLI2_28 are reported in Table 6. In general, this round of engineering showed more limited improvement compared to the two previous enzyme engineering rounds.

[0168] Table 6. Summary of the FIOP of the best enzymes from the 3rdround of enzyme engineering. Reactions contained 0.25% (v / v) lysate and were incubated for 30 minutes at 23 °C.

[0169] In the 4thround of enzyme engineering, the triple mutant identified in the 2ndround of engineering in which Serine 57, Serine 62 and Asparagine 65 are mutated to a Threonine, Proline and Tryptophan, respectively, was chosen as the new parent.

[0170] In this engineering round, error prone PCR was used to evolve the ligase further.

[0171] Error prone PCR was performed using Casting Error Prone PCR (cepPCR) (Y ang el al. (2017)). The ligase gene was divided into 5 parts, each approximately 200 bp long, and cepPCR libraries were generated for each of the 5 gene parts as follows. Error-prone PCR amplification with non- mutagenic primers using Taq DNA polymerase and different MnCT concentrations (0.2 mM, 0.55 mM and 0.9 mM) was performed using the triple mutant template. The forward non-mutagenic primer of the first fragment and the reverse non-mutagenic primer of the fifth fragment included the start codon and the C-terminal His-tag and stop codon, respectively, to avoid introducing undesired mutations on these sites that would otherwise lead to non-functional sequences. The generated megaprimer was used for vector amplification based on the MEGAWHOP technique (Miyazaki (2002)), using the triple mutant as template and Q5 DNA polymerase, followed by l) n\ digestion of the template prior to transformation.

[0172] The best enzyme variants identified during the 4thround of enzyme engineering are summarized in Table 7. Table 7. Summary of the FIOP of the best enzymes from the 4thround of enzyme engineering.

[0173] Reactions contained 0.25% (v / v) lysate and were incubated for 30 minutes at 23 °C. a Calculated

[0174] In the 5thround of enzyme engineering, new variants were generated by error prone PCR on the triple mutant identified in the 2ndround of engineering in which Serine 57, Serine 62 and Asparagine 65 are mutated to a Threonine, Proline and Tryptophan, respectively.

[0175] Based on the 4thround results, MnCF concentration was lowered to 0.2 mM, and more plates from error prone PCR enzyme engineering were tested. The best enzyme variants identified during the 5thround of enzyme engineering are summarized in Table 8.

[0176] Table 8. Summary of the FIOP of the best enzymes from the 5thround of enzyme engineering.

[0177] Reactions contained 0. 125% (v / v) lysate and were incubated for 30 minutes at 23 °C. a Calculated

[0178] During the 6thround of enzyme engineering, a restricted library was built around the engineering of residues that were identified during round 5 as hotspot. Five residues, namely Aspartate 277, Threonine 279, Lysine 281, Glutamine 282 and Glycine 284, located in the C-terminal domain of the enzyme, were selected. The parent used for this round of engineering is the sequence containing the seven mutations S57T S62P N65W M287V G288D Q319H V322I, identified during the 4thenzyme engineering round.

[0179] Given the high number of possible combinations for saturation at five positions simultaneously, only eight residues at each amino acid site were chosen. From this theoretical library (8A5 = 32,768 variants), only 4.4% of the total possible variants were pre-screened and sequenced by Na- nopore-based consensus sequencing. The sequencing and FIOP results of the pre-screened variants were used for machine learning and a second library was designed.

[0180] Among all the enzyme variants screened during this engineering round, two mutants showed improvements over the parent (Table 9).

[0181] Table 9. Summary of the FIOP of the best enzymes from the 6thround of enzyme engineering. Reactions contained 0.2% (v / v) lysate and were incubated for 30 minutes at 23 °C. a Calculated

[0182] During round 6 of enzyme engineering, the improvement obtained despite multiple mutations was quite limited. Therefore, in the 7thround of enzyme engineering co-evolution analysis was conducted on two different scaffolds: the best scaffold identified in the 4thround of enzyme engineering (S57T S62P N65W M287V G288D Q319H V322I) and the scaffold identified in the 5thround of enzyme engineering (S57T S62P N65W K285E). The improvement of the mutants identified during this round of enzyme engineering were calculated on the parent with seven mutations.

[0183] In this round, amino acids that showed beneficial effects in previous enzyme engineering rounds and other amino acids that show co-evolution in the ligase families were tested (Table 10).

[0184] The greater improvements were observed when the parent enzyme was the least evolved enzyme sequence (S57T S62P N65W K285E).

