EP4149563A1 - Peptidic scaffolds, processes for manufacturing the same, and uses thereof as soluble supports - Google Patents
Peptidic scaffolds, processes for manufacturing the same, and uses thereof as soluble supportsInfo
- Publication number
- EP4149563A1 EP4149563A1 EP21724701.4A EP21724701A EP4149563A1 EP 4149563 A1 EP4149563 A1 EP 4149563A1 EP 21724701 A EP21724701 A EP 21724701A EP 4149563 A1 EP4149563 A1 EP 4149563A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- amino acid
- seq
- group
- peptidic scaffold
- peptidic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
Definitions
- the present invention relates to functionalized peptidic scaffolds comprising a series of proline (Pro, P) and reactive amino acids, processes for manufacturing the same, and uses thereof as a soluble support in any methods which require or else could be carried out using a support, such as for synthesis processes or molecular biology and biochemistry assays.
- Pro proline
- reactive amino acids processes for manufacturing the same, and uses thereof as a soluble support in any methods which require or else could be carried out using a support, such as for synthesis processes or molecular biology and biochemistry assays.
- solid supports are commonly used in the art.
- functionalized magnetic beads are commonly used as carriers of antigens, antibodies, catalyzers, proteins and nucleic acids; and offer the advantage to be easily operable with automated system including electromagnets.
- Sepharose beads are also commonly used in biochemistry, for binding antibodies and other proteins in various biochemical assays. Because of their large size, such solid supports allow to perform synthesis and biochemical assays using filtration systems, such as vacuum filtration, for easily filtering and washing or eluting reagents and by-products.
- solid supports are not devoid of drawbacks.
- they may be involved in non-specific interactions with some compounds of the reaction mixture, leading to their accumulation on and close to the support, and formation of undesired aggregates in the reaction mixture.
- solid supports have a steric hindrance which prevents large compounds of the reaction mixture, such as enzymes for example, to reach the support- bound molecules closer to the support.
- initiator oligonucleotide primers are typically bound to a solid support.
- DNA polymerization is not as efficient using such initiator oligonucleotide primers attached to a support, as compared to free initiator oligonucleotide primers; but most importantly, only 15 to 20 % of the synthetized oligonucleotides are ultimately recovered, because cutting enzymes fail to access the oligonucleotides close to the support to release them. The loss is therefore substantial.
- a peptidic scaffold comprising a poly lysine/proline motif could readily serve as an efficient and reusable soluble support for carrying out assays and synthesis, while overcoming the drawbacks identified in current means and methods. While the present application demonstrates the benefits of such peptidic scaffold in an enzymatic nucleic acid synthesis process, it is readily apparent for one skilled in the art that such peptidic scaffold is usable in any assay and synthesis method which would alternatively be carried out using a solid support.
- the present invention relates to a peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 2, with: “n” being an integral number ranging from 2 to 20, and
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E); preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), aspartic acid (Asp, D) and glutamic acid (Glu, E); more preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), and cysteine (Cys, C); even more preferably Xi is lysine (Lys, K); and X2, X3, X4 and X5, independently from each other and independently for each “n” repeat,
- said at least one nucleotide, oligonucleotide or polynucleotide is covalently bound to the e-amino group of the at least one lysine residue, optionally through a linker or spacer.
- the peptidic scaffold further comprises at least one aromatic amino acid selected from the group consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine (His, H), preferably selected from the group consisting of tryptophan (Trp, W), and tyrosine (Tyr, Y).
- said peptidic scaffold comprises a (Lys-Pro-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 3, or a (Pro-Ly S-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 4, with “n”, X2, X 3 , X4 and Xs being as defined above.
- said peptidic scaffold comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5 to 22.
- said peptidic scaffold comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 25, 48 or 49.
- said peptidic scaffold comprises a (Cy s-Pro-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 26, or a (Pro-Cys-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 27, with “n”, X2, X 3 , X4 and X5 being as defined above.
- the present invention also relates to a process for manufacturing a peptidic scaffold according to the invention, comprising the steps of: a. producing a peptide comprising a (Xi-Pro-X2-X 3 -X4-X5-)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X 3 -X4-X5-)n amino acid sequence with SEQ ID NO: 2, with
- n being an integral number ranging from 2 to 20;
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E); preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), aspartic acid (Asp, D) and glutamic acid (Glu, E); more preferably preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), and cysteine (Cys, C); even more preferably Xi is lysine (Lys, K); and
- X2, X3, X4 and Xs independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid, preferably selected from the group consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (lie, I), leucine (Leu, L), valine (Yal, V), and proline (Pro, P); b. purifying the peptide produced at step a.; thereby obtaining a peptidic scaffold, c. functionalizing the peptidic scaffold, wherein functionalization of the peptidic scaffold comprises:
- the nucleotide, oligonucleotide or polynucleotide, or the linker or spacer comprises a protein-linking group capable of reacting directly or indirectly with the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold.
