CN112746063B - New function and application of nucleoside transferase - Google Patents

Abstract

The present invention provides novel functions and applications of nucleoside transferases. The invention provides terminal deoxyribonucleoside transferases from various species, and the terminal deoxyribonucleoside transferases have excellent catalytic activity when used for catalyzing the nucleotide polymerization reaction modified by a blocking group, and are beneficial to realizing the synthesis of efficient and controllable nucleic acid molecules.

Description

New function and application of nucleoside transferase

Cross Reference to Related Applications

This application claims priority and benefit to the chinese patent application No. 201911034444.6 filed on 29/10/2019, the entire contents of which are incorporated herein by reference.

Technical Field

The invention belongs to the field of genetic engineering, and relates to a new function and application of terminal deoxyribonucleoside transferase.

Background

DNA is a carrier of life information, and obtaining DNA is the first step in research, modification and creation of life. DNA synthesis technology is the most important common basic security technology in the field of life science. There are two main methods for de novo oligonucleotide synthesis: chemical synthesis (solid phase phosphoramidite triester synthesis) and biological synthesis (enzymatic synthesis). In the 50 s of the 20 th century, chemical and enzymatic methods were developed, and the key to oligonucleotide synthesis became controllable, efficient, and sustainable synthesis. In 1981, a solid phase phosphoramidite synthesis method was invented, which uses porous glass (CPG) or Polystyrene (PS) as a solid phase carrier, and adds nucleotide monomers with phosphoramidite onto a synthesis chain one by one through four steps of deprotection, coupling, capping and oxidation, and synthesizes oligonucleotide by extending from 3 'end → 5' end, thereby obtaining an increasing oligonucleotide chain. The synthesis platform developed based on the classical solid phase phosphoramidite synthesis method mainly comprises column synthesis and chip synthesis. All major synthesis platforms have adopted the synthesis method so far, and both platforms have advanced with the development of the technology. However, the chemical synthesis (solid phase phosphoramidite triester synthesis) involves four cycles, which is complicated in reaction steps, long in time (6-8 minutes) for a single cycle, high in chemical reagent consumption and cost, and large in pollution due to the use of a large amount of toxic and flammable organic reagents. In the phosphoramidite four-step method, phosphoramidite of trivalent phosphorus is used as a synthetic monomer, and the molecular structure of the trivalent phosphorus in the valence state is easy to oxidize and react with water, so in order to ensure high coupling efficiency and low synthesis error rate, the whole coupling reaction process needs strict anhydrous and anaerobic environment, i.e. the reaction process needs inert gas protection, and reagents and cleaning solvents used in the reaction need strict control of moisture content, usually the moisture content in the reagents needs to be less than or equal to 30ppm, which inevitably increases the synthesis difficulty and synthesis cost of oligonucleotide.

Since trichloroacetic acid (TCA) is required for the deprotection step in the chemical synthesis to remove the DMT protecting group for the next condensation, when the continuously extending oligonucleotide chain is repeatedly exposed to the protonic acid, side reactions such as depurination residues of deoxyadenosine and deoxyguanosine occur, resulting in an increase in the error rate. In addition to depurination, another problem associated with acid-depurination of DMT protecting groups is the reversible formation of DMT carbenium ions. To completely remove the DMT group, the carbenium ion must be washed off the solid support surface. Otherwise, the 5' -hydroxyl of the deoxynucleotide is re-protected by residual carbenium ion resulting in the generation of a series of failed sequences that continue to extend during each cycle of synthesis. Due to the influence of the above factors, the chemical method for synthesizing oligonucleotides has the problems of short synthesis length, high error rate of longer chain fragments and the like.

Therefore, how to realize the controlled synthesis of oligonucleotides by using biological enzymes is becoming the focus of research. With the development of DNA sequencing, nucleotides with modified blocking groups, which were used for sequencing in the past, were increasingly used for enzymatic oligonucleotide synthesis, and incorporation of only one nucleotide per reaction was achieved. In recent years, various methods have been developed, in which a nucleotide monomer having a chemical group modified at different sites is used to achieve the effect of terminating a reaction by incorporating a nucleotide, thereby achieving the purpose of synthesizing an oligonucleotide in a controlled manner. At present, there are two main methods for enzymatic oligonucleotide synthesis: (1) adopting nucleotide with a blocking group added at the 3 'end as a substrate, only catalyzing the incorporation of one nucleotide with a modification group through enzymatic reaction, and then removing the modification group by adopting a chemical or biological method to ensure that the 3' end is restored to OH; (2) the addition of blocking groups at the remaining sites of the nucleotide, again only catalyzes one modified nucleotide per enzymatic reaction, which then requires de-blocking to allow the catalytic reaction to continue. However, the existing DNA polymerase has low activity, and how to realize high-efficiency controllable enzymatic oligonucleotide synthesis is still a great problem.

Disclosure of Invention

In view of the above-mentioned drawbacks, the present invention provides a Terminal Deoxyribonucleoside Transferase (TDT) selected from any one of the following amino acid sequences:

(a) comprises an amino acid sequence shown by any one of SEQ ID NO 2, 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 and SEQ ID NO 11; or

(b) A functional equivalent or fragment thereof having at least 60% sequence homology with (a), e.g. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% homology.

According to the present invention, the terminal deoxyribonucleoside transferases are derived from the following organisms, respectively: duckbilled, rat ear bat, nudzokor, chimpanzee, gecko, emu, jungle, diphtheria finch, tennons, golden carving.

In one embodiment, the terminal deoxyribonucleoside transferase can synthesize a nucleic acid molecule without a template strand, such as a DNA strand or an RNA strand, including synthetic DNA/RNA cycles iterations useful for catalyzing the synthesis of oligonucleotides, or for other cycles of synthesizing functional DNA and/or RNA fragments, or for catalyzing a DNA synthesizer. Specifically, the terminal deoxyribonucleoside transferase can be efficiently coupled with a 3-terminal modified nucleotide to synthesize a nucleic acid molecule. Adding a reversible blocking group to the 3' end of the nucleotide for modification; preferably, the reversible blocking group is an amino, O-allyl, O-azido, O-phosphate group. Further, the 5' end of the nucleotide optionally contains 2 phosphate groups or 3 phosphate groups.

In a second aspect, the invention also provides a nucleic acid sequence encoding said terminal deoxyribonucleoside transferase. In one embodiment, the nucleic acid sequence is derived from a gene encoding the terminal deoxyribonucleoside transferase in the above-mentioned organism.

In one embodiment, the terminal deoxyribonucleoside transferase has greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% sequence homology to the sequence of (a).

In a third aspect, the invention also relates to an expression cassette comprising said nucleic acid sequence, a vector comprising said nucleic acid or expression cassette. Further, the present invention also provides a transformant comprising the nucleic acid sequence, the expression cassette or the vector, for example, an original host cell transformed or transfected with the nucleic acid sequence, the expression cassette or the vector to form a transformant.

In a specific embodiment, the expression cassette comprises all elements for expressing the terminal deoxyribonucleoside transferase, including elements necessary for transcription and translation in a host cell, for example, the expression cassette comprises a promoter and a terminator, which are not particularly limited and may be those known in the art to be capable of effecting expression of the terminal deoxyribonucleoside transferase. For example, the promoter may be prokaryotic or eukaryotic and may be selected from, for example, the Lacl, LacZ, pclact, ptac, T3 or T7 phage RNA polymerase promoters, CMV promoter, HSV thymidine kinase promoter, SV40 promoter, mouse metallothionein-L promoter, and the like. The expression cassette of the present invention may optionally further comprise an enhancer or other necessary elements.

In a particular embodiment, the host cell may be a prokaryote, such as E.coli, or a eukaryote. The eukaryote may be a lower eukaryote such as a yeast (e.g. pichia pastoris or kluyveromyces lactis) or a fungus (e.g. Aspergillus) or a higher eukaryote such as an insect cell (e.g. Sf9 or Sf21), a mammalian cell or a plant cell. The cell may be a mammalian cell, such as COS (green monkey cell line), CHO (Chinese hamster ovary cell line), mouse cell, human cell, and the like.

In a specific embodiment, the vector may be a plasmid, phage, phagemid, cosmid, virus, YAC, BAC, Agrobacterium (Agrobacterium) pTi plasmid or the like. The vector may preferably comprise one or more elements selected from the group consisting of: an origin of replication, a multiple cloning site and an optional gene. Preferably, the vector is a plasmid. Some non-exhaustive examples of prokaryotic vectors are as follows: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH 46A; ptrc99a, pKK 223-3, pKK 233-3, pDR540, pBR322, pRIT5, pET-28 a. Preferably, the vector is an expression vector, preferably pET-28 a.

In a fourth aspect, the present invention also provides a method for producing the terminal deoxyribonucleoside transferase, comprising culturing the transformant of the third aspect comprising the nucleic acid sequence, the expression cassette or the vector, and optionally collecting the terminal deoxyribonucleoside transferase produced by the transformant in a culture solution.

In a fifth aspect, the invention also provides the use of the terminal deoxyribonucleoside transferase in catalyzing a polymerization reaction of nucleotides, or the use of the terminal deoxyribonucleoside transferase in synthesizing a nucleic acid molecule.

In one embodiment, the terminal deoxyribonucleoside transferase synthesizes a nucleic acid molecule without a template strand, e.g., the terminal deoxyribonucleoside transferase can synthesize a DNA strand or an RNA strand without a template strand.