[0185] Table 10. Summary of the FIOP of enzyme variants evaluated in the 7thround of enzyme engineering. Reactions contained 0.2% (v / v) lysate and were incubated for 30 minutes at 23 °C. a Calculated

[0186] In the 8thand final round of enzyme engineering, the parent chosen as scaffold contained five mutations (S57T S62P N65W K237H K285E). In this library, eight amino acid sites were targeted simultaneously with a reduced panel of amino acid mutations based on MD simulations, co-conserva- tion analysis and from the error prone PCR experiments conducted in the 5thround of engineering.

[0187] The best results from this enzyme engineering round are reported in Table 11.

[0188] Table 11. Summary of the FIOP of the best enzymes from the 8thround of enzyme engineering. Reactions contained 0.125% (v / v) lysate and were incubated for 30 minutes at 23 °C.

[0189] a Calculated

[0190] Verification of the final variants from Round 8 engineering

[0191] The lysates from the best mutants obtained from Round 8 of enzyme engineering were tested and compared in a time course reaction according to the following protocol (Table 12).

[0192] The reactions contained 50 mM Tris-HCl, 1.5 mM MgCT. 1 mM DTT, 0.4 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were: 0. 1 mM 3’0H-49-mer RNA substrate, 0. 1 mM 5’PO4-51-mer RNA substrate, and 0. 12 mM of 28-mer DNA adapter. Reactions were performed with 0. 125% (v / v) fresh clarified lysate in a final reaction volume of 40 pL. Reactions were quenched after 30 minutes or 4 hours.

[0193] Reactions were quenched by adding 40 pL phenol / chloroform / isoamyl alcohol (PCI) and diluted with 60 pL water. Samples were mixed and centrifuged at 3,300 g for 2 minutes for phase separation. Afterwards, 50 pL of the upper aqueous phase were collected, further diluted with 50 pL water, and analyzed by HPLC-UV. Table 12. Comparison of the conversion obtained with the best variants from the 8thround of enzyme engineering. Reactions contained 0.125% (v / v) lysate and were incubated for 30 minutes or 4 hours at 23 °C.

[0194] Characterization of a selection of RNA ligases in purified form

[0195] Further characterization was performed on a selection of engineered RNA ligases to verify if these enzymes maintained an advantage over the parent enzyme when they were reacted with other RNA sequences.

[0196] The engineered RNA ligases tested are reported in Table 13.

[0197] Table 13. List of engineered ligase variants selected for further tests.

[0198] Ligation of a siRNA containing 2’-F at the 3’-OH fragment and 2’-F at the 5’-PC>4 fragment by a selection of purified engineered ligases

[0199] The purified ligases were tested and compared in a time course reaction (Table 14). The reactions were performed according to the following protocol.

[0200] The reactions contained 50 mM Tris-HCl, 15 mM MgCL. 1 mM DTT, 2 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were: 0.25 mM 3’OH fragment with 2’-F, 0.275 mM 5 ’PCL fragment with 2’-F and 0.3 mM of sense strand with lipocap. Reactions were performed with 0.0025 mg / mL of purified ligase in a final reaction volume of 40 pL. Reactions were quenched at different time points.

[0201] Reactions were sampled by withdrawing 10 pL from the reaction solution and mixing them with 1 pL EDTA 0.5 M and 10 pL water. After 10 minutes incubation, the solution was diluted with 180 pL water and the solution was filtered over Ami con Ultra Centrifugal Filter (3 kDa MWCO, 500 pL) by centrifuging the filter for 3 minutes at 13,000 rpm. The filter washed twice with 100 pL water and finally the oligonucleotides were eluted by inverting the filter and centrifuging again at 1000 ref for 3 minutes. The samples were injected directly on the LC-MS for quantification of the reaction outcome.

[0202] Table 14. Summary of the FIOP overrLI2_28 for a selection of engineered ligase variants for the ligation of a siRNA containing 2’-F at the 3’-OH fragment and 2’-F at the 5'-PO i fragment

[0203] Ligation of a siRNA containing 2’-F at the 3’-OH fragment and 2’-0Me at the 5’-PC>4 fragment by a selection of purified engineered ligases

[0204] The purified ligases were tested and compared in a time course reaction (Table 15). The reactions were performed according to the following protocol.

[0205] The reactions contained 50 mM Tris-HCl, 15 mM MgCf. 1 mM DTT, 2 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were: 0.25 mM 3’OH fragment with 2’-F, 0.275 mM 5 ’PCL fragment with 2’-OMe and 0.3 mM of sense strand with lipocap. Reactions were performed with 0.0025 mg / mL of purified ligase in a final reaction volume of 40 pL. Reactions were quenched at different time points.