- the present invention also relates to the use of a peptidic scaffold according to the invention, as a soluble support.
- the use as a soluble support is in a synthesis process of nucleic acids, preferably a liquid-phase synthesis process of nucleic acids.
- the synthesis process of nucleic acids is a de novo nucleic acid synthesis process, a template-dependent nucleic acid synthesis process or a nucleic acid assembly process.
- the use as a soluble support is in molecular biology and biochemistry assays.
- the molecular biology and biochemistry assay is a nucleic acid hybridization assay.
- the present invention also relates to the use of a peptidic scaffold comprising a (Xi-Pro- X 2 -X3-X4-X 5 -)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 2, with:
- n being an integral number ranging from 2 to 20
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E);
- Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), aspartic acid (Asp, D) and glutamic acid (Glu, E); more preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), and cysteine (Cys, C); even more preferably Xi is lysine (Lys, K); and X2, X3, X4 and X
- said at least one organic molecule is covalently bound to the e-amino group of the at least one lysine residue through a linker or spacer.
- the peptidic support further comprises at least one aromatic amino acid selected from the group consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine (His, H), preferably selected from the group consisting of tryptophan (Trp, W), and tyrosine (Tyr, Y).
- aromatic amino acid selected from the group consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine (His, H), preferably selected from the group consisting of tryptophan (Trp, W), and tyrosine (Tyr, Y).
- said peptidic scaffold comprises a (Lys-Pro-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 3, or a (Pro-Ly S-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 4, with “n”, X2, X3, X4 and X5 being as defined above.
- said peptidic scaffold comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5 to 22; preferably comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.
- said peptidic scaffold comprises a (Cy s-Pro-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 26, or a (Pro-Cys-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 27, with “n”, X2, X3, X4 and Xs being as defined above.
- the synthesis process of organic molecules is a synthesis process of peptides, carbohydrates, or conjugates thereof.
- the present invention also relates to a kit-of-parts comprising: a peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-X5-)n amino acid sequence with
- SEQ ID NO: 1 or a (Pro-Xi -X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 2, with
- n being an integral number ranging from 2 to 20,
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E); preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), aspartic acid (Asp, D) and glutamic acid (Glu, E); more preferably Xi is a reactive amino acid selected from the group consisting of lysine (Lys, K), and cysteine (Cys, C); even more preferably Xi is lysine (Lys, K); and
- X2, X3, X4 and Xs independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid, preferably selected from the group consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (lie, I), leucine (Leu, L), valine (Val, V), and proline (Pro, P); a nucleotide, oligonucleotide or polynucleotide, optionally bound to a linker or spacer; wherein the nucleotide, oligonucleotide or polynucleotide, or the linker or spacer, comprises a protein-linking group capable of reacting directly or indirectly with the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold; instructions to couple the nucleotide, oligonucleotide or polynucleotide to the peptidic scaffold.
- alanine Al, A
- the present invention relates to a peptidic scaffold comprising a series of proline (Pro, P) and reactive amino acids.
- reactive amino acid refers to amino acid residues others that aliphatic amino acids, i.e., amino acid residues comprising a reactive sidechain group.
- reactive amino acids include, but are not limited to, lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E).
- the present invention relates to a peptidic scaffold comprising a (Lys-Pro) or (Pro-Lys) amino acid sequence motif.
- peptidic scaffold comprising a (Cys-Pro) or (Pro-Cys) amino acid sequence motif.
- peptidic scaffold comprising a (Ser-Pro) or (Pro- Ser) amino acid sequence motif.
- a peptidic scaffold comprising a (Thr-Pro) or (Pro- Thr) amino acid sequence motif. It also relates to a peptidic scaffold comprising a (Tyr-Pro) or (Pro-Tyr) amino acid sequence motif.
- It also relates to a peptidic scaffold comprising a (His-Pro) or (Pro-Hi s) amino acid sequence motif.