In one embodiment, the terminal deoxyribonucleoside transferase is coupled to a 3 'modified nucleotide to synthesize a nucleic acid molecule, wherein the 3' modified nucleotide is a nucleotide to which a reversible blocking group, specifically exemplified by amino, O-allyl, O-azido, O-phosphate group, is added to the 3 'end, and further, the 5' end of the nucleotide optionally contains 2 phosphate groups or 3 phosphate groups. The terminal deoxyribonucleoside transferase can improve the polymerization efficiency of a nucleotide modified at the 3' end.

In a sixth aspect, a method of synthesizing a nucleic acid molecule without a template strand is presented, comprising the steps of: a) providing a starting sequence; b) adding a nucleotide provided by the invention with a reversible blocking group at the 3' end in the presence of Terminal Deoxyribonucleoside Transferase (TDT) provided by the first aspect of the invention to the starting sequence; c) removing the TDT (or removing the solvent); d) cleaving the reversible blocking group in the presence of a cleaving agent; e) the cleavage agent is removed. The synthesis process may be carried out in one or more cycles.

It will be appreciated that steps (b) to (e) of the method may be repeated a plurality of times to produce a DNA or RNA strand of the desired length. Thus, in one embodiment, more than 1 nucleotide is added to the starting sequence, for example more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides are added to the starting sequence by repeating steps (b) to (e). In another embodiment, more than 200 nucleotides are added, such as more than 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides.

In one embodiment, in the method for synthesizing a nucleic acid molecule, the reversible blocking group carried by the nucleotide having the reversible blocking group at the 3' end is X. In one embodiment, X represents-OR, and R represents C_1-6Alkyl radical, CH₂N₃、N₃、NH₂Allyl or phosphate groups.In an alternative embodiment, X represents C_1-6Alkyl radical, CH₂N₃、N₃、NH₂. In one embodiment, the nucleotide with a reversible blocking group at the 3' end is selected from the group consisting of amino, O-allyl, O-azido, O-phosphate groups.

In one embodiment, the lysing agent is a chemical lysing agent. In one embodiment, the cleavage agent is an enzymatic cleavage agent. In one embodiment, the cleavage agent is selected from tris (2-carboxyethyl) phosphine (TCEP), palladium complexes, sodium nitrite, or T4 polynucleotide kinase (PNK).

In a seventh aspect, the present invention provides the use of a kit in a method of nucleic acid synthesis, wherein the kit comprises a terminal deoxyribonucleoside transferase as defined in the first or second aspect of the invention, a starting sequence, one or more 3 'modified nucleotides, the 3' end bearing a reversible blocking group, specifically exemplified by amino, O-allyl, O-azido, O-phosphate group modification, optionally a cleavage agent. In one embodiment, the kit further optionally comprises instructions for using the kit according to any of the methods defined herein. The nucleotides, starting sequences have the meaning described above.

Advantageous effects

The present inventors have obtained Terminal Deoxyribonucleoside Transferase (TDT) from various sources, and have unexpectedly found that the TDT enzyme of the present invention has very high catalytic activity and substrate specificity for deoxyribonucleotides modified at the 3' end with amino, O-allyl, O-azido, or O-phosphate groups. Therefore, the mild conditions of action can reduce the formation of by-products and DNA damage, such as depurination, thereby directly synthesizing longer-chain oligonucleotides, and being used for synthesizing oligonucleotides with high efficiency and controllability.

Furthermore, the reaction is carried out under aqueous conditions, avoiding the need to use strong organic solvents which may be harmful to the environment.

Drawings

FIG. 1: plasmid map of the TDT-encoding gene sequence constructed into expression vector pET-28a in example 1.

FIG. 2: schematic coupling reaction of TDT with X-modified nucleotides at the 3' end. Wherein x represents an amino group, an O-allyl group, an O-azido group, or an O-phosphoric acid group.

FIG. 3: various TDTs add different substrates to the comparison of the starting sequences. A: the substrate is deoxyribonucleotide; b: the substrate is deoxyribonucleotide with amino-modified 3' end; c: the substrate is deoxyribonucleotide modified by azido at the 3' end. CK: no enzyme blank control; TDT-1-TDT-11 corresponds to TDT enzyme with SEQ ID NO of 1-11 and represents the catalytic result of the TDT enzyme from different species; dotted gray line: an oligonucleotide chain (n); black dotted line: the oligonucleotide chain (n + 1).

FIG. 4: efficiency of mouse TDT catalysis of reversible blocking group at 3' end. C: initial sequence control (16 nt); 1,2,3 and 4 respectively show that the mouse TDT is used to insert various deoxyribonucleotides with reversible blocking groups at the 3' end shown in figure 2.

FIG. 5: efficiency of catalyzing 3' end with reversible blocking group by using gold carving TDT. C: initial sequence control (16 nt); 1,2,3 and 4 respectively show that the deoxyribonucleotides with the 3' end provided with the reversible blocking groups shown in the figure 2 are inserted by using gold carving TDT.

FIG. 6: TDT of different species catalyzes the efficiency of the reversible blocking group at the 3' end. n: initial sequence control (16 nt); the substrates 1,2,3,4 correspond to the deoxyribonucleotides of FIG. 2 with a reversible blocking group, respectively. The ratio of n to n +1 is: after TDT catalysis, the ratio of the band (n +1) concentration of one deoxyribonucleotide divided by the total DNA (the sum of the intensity values of the 16bp and 17bp DNA bands of the lane)) concentration was added.

FIG. 7: the DNA strand is synthesized without the template strand. n, the start sequence; n + 1: a DNA sequence incorporating 1 nucleotide; n + 2: a 2 nucleotide DNA sequence was incorporated.

Detailed Description

Definitions and explanations

Amino acids in the present invention are represented by a single or three letter code and have the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).

In the present invention, "homology" has the conventional meaning in the art and refers to "identity" between two nucleic acid or amino acid sequences, the percentage of which represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment (best alignment), the differences between the two sequences being randomly distributed over their entire length. Percent homology or percent identity means the percentage of identical nucleotides or amino acid residues between two sequences to be compared, obtained after optimal alignment (best alignment), which percentage is purely statistical and the differences between the two sequences are randomly distributed and distributed over their entire length. Generally, an optimal alignment (best alignment) is one in which the percentage of identity between two sequences to be compared is highest, e.g., a sequence comparison between two nucleic acid or amino acid sequences is made by comparing the sequences after optimal alignment, the comparison being made over a segment or comparison window to identify and compare local regions of sequence similarity; such comparison of sequences can be performed manually or using sequence alignment tools (e.g., online sequence alignment software). For example: polypeptide SEQ ID NO: x and SEQ ID NO: y, which is the amino acid sequence in the aligned sequence SEQ ID NO: x and SEQ ID NO: the sum of common amino acids between Y divided by SEQ ID NO: x or SEQ ID NO: the shorter length of Y, expressed as a percentage.

"functional equivalents or fragments" in the context of the present invention mean polypeptides which differ from the exact sequence of TdT of SEQ ID NO 2-11 according to the invention, but which can exert the same function, i.e.catalyze the addition of nucleotide triphosphates to the 3' end of a DNA strand in a template-independent manner.

In the context of the present invention, the terms "primer" and "initiation sequence" are used interchangeably and refer to an initial nucleic acid fragment, typically an RNA oligonucleotide, DNA oligonucleotide or chimeric sequence that is complementary to a primer binding site formed by all or part of a target nucleic acid molecule. The starting sequence may comprise natural, synthetic or modified nucleotides. The lower limit of the primer length is the minimum length required for a stable duplex to form under the conditions of the nucleic acid amplification reaction. The term "starting sequence" as used herein refers to an oligonucleotide having a free 3 'end to which a 3' modified nucleotide can be ligated. In one embodiment, the initiation sequence is a DNA promoter sequence. In another embodiment, the initiation sequence is an RNA promoter sequence.

In the present invention, the term "substitution" with respect to an amino acid position or residue means that the amino acid at a specific position has been replaced with another amino acid. Substitutions may be conservative or non-conservative.

In the present invention, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA may be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the alteration in the synthesis of a gene caused by the introduction of foreign DNA into a cell. The term "terminal deoxyribonucleoside transferase produced from a transformant" refers to a product obtained by culturing the transformant according to a known method for culturing a microorganism.

In the present invention, "synthesis of nucleic acid molecules" includes a method of synthesizing a length of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), in which a strand of nucleic acid (n) is extended by adding additional nucleotides (n + 1). In one embodiment, the nucleic acid is DNA. In an alternative embodiment, the nucleic acid is RNA. For example, a DNA strand synthesis method in which the DNA strand (n) is extended by adding additional nucleotides (n + 1). The methods described herein provide for the novel use of terminal deoxynucleotidyl transferases of the invention and 3' reversibly modified nucleotides to add nucleotides sequentially in de novo DNA strand synthesis.