[0206] Reactions were sampled by withdrawing 10 pL from the reaction solution and mixing them with 1 pL EDTA 0.5 M and 10 pL water. After 10 minutes incubation, the solution was diluted with 180 pL water and the solution was filtered over Ami con Ultra Centrifugal Filter (3 kDa MWCO, 500 pL) by centrifuging the filter for 3 minutes at 13,000 rpm. The filter washed twice with 100 pL water and finally the oligonucleotides were eluted by inverting the filter and centrifuging again at 1000 ref for 3 minutes. The samples were injected directly on the LC-MS for quantification of the reaction outcome.

[0207] Table 15. Summary of the FIOP overrLI2_28 for a selection of engineered ligase variants for the ligation of a siRNA containing 2’-F at the 3’-OH fragment and 2’-OMe at the 5'-PO i fragment

[0208] Ligation of a siRNA containing 2’-0Me at the 3’-OH fragment and 2’-F at the 5’-PC>4 fragment by a selection of purified engineered ligases

[0209] The purified ligases were tested and compared in a time course reaction (Table 16). The reactions were performed according to the following protocol.

[0210] The reactions contained 50 mM Tris-HCl, 15 mM MgCh, 1 mM DTT, 2 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were: 0.25 mM 3’OH fragment with 2’-OMe, 0.275 mM 5’PC>4 fragment with 2’-F and 0.3 mM of sense strand with lipocap. Reactions were performed with 0.01 mg / mL of purified ligase in a final reaction volume of 40 pL. Reactions were quenched at different time points.

[0211] Reactions were sampled by withdrawing 10 pL from the reaction solution and mixing them with 1 pL EDTA 0.5 M and 10 pL water. After 10 minutes incubation, the solution was diluted with 180 pL water and the solution was filtered over Ami con Ultra Centrifugal Filter (3 kDa MWCO, 500 pL) by centrifuging the filter for 3 minutes at 13,000 rpm. The filter washed twice with 100 pL water and finally the oligonucleotides were eluted by inverting the filter and centrifuging again at 1000 ref for 3 minutes. The samples were injected directly on the LC-MS for quantification of the reaction outcome.

[0212] Table 16. Summary of the FIOP overrLI2_28 for a selection of engineered ligase variants for the ligation of a siRNA containing 2’-OMe at the 3’-OH fragment and 2’-F at the 5’-PO4 fragment. Ligation of a siRNA containing 2’-0Me at the 3’-OH fragment and 2’-0Me at the 5’-PC>4 fragment by a selection of purified engineered ligases

[0213] The purified ligases were tested and compared in a time course reaction (Table 17). The reactions were performed according to the following protocol.

[0214] The reactions contained 50 mM Tris-HCl, 15 mM MgCf. 1 mM DTT, 2 mM ATP; adjusted to pH 7.5. Final concentrations of the RNA oligomers were: 0.25 mM 3’OH fragment with 2’-OMe, 0.275 mM 5 ' PO i fragment with 2’-OMe and 0.3 mM of sense strand with lipocap. Reactions were performed with 0.01 mg / mL of purified ligase in a final reaction volume of 40 pL. Reactions were quenched at different time points.

[0215] Reactions were sampled by withdrawing 10 pL from the reaction solution and mixing them with 1 pL EDTA 0.5 M and 10 pL water. After 10 minutes incubation, the solution was diluted with 180 pL water and the solution was filtered over Ami con Ultra Centrifugal Filter (3 kDa MWCO, 500 pL) by centrifuging the filter for 3 minutes at 13,000 rpm. The filter washed twice with 100 pL water and finally the oligonucleotides were eluted by inverting the filter and centrifuging again at 1000 ref for 3 minutes. The samples were injected directly on the LC-MS for quantification of the reaction outcome.

[0216] Table 17. Summary of the FIOP overrLI2_28 for a selection of engineered ligase variants for the ligation of a siRNA containing 2’-OMe at the 3’-OH fragment and 2’-OMe at the 5'-PO i fragment.

[0217] Engineered rLI2_28 sequences

[0218] The sequences of the engineered ligases are reported below.

[0219] REFERENCES

[0220] G. F. Deleavey, M. J. Damha, Chem. Biol. 2012, 19, 937-54.

[0221] P. Eastman, V. Pande, Comput. Sci. Eng. 2010, 12, 4, 34-9.

[0222] P. Eastman, R. Galvelis, et al., T. E. Markland, J. Phys. Chem. B 2024, 128, 1, 109-16.

[0223] S. M. Freier, K. H. Altmann, Nucl. Acid Res. 1997, 25, 4429-43.