- a peptidic scaffold comprising a (Arg-Pro) or (Pro- Arg) amino acid sequence motif. It also relates to a peptidic scaffold comprising a (Asp-Pro) or (Pro- Asp) amino acid sequence motif.
- the peptidic scaffold comprises a (Xi-Pro-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X 3 -X4-X5-)n amino acid sequence with SEQ ID NO: 2, with:
- n being an integral number equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, Xi being a reactive amino acid, and
- X2, X3, X4, and X5 independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid.
- n is an integral number ranging from 2 to 20. In one embodiment, “n” equals to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
- aliphatic amino acid refers to an amino acid that comprises an aliphatic side chain functional group. Aliphatic amino acids are non-polar and hydrophobic. Examples of aliphatic amino acids include, but are not limited to, alanine (Ala, A), glycine (Gly, G), isoleucine (He, I), leucine (Leu, L), valine (Yal, V) and proline (Pro, P).
- X2, X3, X4 and X5 are, independently from each other and independently for each “n” repeat, absent or selected from the group comprising or consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (lie, I), leucine (Leu, L), valine (Yal, V), and proline (Pro, P).
- X2, X3, X4 and X5 are, independently from each other and independently for each “n” repeat, absent or selected from the group comprising or consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (He, I), leucine (Leu, L) and valine (Yal, V).
- alanine Al, A
- Gly G
- isoleucine He, I
- Leu, L leucine
- valine Yal, V
- aliphatic amino acids are non-reactive, and can therefore be useful when introduced into the peptidic scaffold to reduce steric hindrance between two reactive amino acids, for example, as detailed below, between two functionalized reactive amino acids.
- at least one of X2, X3, X4 and X5 is not absent.
- the peptidic scaffold further comprises at least one aromatic amino acid, such as 1, 2, 3, 4, 5 aromatic amino acids or more.
- aromatic amino acid refers to an amino acid that includes an aromatic ring.
- aromatic amino acids include, but are not limited to, tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine (His, H).
- Other examples of aromatic amino acids include, but are not limited to, the non-coded (or non-proteinogenic) amino acids thyroxine, 5 -hydroxy try ptophan and L-DOPA.
- aromatic amino acids absorb ultraviolet (UV) light, and can therefore be useful when introduced into the peptidic scaffold to monitor, e.g., its concentration in solution.
- UV ultraviolet
- the peptidic scaffold further comprises at least one aromatic amino acid selected from the group comprising or consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine (His, H), such as 1, 2, 3, 4, 5 aromatic amino acids or more selected from the group comprising or consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine
- aromatic amino acid selected from the group comprising or consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine
- the peptidic scaffold further comprises at least one aromatic amino acid selected from the group comprising or consisting of tryptophan (Trp, W) and tyrosine (Tyr, Y), such as 1, 2, 3, 4, 5 aromatic amino acids or more selected from the group comprising or consisting of tryptophan (Trp, W) and tyrosine (Tyr, Y).
- aromatic amino acid selected from the group comprising or consisting of tryptophan (Trp, W) and tyrosine (Tyr, Y), such as 1, 2, 3, 4, 5 aromatic amino acids or more selected from the group comprising or consisting of tryptophan (Trp, W) and tyrosine (Tyr, Y).
- the peptidic scaffold comprises more than 1 aromatic amino acid, such as at least 2, the at least two aromatic amino acids can be consecutive in the sequence ( i.e ., with no non-aromatic amino acids in-between); or can be separated by at least one non-aromatic amino acid.
- non-aromatic amino acid refers to those amino acids which are not tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), or histidine (His, H); preferably which are not tryptophan (Trp, W), or tyrosine (Tyr, Y).
- the at least one non-aromatic amino acid is an aliphatic amino acid. In one embodiment, the at least one non-aromatic amino acid is a proline (Pro, P).
- the peptidic scaffold comprises at least one tryptophan (Trp, W) and at least one tyrosine (Tyr, Y) residue. In one embodiment, the peptidic scaffold comprises one tryptophan (Trp, W) and one tyrosine (Tyr, Y) residue.
- Xi is selected from the group comprising or consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E).
- Xi is selected from the group comprising or consisting of lysine (Lys, K), cysteine (Cys, C), aspartic acid (Asp, D) and glutamic acid (Glu, E).
- Xi is selected from the group comprising or consisting oflysine (Lys, K) and cysteine (Cys, C).
- Xi is lysine (Lys, K).