In the present invention, the term "nucleotide" may be deoxyribonucleotides (dNTPs) or dideoxyribonucleotides (ddNTPs); in one embodiment, the nucleotide may be a nucleotide triphosphate, meaning a molecule that contains a nucleoside bound to three phosphate groups (i.e., a base linked to a deoxyribose or ribose molecule). Examples of deoxyribonucleotide-containing nucleotide triphosphates are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP). Examples of ribose-containing nucleotide triphosphates are: adenosine Triphosphate (ATP), Guanosine Triphosphate (GTP), Cytidine Triphosphate (CTP) or Uridine Triphosphate (UTP), but also other nucleosides can be combined with the three phosphate esters to form nucleotide triphosphates, such as naturally occurring modified and artificial nucleosides. "3 ' modified nucleotide" as used herein refers to a nucleotide having an additional group at the 3' terminus (e.g., dATP, dGTP, dCTP, or dTTP) which prevents further addition of nucleotides during the reaction, i.e., by replacing the 3' group with a blocking group. It will be appreciated that the present method uses a reversible 3' blocking group which can be removed by cleavage (e.g. a cleavage agent) to allow for the addition of additional nucleotides.

There are several proven reversible protecting groups, such as amino, O-allyl, O-azido, O-phosphate groups, which can be applied in the methods of the present invention. Examples of suitable protecting Groups are described in Greene's Protective Groups in Organic Synthesis, (Wuts, P.G.M. & Greene, T.W. (2012), 4 th edition, John Wiley & Sons).

"TDT" in the present invention refers to Terminal Deoxynucleotidyl Transferase (TDT) and includes references to purified and recombinant forms of the enzyme. TDT is also commonly referred to as DNTT (DNA nucleotidyl transferase), and any such terms should be used interchangeably.

"cleavage agent" as referred to herein refers to a substance capable of cleaving off a blocking group from a 3' end modified nucleotide, which can be removed quantitatively in aqueous solution entirely with compounds that have been documented to be useful as cleavage agents. The 3' terminal blocking groups described in the present invention can all be removed quantitatively in aqueous solution with compounds which have been documented to be useful as lytic agents (see, for example, Wuts, PGM & Greene, TW (2012) fourth edition, John Wiley & Sons; Hutter, D. et al (2010) Nucleic Acids 29,879-895; EP 1560838 and US 7,795,424). In one embodiment, the cleavage agent of the invention is a chemical cleavage agent or an enzymatic cleavage agent. It will be appreciated by those skilled in the art that the choice of cleavage agent will depend on the type of-OH nucleotide blocking group used. Those skilled in the art will appreciate that the choice of cleavage agent will depend on the type of 3' -nucleotide blocking group used. For example, palladium complexes can be used to cleave 3 '-O-allyl groups, or sodium nitrite can be used to cleave 3' -O-amino groups, tris (2-carboxyethyl) phosphine (TCEP) can be used to cleave 3 '-O-azido groups, and T4 polynucleotide kinase (PNK) can be used to cleave 3' -O-phospho groups. Thus, in one embodiment, the lysing agent is selected from: tris (2-carboxyethyl) phosphine (TCEP), palladium complex, sodium nitrite or T4 polynucleotide kinase (PNK). "method for synthesizing nucleic acid" of the present invention "

In one embodiment, the Terminal Deoxynucleotidyl Transferase (TDT) of the present invention is used in a kit comprising one or more buffers (e.g., Tris, Hepes, Mops, phosphate, carbonate, or dimethylarsinate, etc.), one or more salts (e.g., Na)⁺、K⁺、Mg²⁺、Mn²⁺、Cu²⁺、Zn²⁺、Co²⁺Etc. all with appropriate counterions, e.g. Cl^-) One or more solutions stabilizing the protein structure (e.g.glycerol, PMSF or Triton etc.) and an extension solution of an inorganic pyrophosphatase (e.g.Saccharomyces cerevisiae homologue). It will be appreciated that the choice of buffer and salt will depend on optimal enzyme activity and stability.

In one embodiment, step (b) is performed at a pH range between 5 and 10. Thus, it will be appreciated that any buffer having a buffer range of pH 5-10 may be used, for example, Tris, Hepes, Mops, phosphate, carbonate, dimethylarsinate or the like.

In one embodiment, the pH of the lysing agent is not limited, it being understood that any buffer comprising any pH of the lysing agent may be used, it being understood that the optimum temperature will depend on the lysing agent used. The temperature used helps to aid cutting.

In one embodiment, step (d) is performed at a temperature of less than 99 ℃, e.g., less than 95 ℃, 90 ℃, 85 ℃, 80 ℃, 75 ℃, 70 ℃, 65 ℃, 60 ℃, 55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃ or 30 ℃. It will be appreciated that the optimum temperature will depend on the cracking agent used. The temperature used helps to aid cleavage and destroy any secondary structure formed during nucleotide addition.

In one embodiment, steps (c) and (e) are performed by applying a wash solution. In one embodiment, the wash solution comprises the same buffers and salts as used in the extension solution described herein. This has the advantage of allowing the wash solution to be collected after step (c) and recycled as an extension solution in step (b) when repeating the method steps.

The template-independent nucleic acid synthesis methods described herein have the ability to add nucleic acid sequences of defined composition and length to the starting sequence. Thus, one skilled in the art will appreciate that the methods described herein can be used as novel methods for introducing linker sequences into nucleic acid libraries.

If the starting sequence is not a defined sequence, but a library of nucleic acid fragments (e.g.produced by sonication of genomic DNA, or e.g.messenger RNA), the method enables de novo synthesis of "adaptor sequences" on each fragment. The installation of linker sequences is an integral part of the library preparation of next generation library nucleic acid sequencing methods, as they contain sequence information that allows hybridization to flow cell/solid support as well as hybridization to sequencing primers.

Currently used methods include single-stranded ligation, but this technique is limited because ligation efficiency decreases strongly with increasing fragment length. Thus, current methods are not capable of binding sequences that are over 100 nucleotides in length. Thus, the methods described herein allow for improved library preparation in a manner that is currently possible.

Thus, in one embodiment, a linker sequence is added to the starting sequence. In another embodiment, the starting sequence may be a nucleic acid from a library of nucleic acid fragments.

Reagent kit

According to a further aspect of the invention there is provided the use of a kit in a method of nucleic acid synthesis, wherein the kit comprises a TDT as defined in the first or second aspects of the invention, optionally in combination with one or more components selected from: a start sequence, one or more 3' -blocked nucleotide triphosphates, an inorganic pyrophosphatase, and a cleaving agent; further optionally comprising instructions for using the kit according to any of the methods defined herein.

Suitably, the kit according to the invention may further comprise one or more components selected from: an extension solution, wash solution and/or lysis solution as defined herein; optionally instructions for using the kit according to any of the methods defined herein.

The present invention is further illustrated in the following examples, which are not intended to limit the scope of the invention. The details of the partial molecular cloning method vary depending on the reagents, enzymes or kits provided by the supplier, and should be conducted according to the product instructions, and will not be described in detail in the examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 obtaining of TDT protein

1. TDT protein amino acid sequence of different species

The amino acid sequences are respectively as follows: SEQ ID NO 1 (mouse), SEQ ID NO 2 (duckbilled), SEQ ID NO 3 (Mylabris, Euzokor), SEQ ID NO 4 (nudzokor), SEQ ID NO 5 (chimpanzee), SEQ ID NO 6 (gecko), SEQ ID NO 7 (emu), SEQ ID NO 8 (rooster), SEQ ID NO 9 (diphtheria finches), SEQ ID NO 10 (falcon), SEQ ID NO 11 (gold carvings). Homology is shown in table 1:

TABLE 1 analysis of amino acid sequence homology of TDT proteins from different species

2. Construction of expression vectors

Can be synthesizedAll gene sequences of the amino acid sequences shown in the list can be used for the construction of the expression vector. This example constructed the gene sequence encoding TDT protein in the above species described in NCBI into expression vector pET-28a (Novagen, Kan)⁺See figure 1), and the recombinant plasmid is obtained and named pET-28a-TDT between the enzyme cutting sites NdeI and XhoI. pET-28a is described as an example, and the expression vector is not limited thereto, and all vectors that can be used to express a protein are included. In the examples, NdeI and XhoI cleavage sites are mentioned, the cleavage sites are not limited thereto, and the cleavage sites which can be used for constructing the expression gene fragment in the expression vector are all included.

3. Expression of genes

For the in vitro detection of the TDT enzyme activity, the enzyme was exogenously expressed and purified in E.coli. The host bacterium described in the examples is e.coli BL21(DE3), but the host bacterium is not limited thereto, and all hosts that can be used for expressing proteins are included.

(1) Transferring the escherichia coli expression recombinant plasmid pET-28a-TDT into E.coli BL21(DE3) to obtain a recombinant bacterium. Positive clone screening using kanamycin-resistant plates (Kan)⁺100mg/mL), cultured overnight at 37 ℃;

(2) selecting single clone to 5mL LB liquid culture medium (Kan)⁺100mg/mL), culturing at 37 deg.C and 220r/min to OD₆₀₀Is 0.6-0.8. Transfer 5mL of LB medium to 800mL of 2YT medium (Kan)⁺100mg/mL), cultured at 37 ℃ and 220rpm to OD₆₀₀When the concentration is 0.6-0.8 ℃, cooling to 16 ℃, adding IPTG (isopropyl thiogalactoside) to the final concentration of 0.5mM, and carrying out induced expression for 16 h;

(3) collecting the culture bacteria liquid into a bacteria collection bottle, and centrifuging at 5500r/min for 15 min;

(4) the supernatant was discarded, and the resulting pellet was suspended in 35mL of protein buffer (50mM Tris-HCl, 2mM EDTA, 0.1% Triton X-100, pH7.4) and poured into a 50mL centrifuge tube and stored in a freezer at-80 ℃.