[0224] I. Hirao, M. Kimoto, R. Yamashige, Acc. Chem. Res. 2012, 45, 2055-65.

[0225] C. K. Ho, S. Shuman, Proc. Natl. Acad. Sci. USA 2002, 99, 12709-14.

[0226] T. M. Miyazaki K, Biotechniques 2002, 1038, 33, 5, 1033 4. 1036-8.

[0227] J. Nandakumar, C. K. Ho, C. D. Lima, S. Shuman, J. Biol. Chem. 2004, 279, 31337-47.

[0228] J. Nandakumar, S. Shuman, J. Biol. Chem. 2005, 280, 23484-9.

[0229] J. Nandakumar, S. Shuman, C. D. Lima, Cell 2006, 127, 71-84.

[0230] E. Uhlmann, Curr. Opin. Drug Discov. Dev. 2000, 3(2), 293-13.

[0231] A. Waterhouse, M. Bertoni, et al., T. Schwede, Nucleic Acids Res. 2018, 46, W296-303.

[0232] Y. Yan, J. He, S.-Y. Huang, Nat. Protoc. 2020, 15, 1829-52.

[0233] J. Yang, A. J. Ruff, et al., U. Schwaneberg, Biotechnol. Bioeng. 2017, 114, 1921-7.

Claims

Claims1. A RNA ligase variant comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of Seq. Id. No. 2 and wherein the RNA ligase variant comprises at least three amino acid substitutions relative to Seq. Id. No. 2, wherein the amino acid substitutions are selected from the group consisting of S57T, S62P, N65T and N65W.

2. The RNA ligase variant of claim 1, wherein the at least 3 amino acid substitutions are S57T, S62P, N65W.

3. The RNA ligase variant of claim 1, wherein the at least 3 amino acid substitutions are S57T, S62P, N65T.

4. The RNA ligase variant of claims 1 - 3, wherein the variant comprises an additional amino acid substitution selected from the group consisting of F67Y, H89R, D91N, K93E, F101L, Y126H, D140A, Y145F, T187A, E189G, F195S, S204A, S204C, S230T, K237T, S233T, K237H, S238G, T257G, NT11 , E277G, E277P, E277V, T279K, T279S, A280T, A280V, K281E, K281N, D282N, G284D, G284A, K285E, K285E, K285M, V286I, M287V, G288D, L289F, V296A, H307L, L313H, K315E Q317H, G319H, V322I, I326T, I326V, R327Q, T331S, and H339N.

5. The RNA ligase variant of claims 1 - 4, wherein the variant comprises amino acid substitutions:S57T S62P N65W H89R (Seq. Id, No. 5) orS57T S62P N65W D91N (Seq. Id, No. 6) orS57T S62P N65W K93E (Seq. Id, No. 7) orS57T S62P N65W F101L (Seq. Id, No. 8) orS57T S62P N65W S131G (Seq. Id, No. 9) orS57T S62P N65W D140N (Seq. Id, No. 10) orS57T S62P N65W D140A E189G (Seq. Id, No. 11) orS57T S62P N65W F195S (Seq. Id, No. 12) orS57T S62P N65W E277V K285E (Seq. Id, No.13) orS57T S62P N65W K285E (Seq. Id, No. 14) orS57T S62P N65W E277G G284D (Seq. Id, No. 15) orS57T S62P N65W Q317H I326T (Seq. Id, No. 16) orS57T S62P N65W V286I (Seq. Id, No. 17) orS57T S62P N65W R327Q (Seq. Id, No. 19) orS57T S62P N65W K281E (Seq. Id, No. 20) orS57T S62P N65W V272A A280V (Seq. Id, No. 21) orS57T S62P N65W M287V (Seq. Id, No. 22) orS57T S62P N65W K285E G288D H307L (Seq. Id, No. 23) orS57T S62P N65W T279S (Seq. Id, No. 24) orS57T S62P N65W L289F (Seq. Id, No. 25) orS57T S62P N65W T279K D282N M287V G288D Q319H V322I (Seq. Id, No. 27) orS57T S62P N65W T279K K281N G284A M287V G288D Q319H V322I H339N (Seq. Id,No. 30).