- the peptidic scaffold comprises a (Lys-Pro-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 3, or a (Pro-Lys-X2-X 3 -X4-X5-)n amino acid sequence with SEQ ID NO: 4, with:
- n being an integral number equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, and
- X2, X3, X4 and X5 independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid.
- X2, X3, X4 and X5 may, independently from each other, be identical in each “n” repeat of SEQ ID NO: 3 or SEQ ID NO: 4.
- X2, X3, X4 and X5 may, independently from each other, be identical in at least two successive “n” repeats of SEQ ID NO: 3 or SEQ ID NO: 4
- X2, X3, X4 and X5 may, independently from each other, be different in each “n” repeat of SEQ ID NO: 3 or SEQ ID NO: 4.
- X2, X3, X4 and X5 may, independently from each other, be different in at least two successive “n” repeats of SEQ ID NO: 3 or SEQ ID NO: 4
- the present disclosure encompasses sequences wherein the first and/or last amino acid of the (Lys-Pro) or (Pro-Lys) amino acid sequence motif can be removed, i.e., the peptidic scaffold can comprise a proline in N or C-term and/or a lysine in N or C-term.
- At least one aromatic amino acid is located in N-term or in C-term of the (Lys-Pro-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 3 or (Pro-Lys-X2- X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 4.
- At least one aromatic amino acid such as 1, 2, 3, 4, or 5 aromatic amino acids, is/are located in N-term of the (Ly s-Pro-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 3 or (Pro-Ly S-X2-X3 -X4-X5 -)n amino acid sequence with
- At least one aromatic amino acid such as 1, 2, 3, 4, or 5 aromatic amino acids, is/are located in C-term of the (Lys-Pro-X2-X 3 -X4-X5-)n amino acid sequence with SEQ ID NO: 3 or (Pro-Ly S-X2-X3 -X4-X5 -)n amino acid sequence with
- At least one aromatic amino acid may be located between two (Lys-Pro-X2-X 3 -X4-X5-) or (Pro-Ly S-X2-X3 -X4-X5 -) repeats in the amino acid sequences with SEQ ID NO: 3 or SEQ ID NO: 4.
- At least one aromatic amino acid may be located within at least one (Ly s-Pro-X2-X 3 -X4-X5 -) or (Pro-Ly S-X2-X3 -X4-X5 -) repeat in the amino acid sequences with SEQ ID NO: 3 or SEQ ID NO: 4 (i.e., between any one of Lys-Pro, Pro-Lys, Pro-X 2 , Lys-X 2 , X 2 -X 3 , X3-X4 and/or X4-X5).
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in Table 1 below.
- Table 1 with:
- X 2 , X3, X4, and X5 being as defined hereinabove;
- the peptidic scaffold comprises or consists of the amino acid sequence PKPKPKPKPKPKPKPKPKPYPWP (SEQ ID NO: 24).
- the peptidic scaffold comprises or consists of the amino acid sequence AKPGKPLKPAKPGKPLKPGKPYPWP (SEQ ID NO: 25). In an exemplary embodiment, the peptidic scaffold comprises or consists of the amino acid sequence APKAPKAPKAPKAPKAPKAPKWPW (SEQ ID NO: 48).
- the peptidic scaffold comprises or consists of the amino acid sequence APKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKWPW
- Xi is cysteine (Cys, C).
- the peptidic scaffold comprises a (Cys-Pro-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 26, or a (Pro-Cys-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 27, with:
- n being an integral number equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, and
- X2, X3, X4 and X5 independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid.
- X2, X3, X4 and X5 may, independently from each other, be identical in each “n” repeat of SEQ ID NO: 26 or SEQ ID NO: 27. In one embodiment, X2, X3, X4 and X5 may, independently from each other, be identical in at least two successive “n” repeats of SEQ ID NO: 26 or SEQ ID NO: 27.
- X2, X3, X4 and X5 may, independently from each other, be different in each “n” repeat of SEQ ID NO: 26 or SEQ ID NO: 27. In one embodiment, X2, X3, X4 and X5 may, independently from each other, be different in at least two successive “n” repeats of SEQ ID NO: 26 or SEQ ID NO: 27.
- the present disclosure encompasses sequences wherein the first and/or last amino acid of the (Cys-Pro) or (Pro-Cys) amino acid sequence motif can be removed, i.e ., the peptidic scaffold can comprise a proline in N or C-term and/or a cysteine in N or C-term.