4. Protein purification

(1) Breaking the bacteria: and (3) breaking the bacterial precipitation obtained in the step (3) for 2 times by adopting a high-pressure low-temperature breaker under the conditions of the pressure of 1200bar and the temperature of 4 ℃. Centrifuging at 4 deg.C and 10000r/min for 45min, collecting precipitate and supernatant, and sampling;

(2) and (3) purification: filtering the supernatant with a 0.45 μm microporous membrane, and purifying by nickel affinity chromatography, which comprises the following steps:

a: column balancing: before hanging the supernatant, ddH is firstly used₂ Washing 2 column volumes with O, and balancing 1 column volume of the Ni affinity chromatography column with protein buffer solution;

b: loading: the supernatant was passed through the Ni affinity column slowly at a flow rate of 0.5mL/min and repeated again;

c: and (3) eluting the hybrid protein: washing 1 column volume by using a protein buffer solution, eluting and binding stronger hybrid protein by using 50mL of protein buffer solution containing 50mM imidazole, dripping a few previous samples to prepare samples;

d: eluting the target protein: the target protein was eluted with 20mL of a buffer containing 100mM, 200mM, and 300mM of imidazole protein, and the first few samples were run through the column and prepared, and the results of 12% SDS-PAGE are shown in FIG. 2.

(3) Concentrating and replacing liquid: the collected target protein was concentrated by centrifugation (4 ℃ C., 3400r/min) using a 50mL Amicon ultrafiltration tube (30kDa, Millipore Co.) to 1 mL. 10mL of protein buffer was added, the mixture was concentrated to 1mL, and the process was repeated 1 time to obtain the purified protein TDT.

(4) The concentration of the concentrated protein was measured by a Nondrop 2000 microspectrophotometer and found to be 4 mg/mL. Thus obtaining the purified and concentrated TDT protein.

Example 2 functional verification

The adopted substrates in the embodiment of the invention are as follows:

(1) deoxyribonucleotides (dNTPs) including adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, thymine deoxyribonucleotide;

(2) based on the deoxyribonucleotide of (1), the 3'-OH terminal is changed to a reversible blocking group, and preferably, the modifying group at the 3' -terminal is an amino group.

The in vitro pure enzyme reaction, the reaction scheme is shown in FIG. 2.

Reaction system: 100mM NaCl, 0.25mM CoCl₂，50mM KAc，10mM Mg(Ac)₂pH 6.8. Substrate: mu.M of the starting sequence 1(14bp), 100. mu.M of deoxyribonucleotide or 100. mu.M of deoxyribonucleotide having an amino group at the 3' -end. Enzyme: 50 μ M TDT protein from different species prepared in example 1. Starting sequence 1: TAATACGACTCACT

Reaction conditions are as follows: reacting at 30 ℃ for 30s, inactivating protein at 95 ℃, and centrifuging to obtain supernatant. The results of the detection using the Qsep100 full-automatic nucleic acid analysis system of bioptic are shown in FIG. 3.

As can be seen from the results in FIG. 3, TDT1-11 derived from different species has the function of catalyzing deoxyribonucleotides (FIG. 3A); and also has a function of catalyzing deoxyribonucleotides whose 3' -end is modified with an amino group (FIG. 3B); it does not have a function of catalyzing a deoxyribonucleotide modified with an azido group at the 3' end (FIG. 3C).

Example 3 functional verification

The adopted substrates in the embodiment of the invention are as follows:

(1) deoxyribonucleotides (dNTPs) including adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, thymine deoxyribonucleotide;

(2) based on the deoxyribonucleotide of (1), the 3 'end is changed into a reversible blocking group, and preferably, the modification group at the 3' end is an oxyamino group; preferably, the modifying group at the 3' end is an oxyallyl group; preferably, the modifying group at the 3' end is an oxazido group; preferably, the modifying group at the 3' end is an oxyphosphate group.

The deoxyribonucleotides with a reversible blocking group at the 3 'end provided above by way of example are thymine deoxyribonucleotides, and the remaining three deoxyribonucleotides differ from this example only in the base end, with the reversible blocking group at the 3' end being identical.

Reaction system: 100mM NaCl, 0.25mM CoCl₂，50mM KAc，10mM Mg(Ac)₂pH 6.8. Substrate: 0.2. mu.M of the starting sequence 2(16nt),100 μ M deoxyribonucleotide with a reversible blocking group at the 3' end. Enzyme: 50 μ M TDT protein from different species. Starting sequence 2: TAATACGACTCACTAC are provided. For deoxyribonucleotides with an oxyphosphate group at the 3' end, after the reaction is finished, T4 polynucleotide kinase (PNK) is adopted to remove a phosphate protecting group, and then deformed polyacrylamide gel analysis is carried out, wherein the PNK reaction condition refers to the commercial PNK enzyme reaction condition.

Reaction conditions are as follows: reacting at 30 deg.C for 60min, inactivating protein at 95 deg.C, centrifuging, and collecting supernatant. Samples were analyzed on a 20% denaturing polyacrylamide gel (Biorad) running at 300V for 3 h. The gel was stained with 1 × SYBR Gold for 20min, and the brightness of each DNA band in the picture was further analyzed by a fluorescent gel imager of Tanon to obtain a brightness value, and the nucleotide incorporation efficiency was obtained by dividing the brightness value of the DNA band after incorporation of the nucleotide (17bp corresponding to the DNA band brightness value) by the brightness value of the total DNA band (the sum of the brightness values of 16bp and 17bp corresponding to the DNA bands).

FIG. 4 shows that in the presence of the starting sequence (C control), with the 3 'terminal carrying an oxyamino group (1), an oxyallyl group (2), an oxyazide group (3) and an oxyphosphate group (4) as substrates, the mouse TDT has no significant function of incorporating the starting sequence into nucleotides, i.e., the mouse TDT has no function of catalyzing deoxyribonucleotides with reversible blocking groups at the 3' terminal.

FIG. 5 shows that, in the presence of the initiation sequence (C control), the gold carving TDT has a significant function of enabling the initiation sequence to be doped into nucleotide by using an oxyamino group (1), an oxyallyl group (2), an oxyazide group (3) and an oxyphosphate group (4) at the 3 'end as substrates, namely the gold carving TDT has a function of catalyzing deoxyribonucleotide with a reversible blocking group at the 3' end.

FIG. 6 efficiency of TDT catalysis of 3' end with reversible blocking group of different species

FIG. 6 shows that TDT from different species has different catalytic efficiency for deoxyribonucleotides with reversible blocking group at the 3 'end, using as substrates the oxyamino group (substrate 1), oxyallyl group (substrate 2), oxyazide group (substrate 3) and oxyphosphate group (substrate 4) at the 3' end in the presence of the starting sequence (C control).

Example 4 example of synthesizing DNA strand without template strand

a) Providing an immobilized start sequence;

b) adding TDT from different species, deoxyribonucleotide with reversible blocking group at 3' end, buffer solution (such as phosphate), and salt ion (such as Mg)²⁺、Co²⁺) The solution is extended and reacted at optimized concentration, time and temperature. The deoxyribonucleotide with the reversible blocking group at the 3' end contains one of nitrogenous base adenine, guanine, cytosine or thymine;

c) the extension mixture is removed with an elution solution and recovered. The elution solution is an extension mixture which does not contain TDT and contains deoxyribonucleotides with reversible blocking groups at the 3' ends;

d) the immobilized DNA strand (n +1) is treated with a lysis mixture of a suitable buffer, a denaturing agent (e.g. urea, formamide, betaine, etc.) and a lysis agent (e.g.: palladium complexes can be used for cleaving 3 '-O-allyl groups, or sodium nitrite can be used for cleaving 3' -O-amino groups, tris (2-carboxyethyl) phosphine (TCEP) can be used for cleaving 3 '-O-azido groups, and T4 polynucleotide kinase (PNK) can be used for cleaving 3' -O-phospho groups. The temperature depends on the cracking agent used;

e) the immobilized deblocked DNA strand (n +1) is treated with a washing solution to remove the cleavage mixture.

f) Repeating steps (b) to (e) of the method to produce an oligonucleotide strand of the desired length.