6. The RNA ligase variant of claims 1 - 4, wherein the variant comprises amino acid substitutions:S57T S62P N65W E277V K285E (Seq. Id. No. 13) orS57T S62P N65W E277P K285E (Seq. Id. No. 42) orS57T S62P N65W A280T K285E (Seq. Id. No. 44) orS57T S62P N65W K237T K285E (Seq. Id. No. 46) orS57T S62P N65W K237H K285E (Seq. Id. No. 47) orS57T S62P N65W S204A K285E (Seq. Id. No. 48) orS57T S62P N65W S204C K285E (Seq. Id. No. 49) orS57T S62P N65W S204C (Seq. Id. No. 58) orS57T S62P N65W S204A T257G K285E (Seq. Id. No. 50) orS57T S62P N65W Y145F S204A K285E (Seq. Id. No. 51) orS57T S62P N65T S204C M287V G288D Q319H V322I (Seq. Id. No. 54) orS57T S62P N65W T279K D282N M287V G288D Q319H V322I (Seq. Id. No. 27) orS57T S62P N65W T279K K281N G284A M287V G288D Q319H V322I H339N (Seq. Id.No. 30)7. The RNA ligase variant of claims 1 - 4, wherein the variant comprises amino acid substitutions:S57T S62P N65W F67Y S230T K237H S238G K285E Q317H (Seq. Id. No. 63) orS57T S62P N65W K237H S238G E277P K285E Q317H (Seq. Id. No. 64) orS57T S62P N65W K237H E277P K285E Q317H (Seq. Id. No. 65) orS57T S62P N65W F67Y S233T K237H S238G K285E Q317H I326V R327Q (Seq. Id. No. 66) orS57T S62P N65W F67Y K237H S238G E277P K285E (Seq. Id. No. 67) orS57T S62P N65W F67Y S230T K237H S238G K285E Q317H R327Q (Seq. Id. No. 68) orS57T S62P N65W K237H S238G E277P K285E Q317H R327Q (Seq. Id. No. 69) orS57T S62P N65W F67Y K237H E277P K285E Q317H (Seq. Id. No. 70) orS57T S62P N65W F67Y S233T K237H S238G E277P K285E Q317H R327Q (Seq. Id. No. 73) orS57T S62P N65W F67Y S230T K237H E277P K285E Q317H (Seq. Id. No. 74).

8. The RNA ligase variant of claim 1 - 4, wherein the variant comprises amino acid substitutions:S57T S62P N65W K285E (Seq. Id. No. 14) orS57T S62P N65W T279K K281N G284A M287V G288D Q319H V322I H339N (Seq. Id.No. 30) orS57T S62P N65W K237H K285E (Seq. Id. No. 47) orS57T S62P N65W F67Y S230T K237H S238G K285E Q317H (Seq. Id. No. 63) orS57T S62P N65W K237H S238G E277P K285E Q317H (Seq. Id. No. 64) orS57T S62P N65W K237H E277P K285E Q317H (Seq. Id. No. 63) orS57T S62P N65W F67Y S233T K237H S238G K285E Q317H 1326 V R327Q (Seq. Id. No.74) orS57T S62P N65W F67Y K237H S238G E277P K285E (Seq. Id. No. 67) orS57T S62P N65W F67Y S230T K237H S238G K285E Q317H R327Q (Seq. Id. No. 63) orS57T S62P N65W K237H S238G E277P K285E Q317H R327Q (Seq. Id. No. 69) orS57T S62P N65W F67Y K237H E277P K285E Q317H (Seq. Id. No. 70) orS57T S62P N65W S230T K237H S238G K285E Q317H (Seq. Id. No. 71) orS57T S62P N65W F67Y S233T K237H E277P K285E Q317H (Seq. Id. No. 72) orS57T S62P N65W F67Y S233T K237H S238G E277P K285E Q317H R327Q (Seq. Id. No. 73) orS57T S62P N65W F67Y S230T K237H E277P K285E Q317H (Seq. Id. No. 74) orS57T S62P N65W F67Y S230T K237H S238G K285E (Seq. Id. No. 75).

9. The RNA ligase variant of claims 1 - 8, wherein the amino acid sequence of the RNA ligase variant is at least 90% identical to the amino acid sequence of Seq. Id. No. 2.

10. The RNA ligase variant of claim 9, wherein the amino acid sequence of the RNA ligase variant is at least 95% identical to the amino acid sequence of Seq. Id. No. 2.

11. A RNA ligase variant comprising an amino acid sequence selected from the group consisting of Seq. Id. No. 3 - 27, 30 - 36, 38 - 40 and 42 - 75.

12. The RNA ligase variant of claim 10 consisting of an amino acid sequence selected from the group of Seq. Id. No. 3 - 27, 30 - 36, 38 - 40 and 42 - 75.

13. The RNA ligase variant of claims 1 - 12 for use in a ligation reaction comprising oligonucleotides.

14. The RNA ligase for the use of claim 13, wherein the oligonucleotide is selected from RNA, antisense oligonucleotides, guide RNA, sgRNA, siRNAs and RNA-DNA hybrids.

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