- At least one aromatic amino acid is located in N-term or in C-term of the (Cys-Pro-X 2 -X3-X4-X5-)n amino acid sequence with SEQ ID NO: 26 or (Pro-Cys-X2- X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 27.
- At least one aromatic amino acid such as 1, 2, 3, 4, or 5 aromatic amino acids, is/are located in N-term of the (Cys-Pro-X2-X 3 -X4-X5-)n amino acid sequence with SEQ ID NO: 26 or (Pro-Cys-X2-X3-X4-Xs-)n amino acid sequence with
- At least one aromatic amino acid is/are located in C-term of the (Cy s-Pro-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 26 or (Pro-Cy S-X2-X3 -X4-X5 -)n amino acid sequence with
- At least one aromatic amino acid may be located between two (Cy s-Pro-X2-X 3 -X4-X5 -) or (Pro-Cys-X2-X 3 -X4-Xs-) repeats in the amino acid sequences with SEQ ID NO: 26 or SEQ ID NO: 27.
- At least one aromatic amino acid may be located within at least one (Cy s-Pro-X2-X3 -X4-X5 -) or (Pro-Cys-X2-X3-X4-X5-) repeat in the amino acid sequences with SEQ ID NO: 26 or SEQ ID NO: 27 (i.e., between any one of Cys-Pro, Pro-Cys, Pro-X 2 , Cys-X 2 , X2-X3, X3-X4 and/or X4-X5).
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in Table 2 below.
- X2, X3, X4, and X5 being as defined hereinabove;
- X7, X9, Xu, X13, X15, and X17 independently from each other being absent or being a non aromatic amino acid; such as an aliphatic amino acid; such as a proline (Pro, P); and Xg, X10, X12, X14, and Xi6 independently from each other being absent or being an aromatic amino acid; such as a tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), or histidine (His, H); such as a tryptophan (Trp, W) or a tyrosine (Tyr, Y).
- the peptidic scaffold is linear.
- the peptidic scaffold is cyclized.
- the peptidic scaffold comprises a protecting group on the a-amino group of its N-terminal amino acid, on the a-carboxylic acid group of its C -terminal amino acid, or both.
- such protecting groups can enhance the stability and/or solubility of the peptidic scaffold, in particular the protecting groups can improve protease stability of the peptidic scaffold.
- hydrophobic groups such as acetyl, dansyl, /-butyl oxy carb ony 1 or 9- fluorenylmethoxy-carbonyl groups can be added to the a-amino group of the N-terminal amino acid.
- these hydrophobic groups, or /-butyl, ami do or para-nitrobenzyl ester can be added to the a-carboxylic acid group of the C -terminal amino acid.
- the peptidic scaffold is acetylated at the N-terminus, ami dated at the C -terminus, or both.
- the peptidic scaffold comprises an affinity tag in N-term and/or in C-term.
- affinity tag refers to a ligand or chemical group which is covalently linked to the peptidic scaffold of the invention, and which is capable of interacting with an “affinity partner” in order to be extracted or purified from a solution by affinity or pull-down purification.
- the affinity tag is capable of forming a specific binding interaction with its affinity partner, i.e., a binding interaction which is stronger than any binding interaction that may occur between the affinity partner and any other chemical substance present in a sample.
- affinity tag/ affinity partner pairs include, but are not limited to, albumin binding protein (ABP)/albumin, biotin-carboxy carrier protein (BCCP)/(strep)avidin, calmodulin binding peptide (CBP)/calmodulin, chloramphenicol acetyl transferase (CAT)/ chi orampheni col , cellulose binding domain (CBP)/cellulose, chi tin binding domain (CBD)/chitin, choline-binding domain (CBD)/choline, dihydrofolate reductase (DHFR)/methotrexate, galactose-binding protein (GBP)/galactose, glutathione S-transferase (GST)/ glutathi one, HaloT ag ® /HaloLinkTM, histidine affinity tag (HAT)/ divalent cation (M 2+ , Co 2+ , Cu 2+ or Zn 2+ ), maltose-
- the affinity partner can be an antibody directed to the affinity tag.
- the affinity tag can be selected from alkaline phosphatase (AP), AU1 epitope, AU5 epitope, bacteriophage T7 epitope (T7-tag), bluetongue virus tag (B-tag), E2 epitope, FLAG epitope, Glu-Glu (EE-tag), human influenza hemagglutinin (HA), HSV epitope, KT3 epitope, Myc epitope, Protein C, SI -tag, staphylococcal protein A (protein A), T7 epitope, TrpE, and YSY-G.