FIG. 7 shows that in the presence of the starting sequence (n), the synthesis of a DNA strand (n +1 or n +2) without a template strand can be achieved using TDTs derived from different species provided by the present invention and deoxyribonucleotides having a reversible blocking group at the 3-terminus provided by the present invention as substrates

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

Novel function and application of <120> nucleoside transferase

<150> 201911034444.6

<151> 2019-10-29

<160> 13

<170> SIPOSequenceListing 1.0

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Met Asp Pro Leu Gln Ala Val His Leu Gly Pro Arg Lys Lys Arg Pro

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Arg Gln Leu Gly Thr Pro Val Ala Ser Thr Pro Tyr Asp Ile Arg Phe

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Arg Asp Leu Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg

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Arg Ala Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

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Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

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Gly Ser Asp Val Leu Glu Trp Leu Gln Leu Gln Asn Ile Lys Ala Ser

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Ser Glu Leu Glu Leu Leu Asp Ile Ser Trp Leu Ile Glu Cys Met Gly

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Ala Gly Lys Pro Val Glu Met Met Gly Arg His Gln Leu Val Val Asn

115 120 125

Arg Asn Ser Ser Pro Ser Pro Val Pro Gly Ser Gln Asn Val Pro Ala

130 135 140

Pro Ala Val Lys Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr

145 150 155 160

Leu Asn Asn Tyr Asn Gln Leu Phe Thr Asp Ala Leu Asp Ile Leu Ala

165 170 175

Glu Asn Asp Glu Leu Arg Glu Asn Glu Gly Ser Cys Leu Ala Phe Met

180 185 190

Arg Ala Ser Ser Val Leu Lys Ser Leu Pro Phe Pro Ile Thr Ser Met

195 200 205

Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Ser Ile

210 215 220

Ile Glu Gly Ile Ile Glu Asp Gly Glu Ser Ser Glu Ala Lys Ala Val

225 230 235 240

Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe

245 250 255

Gly Val Gly Leu Lys Thr Ala Glu Lys Trp Phe Arg Met Gly Phe Arg

260 265 270

Thr Leu Ser Lys Ile Gln Ser Asp Lys Ser Leu Arg Phe Thr Gln Met

275 280 285

Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Asn

290 295 300

Arg Pro Glu Ala Glu Ala Val Ser Met Leu Val Lys Glu Ala Val Val

305 310 315 320

Thr Phe Leu Pro Asp Ala Leu Val Thr Met Thr Gly Gly Phe Arg Arg

325 330 335

Gly Lys Met Thr Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu

340 345 350

Ala Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Thr Asp Phe

355 360 365

Trp Lys Gln Gln Gly Leu Leu Leu Tyr Cys Asp Ile Leu Glu Ser Thr

370 375 380

Phe Glu Lys Phe Lys Gln Pro Ser Arg Lys Val Asp Ala Leu Asp His

385 390 395 400

Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu Asp His Gly Arg Val His

405 410 415

Ser Glu Lys Ser Gly Gln Gln Glu Gly Lys Gly Trp Lys Ala Ile Arg

420 425 430

Val Asp Leu Val Met Cys Pro Tyr Asp Arg Arg Ala Phe Ala Leu Leu

435 440 445

Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala

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Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Arg

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Thr Lys Arg Val Phe Leu Glu Ala Glu Ser Glu Glu Glu Ile Phe Ala

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His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala

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Met Asp Arg Phe Ser Ala Thr Ile Ser Asp Leu Lys Lys Lys Arg Arg

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Arg Met Asn Gly Cys Ile Ser Pro Thr Leu Tyr Glu Ile Lys Phe Asn

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Glu Phe Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg Arg

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Ala Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu Ser

50 55 60

Glu Leu Ser Glu Ser Val Thr His Ile Val Ala Glu Asn Asn Ser Cys

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Ser Asp Val Leu Glu Trp Leu Ala Val Gln Asn Val Gly Asp Ser Ser

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Arg Phe Glu Leu Leu Asp Ile Ser Trp Leu Thr Glu Cys Met Lys Val

100 105 110

Gly Lys Pro Val Glu Ala Ile Gly Lys His Gln Leu Met Arg Gly Asn

115 120 125

Cys Leu Thr Asn Ser Ala Pro Ile Asn Cys Met Thr Glu Thr Pro Ser

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Leu Ala Thr Lys Gln Val Ser Gln Tyr Ala Cys Glu Arg Arg Thr Thr

145 150 155 160

Leu Asn Asn Cys Asn Gln Lys Phe Thr Asp Ala Phe Glu Ile Leu Ala

165 170 175

Lys Asp Phe Glu Phe Arg Glu Asn Glu Gly Ile Cys Leu Ala Phe Met

180 185 190

Arg Ala Ile Ser Val Leu Lys Cys Leu Pro Phe Thr Ile Val Arg Met

195 200 205

Lys Asp Ile Glu Gly Val Pro Trp Leu Gly Asp Gln Val Lys Ser Ile

210 215 220

Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Ser Val Lys Ala Val

225 230 235 240

Leu Asn Asp Glu Arg Tyr Arg Ser Phe Gln Leu Phe Thr Ser Val Phe

245 250 255

Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Tyr Arg Arg Gly Phe Arg

260 265 270

Thr Leu Asp Glu Val Lys Ala Asp Asn Ser Leu Lys Leu Thr Lys Met

275 280 285

Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ala

290 295 300

Lys Glu Glu Ala Asp Ala Val Tyr Leu Ile Val Lys Glu Ala Val Arg

305 310 315 320

Ala Phe Leu Pro Glu Ala Leu Val Thr Leu Thr Gly Gly Phe Arg Arg

325 330 335

Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Ser Asp Pro Glu

340 345 350

Ser Gly Gln Asp Glu Gln Leu Leu Pro Asn Ile Ile Lys Leu Trp Glu

355 360 365

Lys Gln Glu Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu

370 375 380

Lys Thr Lys Ile Pro Ser Arg Lys Val Asp Ala Met Asp His Phe Gln

385 390 395 400

Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Lys Val Asp Ser Gly

405 410 415

Arg Tyr Lys Pro Pro Pro Glu Ser Lys Asn His Glu Ala Lys Asn Trp

420 425 430

Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Phe Glu Gln Tyr Ala

435 440 445

Tyr Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu

450 455 460

Arg Arg Tyr Ala Thr His Glu Lys Lys Met Met Leu Asp Asn His Ala

465 470 475 480

Leu Tyr Asp Lys Thr Lys Lys Ile Phe Leu Lys Ala Glu Ser Glu Glu

485 490 495

Asp Ile Phe Thr His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg

500 505 510

Asn Ala

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Met Asp Ser Leu Gln Met Ala His Ser Gly Pro Arg Lys Lys Arg Pro

1 5 10 15

Arg Gln Met Gly Ala Ser Met Ala Ser Pro Pro Gln Asp Ile Lys Phe

20 25 30

Arg Asp Leu Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg

35 40 45

Arg Ala Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Gly Ser Asp Val Leu Glu Trp Leu Gln Val Gln Lys Val Arg Ala Ser

85 90 95

Ser Gln Pro Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Met Ser

100 105 110

Ala Gly Lys Pro Val Ala Thr Thr Gly Gln His Gln Leu Val Val Arg

115 120 125

Arg Asp Tyr Ser Gly Gly Pro Asn Pro Glu Pro Gln Glu Thr Pro Pro

130 135 140

Leu Ala Gly Arg Lys Ile Ser Pro Tyr Ala Cys Gln Arg Arg Thr Thr

145 150 155 160

Leu Asn Asn Arg Asn His Ile Phe Thr Asp Ala Phe Glu Val Leu Ala

165 170 175

Glu Asn Cys Glu Phe Arg Glu Asn Glu Gly Ser Cys Leu Ala Phe Met

180 185 190

Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met

195 200 205

Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Ser Ile

210 215 220

Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val

225 230 235 240

Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe

245 250 255

Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg

260 265 270

Thr Leu Ser Lys Ile Arg Ser Asp Lys Thr Leu Lys Phe Thr Arg Met

275 280 285

Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr

290 295 300

Lys Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp

305 310 315 320

Ala Phe Leu Pro Asp Ala Phe Val Thr Val Thr Gly Gly Phe Arg Arg

325 330 335

Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly

340 345 350

Ser Thr Glu Glu Glu Glu Gln Gln Leu Leu Pro Lys Val Ile Asn Leu

355 360 365

Trp Glu Arg Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr

370 375 380

Phe Glu Lys Phe Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His

385 390 395 400

Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp

405 410 415

Gly Gly Lys Ser Ser Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg

420 425 430

Val Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu

435 440 445

Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala

450 455 460

Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys

465 470 475 480

Thr Lys Arg Ile Phe Leu Glu Ala Glu Ser Glu Glu Asp Ile Phe Ala

485 490 495

His Leu Gly Leu Asp Tyr Ile Asp Pro Trp Glu Arg Asn Ala

500 505 510

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Met Asp Pro Leu Gln Thr Ala His Leu Gly Pro Arg Lys Lys Arg Pro

1 5 10 15

Arg Gln Thr Gly Thr Leu Met Ala Ser Gly Pro His Asn Ile Arg Phe

20 25 30

Gly Asp Leu Val Leu Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg

35 40 45

Arg Ala Phe Leu Met Glu Leu Ala Arg Lys Lys Gly Phe Arg Val Glu

50 55 60

Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Gly Asn Asp Val Leu Glu Trp Leu Gln Val Gln Asn Ile Gln Ala Ser

85 90 95

Ser Gln Leu Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Met Gly

100 105 110

Ala Gly Lys Pro Val Glu Met Thr Gly Arg His Gln Leu Val Val Arg

115 120 125

Asp Pro Pro Ala Ser Pro Asn Pro Gly Pro Gln Lys Thr Pro Ser Leu

130 135 140

Ala Val Gln Lys Ile Pro Glu Tyr Ala Cys Gln Arg Arg Thr Thr Leu

145 150 155 160

Asp Asn Cys Asn Tyr Ile Phe Thr Asn Ala Phe Glu Ile Leu Ala Glu

165 170 175

Asp Cys Glu Phe Arg Glu Asn Glu Gly Phe Tyr Val Thr Tyr Met Arg

180 185 190

Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met Lys

195 200 205

Asp Thr Glu Gly Ile Pro Cys Leu Gly Gly Arg Val Lys Cys Ile Ile

210 215 220

Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu

225 230 235 240

Asn Asn Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly

245 250 255

Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg Ser

260 265 270

Leu Ser Lys Ile Arg Ser Asp Lys Ser Leu Thr Phe Thr Arg Met Gln

275 280 285

Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg

290 295 300

Ala Glu Ala Glu Ala Val Asn Met Leu Val Lys Glu Ala Val Trp Thr

305 310 315 320

Phe Leu Pro Gly Ala Phe Ile Ser Met Thr Gly Gly Phe Arg Arg Gly

325 330 335

Lys Glu Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Glu Val

340 345 350

Thr Glu Asp Glu Glu Gln Gln Leu Leu His Lys Val Ile Asn Leu Trp

355 360 365

Glu Lys Lys Gly Leu Leu Leu Tyr Ser Asp Leu Val Glu Ser Thr Phe

370 375 380

Glu Lys Leu Lys Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe

385 390 395 400

Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Asn

405 410 415

Asp Lys Ser Pro Gln Gln Gly Gly Lys Thr Trp Lys Ala Ile Arg Val

420 425 430

Asp Leu Val Met Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly

435 440 445

Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr

450 455 460

His Glu Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr

465 470 475 480

Lys Lys Thr Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Thr His

485 490 495

Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala

500 505

<210> 5

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<212> PRT

<213> chimpanzee (Pan trogloytes)

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Met Asp Pro Pro Arg Ala Ser His Leu Ser Pro Arg Lys Lys Arg Pro

1 5 10 15

Arg Gln Thr Gly Ala Leu Met Ala Ser Ser Pro Gln Asp Ile Lys Phe

20 25 30

Gln Asp Leu Val Val Phe Ile Leu Glu Lys Lys Met Gly Thr Thr Arg

35 40 45

Arg Ala Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Gly Ser Asp Val Leu Glu Trp Leu Gln Val Gln Lys Ile Gln Val Ser

85 90 95

Ser Gln Pro Glu Leu Leu Asp Val Ser Trp Leu Ile Glu Cys Ile Gly

100 105 110

Ala Gly Lys Pro Val Glu Met Thr Gly Lys His Gln Leu Val Val Arg

115 120 125

Arg Asp Tyr Ser Asp Ser Thr Asn Pro Gly Pro Pro Lys Thr Pro Pro

130 135 140

Ile Ala Val Gln Lys Ile Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr

145 150 155 160

Leu Asn Asn Cys Asn Gln Ile Phe Thr Asp Ala Phe Asp Ile Leu Ala

165 170 175

Glu Asn Cys Glu Phe Arg Glu Asn Glu Asp Ser Cys Val Thr Phe Met

180 185 190

Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Ile Ser Met

195 200 205

Lys Asp Thr Glu Gly Ile Pro Cys Leu Gly Ser Lys Val Lys Gly Ile

210 215 220

Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val

225 230 235 240

Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Phe Thr Ser Val Phe

245 250 255

Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Arg

260 265 270

Thr Leu Ser Lys Val Arg Ser Asp Lys Ser Leu Lys Phe Thr Arg Met

275 280 285

Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr

290 295 300

Arg Ala Glu Ala Glu Ala Val Ser Val Leu Val Lys Glu Ala Val Trp

305 310 315 320

Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg

325 330 335

Gly Lys Lys Met Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly

340 345 350

Ser Thr Glu Asp Glu Glu Gln Leu Leu Gln Lys Val Met Asn Leu Trp

355 360 365

Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe

370 375 380

Glu Lys Leu Arg Leu Pro Ser Arg Lys Val Asp Ala Leu Asp His Phe

385 390 395 400

Gln Lys Cys Phe Leu Ile Phe Lys Leu Pro Arg Gln Arg Val Asp Ser

405 410 415

Asp Gln Ser Ser Trp Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val

420 425 430

Asp Leu Val Leu Cys Pro Tyr Glu Arg Arg Ala Phe Ala Leu Leu Gly

435 440 445

Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg Tyr Ala Thr

450 455 460

His Glu Arg Lys Met Ile Leu Asp Asn His Ala Leu Tyr Asp Lys Thr

465 470 475 480

Lys Arg Ile Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His

485 490 495

Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala

500 505

<210> 6

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Met Asn Met Ile Arg Ala Thr Gly Leu Ile Pro Met Arg Lys Lys Trp

1 5 10 15

Lys Gly Thr Gln Leu Ser Ala Pro Val Ser Ser Tyr Lys Ile Gln Phe

20 25 30

Asn Asp Ile Ile Ile Phe Ile Val Glu Lys Lys Met Gly Met Ser Arg

35 40 45

Arg Thr Phe Leu Met Asp Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Asn Glu Leu Ser Asp Ser Val Thr His Ile Val Thr Glu Asn Asn Ser

65 70 75 80

Cys Ala Glu Ile Leu Lys Trp Leu Gln Ala Gln Lys Val Glu Asp Ser

85 90 95

Ala Arg Phe Arg Ile Leu Asp Val Ser Trp Leu Thr Ala Cys Leu Glu

100 105 110

Ala Gly Arg Pro Val Asp Ser Glu Lys Tyr Arg Leu Val Gly Glu Lys

115 120 125

Cys Ser Ala Thr Ser Ser Met Gln Ser Val Asp Thr Ser Val Leu Gly

130 135 140

Thr Asp Ser Val Ser Gln Tyr Ala Cys Gln Arg Arg Thr Thr Leu Asn

145 150 155 160

Asn Tyr Asn Lys Lys Phe Thr Asp Ala Phe Glu Val Leu Ala Glu Asn

165 170 175

Tyr Glu Phe Arg Glu Asp Glu Gly His Cys Leu Ala Phe Arg Arg Ala

180 185 190

Ala Ser Val Leu Lys Phe Leu Pro Phe Ala Val Val Arg Val Ser Asp

195 200 205

Val Glu Gly Leu Pro Trp Met Gly Glu Gln Val Lys Ala Ile Ile Glu

210 215 220

Asp Ile Ile Glu Asp Gly Glu Ser Pro Arg Val Asn Ala Val Leu Asn

225 230 235 240

Ser Glu Asn Tyr Arg Ser Ile Lys Leu Phe Thr Ser Val Phe Gly Val

245 250 255

Gly Leu Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Phe Arg Thr Leu

260 265 270

Glu Glu Val Lys Cys Asp Lys Asn Leu Lys Leu Thr Arg Met Gln Arg

275 280 285

Ala Gly Phe Leu His Tyr Glu Asp Leu Val Ser Cys Val Ser Lys Ala

290 295 300

Glu Ala Asp Ala Ala Ser Leu Ile Val Lys Glu Ser Val Gln Arg Phe

305 310 315 320

Ser Pro Ser Ala Leu Val Thr Leu Thr Gly Gly Phe Arg Arg Gly Lys

325 330 335

Lys Ala Gly His Asp Val Asp Phe Leu Ile Thr Val Pro Gly Ser Ser

340 345 350

Gln Glu Asp Glu Leu Leu His Leu Val Ile Asp Phe Trp Lys Lys Gln

355 360 365

Gly Leu Val Leu Tyr Tyr Asp Leu Ile Glu Ser Thr Phe Glu Lys Lys

370 375 380

Lys Leu Pro Ser Lys Lys Val Asp Gly Leu Asp Asn Phe Gln Lys Cys

385 390 395 400

Phe Ile Ile Leu Lys Leu Pro Lys Gly Lys Val Asp Phe Gly Asn Ser

405 410 415

Val Ile Ser Val Ala Ser Ala Glu Gly Gly Lys Lys Asp Trp Lys Ala

420 425 430

Ile Arg Val Asp Leu Val Val Ser Pro Phe Glu Gln Tyr Ala Phe Ala

435 440 445

Leu Leu Gly Trp Ser Gly Ser Arg Gln Phe Glu Arg Asp Leu Arg Arg

450 455 460

Tyr Ala Thr His Glu Lys Lys Met Met Leu Asp Asn His Ala Leu Tyr

465 470 475 480

Asp Lys Thr Lys Lys Ile Phe Leu Lys Ala Ala Ser Glu Glu Glu Ile

485 490 495

Phe Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala

500 505 510

<210> 7

<211> 514

<212> PRT

<213> emu (Dromaius novaehollandiae)

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Met Asp Gly Ile Arg Ala Pro Ala Val Leu Ser Gln Arg Lys Arg Gln

1 5 10 15

Lys Gly Met His Ser Pro Asn Pro Pro Cys Ser Tyr Glu Ile Lys Phe

20 25 30

Asn Lys Phe Val Ile Phe Ile Met Gln Arg Lys Met Gly Met Thr Arg

35 40 45

Arg Thr Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Ser Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Tyr Leu Glu Val Leu Asp Trp Leu Arg Gly Gln Ala Val Gly Asp Ser

85 90 95

Ser Arg Phe Glu Leu Leu Asp Ile Ser Trp Phe Thr Ala Cys Met Glu

100 105 110

Ala Gly Arg Pro Val Ala Ser Glu Met Lys Tyr Arg Leu Met Glu Gln

115 120 125

Gln Asp Gln Phe Pro Thr Leu Asn Thr Ser Glu Pro Glu Val Pro Ser

130 135 140

Phe Ala Ala Asn Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr

145 150 155 160

Leu Asn Asn Phe Asn Lys Lys Phe Thr Asp Ala Phe Glu Ile Met Ala

165 170 175

Glu Asn Tyr Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu

180 185 190

Arg Ala Ala Ser Val Leu Lys Phe Leu Pro Phe Pro Val Val Arg Met

195 200 205

Lys Asp Ile Trp Gly Leu Pro Cys Met Gly Asp Gln Val Arg Asp Ile

210 215 220

Ile Glu Glu Ile Ile Glu Glu Gly Glu Ser Ser Arg Ala Lys Asp Val

225 230 235 240

Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Gln Phe Thr Ser Val Phe

245 250 255

Gly Val Gly Val Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg

260 265 270

Thr Leu Glu Glu Val Lys Ala Glu Lys Thr Leu Lys Leu Ser Lys Met

275 280 285

Gln Lys Val Gly Leu Leu His Tyr Glu Asp Leu Val Ser Cys Val Ser

290 295 300

Lys Glu Glu Ala Asp Ala Val Gly Leu Ile Val Lys Lys Thr Val Cys

305 310 315 320

Met Phe Leu Pro Asp Ala Leu Val Thr Val Thr Gly Gly Phe Arg Arg

325 330 335

Gly Lys Lys Ile Gly His Asp Ile Asp Phe Leu Ile Thr Asn Pro Gly

340 345 350

Pro Arg Glu Asp Asp Asp Leu Leu His Lys Val Val Asp Leu Trp Lys

355 360 365

Lys Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Val

370 375 380

Lys Glu Gln Leu Pro Ser Arg Lys Val Asp Ala Met Asp Asn Phe Gln

385 390 395 400

Lys Cys Phe Ala Ile Leu Lys Leu Tyr Gln Pro Arg Ala Asp Asn Arg

405 410 415

Ser Tyr Asn Ala Ser Lys Glu Phe Asp Met Ala Glu Val Lys Asp Trp

420 425 430

Lys Ala Ile Arg Val Asp Leu Val Ile Ser Pro Phe Glu Gln Tyr Ala

435 440 445

Tyr Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Gly Arg Asp Leu

450 455 460

Arg Arg Tyr Ala Asn His Glu Lys Lys Met Ile Leu Asp Asn His Ala

465 470 475 480

Leu Tyr Asp Lys Arg Lys Arg Ile Phe Leu Lys Ala Gly Ser Glu Glu

485 490 495

Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Val Glu Pro Trp Glu Arg

500 505 510

Asn Ala

<210> 8

<211> 514

<212> PRT

<213> Chicken (Jungle bowl)

<400> 8

Met Glu Arg Ile Arg Pro Pro Thr Val Val Ser Gln Arg Lys Arg Gln

1 5 10 15

Lys Gly Met Tyr Ser Pro Lys Leu Ser Cys Gly Tyr Glu Ile Lys Phe

20 25 30

Asn Lys Leu Val Ile Phe Ile Met Gln Arg Lys Met Gly Met Thr Arg

35 40 45

Arg Thr Phe Leu Met Glu Leu Ala Arg Ser Lys Gly Phe Arg Val Glu

50 55 60

Ser Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Tyr Pro Glu Val Leu Asp Trp Leu Lys Gly Gln Ala Val Gly Asp Ser

85 90 95

Ser Arg Phe Glu Ile Leu Asp Ile Ser Trp Leu Thr Ala Cys Met Glu

100 105 110

Met Gly Arg Pro Val Asp Leu Glu Lys Lys Tyr His Leu Val Glu Gln

115 120 125

Ala Gly Gln Tyr Pro Thr Leu Lys Thr Pro Glu Ser Glu Val Ser Ser

130 135 140

Phe Thr Ala Ser Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr

145 150 155 160

Leu Asn Asn Cys Asn Lys Lys Phe Thr Asp Ala Phe Glu Ile Met Ala

165 170 175

Glu Asn Tyr Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu

180 185 190

Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Pro Val Thr Arg Met

195 200 205

Lys Asp Ile Gln Gly Leu Pro Cys Met Gly Asp Arg Val Arg Asp Val

210 215 220

Ile Glu Glu Ile Ile Glu Glu Gly Glu Ser Ser Arg Ala Lys Asp Val

225 230 235 240

Leu Asn Asp Glu Arg Tyr Lys Ser Phe Lys Glu Phe Thr Ser Val Phe

245 250 255

Gly Val Gly Val Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Leu Arg

260 265 270

Thr Val Glu Glu Val Lys Ala Asp Lys Thr Leu Lys Leu Ser Lys Met

275 280 285

Gln Arg Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ser

290 295 300

Lys Ala Glu Ala Asp Ala Val Ser Ser Ile Val Lys Asn Thr Val Cys

305 310 315 320

Thr Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg

325 330 335

Gly Lys Lys Ile Gly His Asp Ile Asp Phe Leu Ile Thr Ser Pro Gly

340 345 350

Gln Arg Glu Asp Asp Glu Leu Leu His Lys Val Val Asn Leu Trp Lys

355 360 365

Lys Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Val

370 375 380

Lys Glu Gln Ile Pro Ser Arg His Val Asp Ala Met Asp His Phe Gln

385 390 395 400

Lys Cys Phe Ala Ile Leu Lys Leu Tyr Gln Pro Arg Val Asp Asn Ser

405 410 415

Ser Tyr Asn Met Ser Lys Lys Cys Asp Met Ala Glu Val Lys Asp Trp

420 425 430

Lys Ala Ile Arg Val Asp Leu Val Ile Thr Pro Phe Glu Gln Tyr Ala

435 440 445

Tyr Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Gly Arg Asp Leu

450 455 460

Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala

465 470 475 480

Leu Tyr Asp Lys Ser Lys Arg Val Phe Leu Lys Ala Gly Ser Glu Glu

485 490 495

Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Val Glu Pro Trp Glu Arg

500 505 510

Asn Ala

<210> 9

<211> 513

<212> PRT

<213> diphtheria sparrow (Whitethroat)

<400> 9

Met Asp Arg Phe Lys Ala Pro Ala Val Ile Ser Gln Arg Lys Arg Gln

1 5 10 15

Lys Gly Leu His Ser Pro Lys Leu Ser Cys Ser Tyr Glu Ile Lys Phe

20 25 30

Ser Asn Phe Val Ile Phe Ile Met Gln Arg Lys Met Gly Leu Thr Arg

35 40 45

Arg Met Phe Leu Met Glu Leu Gly Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Ser Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Tyr Leu Glu Val Leu Asp Trp Leu Lys Gly Gln Ala Val Gly Asp Ser

85 90 95

Ser Arg Phe Glu Leu Leu Asp Ile Ser Trp Phe Thr Ala Cys Met Glu

100 105 110

Ala Gly Arg Pro Val Asp Ser Glu Val Lys Tyr Arg Leu Met Glu Gln

115 120 125

Ser Gln Ser Leu Pro Leu Asn Met Pro Ala Leu Glu Met Pro Ala Phe

130 135 140

Ile Ala Thr Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr Leu

145 150 155 160

Asn Asn Tyr Asn Lys Lys Phe Thr Asp Ala Phe Glu Val Met Ala Glu

165 170 175

Asn Tyr Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu Arg

180 185 190

Ala Ala Ser Leu Leu Lys Ser Leu Pro Phe Ser Val Thr Arg Met Lys

195 200 205

Asp Ile Gln Gly Leu Pro Cys Val Gly Asp Gln Val Arg Asp Ile Ile

210 215 220

Glu Glu Ile Ile Glu Glu Gly Glu Ser Ser Arg Val Asn Glu Val Leu

225 230 235 240

Asn Asp Glu Arg Tyr Lys Ala Phe Lys Gln Phe Thr Ser Val Phe Gly

245 250 255

Val Gly Val Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg Thr

260 265 270

Val Glu Glu Val Lys Ala Asp Lys Thr Leu Lys Leu Ser Lys Met Gln

275 280 285

Lys Ala Gly Leu Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ser Lys

290 295 300

Ala Glu Ala Asp Ala Val Ser Leu Ile Val Lys Asn Thr Val Cys Thr

305 310 315 320

Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly

325 330 335

Lys Asn Ile Gly His Asp Ile Asp Phe Leu Ile Thr Asn Pro Gly Pro

340 345 350

Arg Glu Asp Asp Glu Leu Leu His Lys Val Ile Asp Leu Trp Lys Lys

355 360 365

Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Val Lys

370 375 380

Glu Gln Leu Pro Ser Arg Lys Val Asp Ala Met Asp His Phe Gln Lys

385 390 395 400

Cys Phe Ala Ile Leu Lys Leu Tyr Gln Pro Arg Val Asp Asn Ser Thr

405 410 415

Cys Asn Thr Ser Glu Gln Leu Glu Met Ala Glu Val Lys Asp Trp Lys

420 425 430

Ala Ile Arg Val Asp Leu Val Ile Thr Pro Phe Glu Gln Tyr Pro Tyr

435 440 445

Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Gly Arg Asp Leu Arg

450 455 460

Arg Tyr Ala Ala His Glu Arg Lys Met Ile Leu Asp Asn His Gly Leu

465 470 475 480

Tyr Asp Arg Arg Lys Arg Ile Phe Leu Lys Ala Gly Ser Glu Glu Glu

485 490 495

Ile Phe Ala His Leu Gly Leu Asp Tyr Val Glu Pro Trp Glu Arg Asn

500 505 510

Ala

<210> 10

<211> 513

<212> PRT

<213> falcon (Falco peregrinus)

<400> 10

Met Asp Arg Ile Arg Ala Pro Gly Val Leu Ser Gln Arg Lys Arg Gln

1 5 10 15

Lys Gly Met His Ser Pro Asn Leu Ser Cys Ser Tyr Glu Ile Lys Phe

20 25 30

Asn Lys Phe Val Ile Phe Ile Met Gln Arg Lys Met Gly Met Thr Arg

35 40 45

Arg Thr Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Ser Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Tyr Pro Glu Val Leu Asp Trp Leu Arg Gly Gln Ala Val Ser Asp Ser

85 90 95

Ser Arg Phe Glu Leu Leu Asp Ile Ser Trp Phe Thr Ala Cys Met Glu

100 105 110

Ala Gly Arg Pro Val Asp Ser Glu Met Lys Tyr Arg Leu Met Glu Gln

115 120 125

Gly Gln Ser Pro Ala Leu Asn Thr Ser Glu Ser Glu Val Pro Ser Phe

130 135 140

Ile Ala Ser Lys Val Ser Glu Tyr Ser Cys Gln Arg Lys Thr Thr Leu

145 150 155 160

Asn Asn Tyr Asn Lys Lys Phe Thr Asp Ala Phe Glu Ile Met Ala Glu

165 170 175

Asn Tyr Glu Phe Lys Glu Asn Glu Ile Ile Cys Leu Glu Phe Leu Arg

180 185 190

Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Pro Val Thr Arg Met Lys

195 200 205

Asp Ile Gln Gly Leu Pro Cys Met Gly Asp Arg Val Arg Asp Val Ile

210 215 220

Glu Glu Ile Ile Glu Glu Gly Glu Ser Ser Arg Val Glu Glu Val Leu

225 230 235 240

Asn Asp Glu Arg Tyr Lys Ser Phe Lys Gln Phe Thr Ser Val Phe Gly

245 250 255

Val Gly Val Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg Thr

260 265 270

Leu Glu Glu Val Lys Ala Asp Lys Thr Leu Lys Leu Ser Lys Met Gln

275 280 285

Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Ser Val Ser Lys

290 295 300

Ala Glu Ala Asp Ala Val Ser Leu Ile Val Lys Asn Thr Val Cys Met

305 310 315 320

Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly

325 330 335

Lys Asn Ile Gly His Asp Ile Asp Phe Leu Ile Thr Asn Pro Gly Pro

340 345 350

Arg Glu Asp Asp Glu Leu Leu His Lys Val Val Asp Leu Trp Lys Lys

355 360 365

Gln Gly Leu Leu Leu Tyr Cys Asp Met Val Glu Ser Thr Phe Val Lys

370 375 380

Glu Gln Leu Pro Ser Arg Lys Val Asp Ala Phe Asp Asn Phe Gln Lys

385 390 395 400

Cys Phe Ile Ile Leu Lys Leu His Gln Pro Arg Val Glu Asn Ser Ser

405 410 415

Tyr Asn Thr Ser Lys Lys Phe Asp Met Ala Glu Val Lys Asp Trp Lys

420 425 430

Ala Ile Arg Val Asp Leu Val Ile Thr Pro Phe Glu Gln Tyr Ala Tyr

435 440 445

Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Gly Arg Asp Leu Arg

450 455 460

Arg Tyr Ala Thr His Glu Arg Lys Met Ile Leu Asp Asn His Ala Leu

465 470 475 480

Tyr Asp Arg Arg Lys Arg Ile Phe Leu Lys Ala Gly Ser Glu Glu Glu

485 490 495

Ile Phe Ala Tyr Leu Gly Leu Asp Tyr Val Glu Pro Trp Glu Arg Asn

500 505 510

Ala

<210> 11

<211> 513

<212> PRT

<213> gold carving (Aquila chrysaestos)

<400> 11

Met Asp Arg Ile Arg Ala Pro Ala Val Leu Ser Gln Arg Lys Arg Gln

1 5 10 15

Lys Gly Met Arg Ser Pro Asn Leu Ser Cys Ser Tyr Glu Ile Lys Phe

20 25 30

Asn Lys Phe Val Ile Phe Ile Met Gln Arg Lys Met Gly Met Thr Arg

35 40 45

Arg Thr Phe Leu Met Glu Leu Ala Arg Arg Lys Gly Phe Arg Val Glu

50 55 60

Ser Glu Leu Ser Asp Ser Val Thr His Ile Val Ala Glu Asn Asn Ser

65 70 75 80

Tyr Leu Glu Val Leu Asp Trp Leu Arg Gly Gln Ala Val Gly Asp Thr

85 90 95

Ser Arg Phe Glu Leu Leu Asp Ile Ser Trp Phe Thr Ala Cys Met Glu

100 105 110

Ala Gly Arg Pro Val Asp Ser Glu Met Lys Tyr Arg Leu Met Glu Gln

115 120 125

Asp Gln Ser Pro Pro Leu Asn Thr Pro Glu Ser Glu Val Pro Ser Phe

130 135 140

Ile Ala Ser Lys Val Ser Gln Tyr Ser Cys Gln Arg Lys Thr Thr Leu

145 150 155 160

Asn Asn Tyr Asn Lys Lys Phe Thr Asp Ala Leu Glu Ile Met Ala Glu

165 170 175

Asn Tyr Glu Phe Lys Glu Asn Glu Ile Phe Cys Leu Glu Phe Leu Arg

180 185 190

Ala Ala Ser Val Leu Lys Cys Leu Pro Phe Pro Val Thr Arg Met Lys

195 200 205

Asp Ile Gln Gly Leu Pro Cys Met Gly Asp Arg Val Arg Asp Val Ile

210 215 220

Glu Glu Ile Ile Glu Glu Gly Glu Ser Ser Arg Ala Lys Glu Val Leu

225 230 235 240

Asn Asp Glu Arg Tyr Lys Ser Phe Lys Leu Phe Thr Ser Val Phe Gly

245 250 255

Val Gly Val Lys Thr Ser Glu Lys Trp Tyr Arg Met Gly Leu Arg Thr

260 265 270

Leu Glu Glu Val Lys Ala Asp Lys Thr Leu Lys Leu Ser Lys Met Gln

275 280 285

Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Ser Lys

290 295 300

Ala Glu Ala Asp Ala Val Ser Leu Ile Val Lys Asn Thr Val Cys Thr

305 310 315 320

Phe Leu Pro Asp Ala Leu Val Thr Ile Thr Gly Gly Phe Arg Arg Gly

325 330 335

Lys Lys Ile Gly His Asp Ile Asp Phe Leu Ile Thr Asn Pro Gly Pro

340 345 350

Lys Glu Asp Asp Glu Leu Leu His Lys Val Val Asp Leu Trp Lys Lys

355 360 365

Gln Gly Leu Leu Leu Tyr Cys Asp Ile Ile Glu Ser Thr Phe Val Lys

370 375 380

Glu Gln Leu Pro Ser Arg Lys Val Asp Ala Met Asp His Phe Gln Lys

385 390 395 400

Cys Phe Ala Ile Leu Lys Leu Tyr Gln Pro Gly Val Asp Asn Ser Ser

405 410 415

Tyr Asn Val Ser Lys Lys Phe Asp Met Ala Glu Val Lys Asp Trp Lys

420 425 430

Ala Ile Arg Val Asp Leu Val Ile Thr Pro Phe Glu Gln Tyr Ala Tyr

435 440 445

Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Gly Arg Asp Leu Arg

450 455 460

Arg Tyr Ala Thr His Glu Arg Lys Met Ile Leu Asp Asn His Ser Leu

465 470 475 480

Tyr Asp Arg Arg Lys Arg Ile Phe Leu Lys Ala Gly Ser Glu Glu Glu

485 490 495

Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Gly Glu Arg Asn

500 505 510

Ala

<210> 12

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

taatacgact cact 14

<210> 13

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

taatacgact cactac 16

Claims (6)

1. Use of a Terminal Deoxyribonucleoside Transferase (TDT) for catalyzing the polymerization of a nucleotide modified at its 3' end by a reversible blocking group, said reversible blocking group being any one of an O-amino, O-allyl, O-azido, O-phosphate group, said terminal deoxyribonucleoside transferase being selected from the group consisting of: 2,3,4, 5, 6, 7, 8, 9, 10, 11.

2. The use of claim 1, wherein the terminal deoxyribonucleoside transferases are derived from the following organisms: duckbilled, rat ear bat, nudzokor, chimpanzee, gecko, emu, jungle, diphtheria finch, tennons, golden carving.

3. Use according to claim 1 or 2, comprising contacting the start sequence with at least one nucleotide in the presence of the terminal deoxyribonucleoside transferase to synthesize a nucleic acid molecule.

4. A method of template strand independent synthesis of a nucleic acid molecule comprising the steps of:

a) providing a starting sequence;

b) adding to said starting sequence a nucleotide with a reversible blocking group at the 3' end in the presence of a Terminal Deoxyribonucleoside Transferase (TDT) of claim 1 or 2;

c) removing the TDT or removing the solvent from the starting sequence;

d) cleaving the reversible blocking group from the nucleotide bearing the reversible blocking group at the 3' end in the presence of a cleaving agent;

e) removing the cracking agent;

wherein the blocking group of the nucleotide with the reversible blocking group at the 3' end is an O-amino group, an O-allyl group, an O-azido group or an O-phosphate group.

5. The method of claim 4, wherein more than 1 nucleotide is added by repeating steps b) through e).

6. A kit for synthesizing a nucleic acid molecule comprising the terminal deoxyribonucleoside transferase of claim 1 or 2, a start sequence, one or more nucleotides having a reversible blocking group at the 3' end, and a cleaving agent; the blocking group of the nucleotide with the reversible blocking group at the 3' end is an O-amino group, an O-allyl group, an O-azido group or an O-phosphate group.

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