- an affinity tag may be particularly suitable for purifying the peptidic scaffold, either before its use (i.e. , after production); but also, after its use, for recycling and reusing purposes.
- the peptidic scaffold is functionalized.
- the peptidic scaffold comprises at least one reactive sidechain group to which an organic molecule may be coupled.
- “Reactive sidechain groups” include, e.g, sidechain groups of a lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) and glutamic acid (Glu, E) amino acid residue, i.e., an amino, a thio, a hydroxyl, an imidazole, a guanidino, or a carboxylic acid group.
- the term “functionalized” also encompasses peptidic scaffolds comprising at least one biomolecule bound thereto.
- biomolecule refers to any organic molecule that is part of, or from, a living organism.
- examples of biomolecules include, but are not limited to, a nucleotide, an oligonucleotide, a polynucleotide, an amino acid residue, a peptide, a polypeptide, a protein, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, and the like.
- the biomolecule is bound to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g, to the -amino group of at least one lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold such as, e.g, to the -amino group of at least one lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the biomolecule is covalently bound to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g, to the e-amino group of at least one lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold such as, e.g, to the e-amino group of at least one lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- a biomolecule is bound to the reactive sidechain group of each reactive amino acid of the peptidic scaffold, such as, e.g, to the e-amino group of each lysine (Lys) residue of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of each cysteine (Cys) residue of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- a biomolecule is covalently bound to the reactive sidechain group of each reactive amino acid of the peptidic scaffold, such as, e.g, to the e-amino group of each lysine (Lys) residue of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of each cysteine (Cys) residue of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the reactive sidechain group of each reactive amino acid of the peptidic scaffold such as, e.g, to the e-amino group of each lysine (Lys) residue of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of each cysteine (Cys) residue of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the biomolecules bound to the reactive sidechain group of each reactive amino acid of the peptidic scaffold such as, e.g, to the e-amino group of each lysine (Lys) residue of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of each cysteine (Cys) residue of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27, are identical with each other.
- the biomolecules bound to the reactive sidechain group of each reactive amino acid of the peptidic scaffold such as, e.g, to the e-amino group of each lysine (Lys) residue of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of each cysteine (Cys) residue of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27, are not identical with each other, i.e., at least two biomolecules bound to the peptidic scaffold are different.
- the biomolecule is a nucleic acid or a fragment thereof, such as, e.g, a nucleotide, an oligonucleotide or polynucleotide.
- the biomolecule is a nucleotide.
- nucleotide refers to molecules comprising a nucleoside and from one to three phosphate groups. They are the building blocks of DNA and RNA.
- nucleotides include, but are not limited to, ribonucleotides (such as adenosine mono-, di- or tri-phosphate [AMP, ADP, ATP]; guanosine mono-, di- or tri-phosphate [GMP, GDP, GTP]; uridine mono-, di- or tri-phosphate [UMP, UDP, UTP]; and cytidine mono-, di- or tri -phosphate [CMP, CDP, CTP]); deoxy rib onucl eoti des (such as deoxyadenosine mono-, di- or tri-phosphate [dAMP, dADP, dATP]; deoxy guanosine mono-, di- or tri -phosphate [dGMP, dGDP, dGTP]; thymidine mono-, di- or tri-phosphate [dTMP, dTDP, dTTP]; and deoxy cytidine mono-, di- or
- the biomolecule is an oligonucleotide.
- oligonucleotide refers to a short-length, single-stranded, polymer of nucleotide monomers. Oligonucleotides typically comprise from two to about one hundred nucleotide monomers.
- nucleotides refers to all combinations of nucleotides as defined above, forming a polymer of nucleotides, such as, e.g., a combination of deoxy rib onucl eoti des forming a DNA oligonucleotide, a combination of ribonucleotides forming an RNA oligonucleotide, or a combination of both deoxyribonucleotides and ribonucleotides forming a mixed DNA/RNA oligonucleotide.
- the biomolecule is a polynucleotide.
- polynucleotide refers to a long-length, single-stranded, polymer of nucleotide monomers. Polynucleotides typically comprise more than about one hundred nucleotide monomers.
- the term refers to all combinations of nucleotides as defined above, forming a polymer of nucleotides, such as, e.g, a combination of deoxyrib onucl eoti des forming a DNA polynucleotide, a combination of ribonucleotides forming an RNA polynucleotide, or a combination of both deoxyrib onucl eoti des and ribonucleotides forming a mixed DNA/RNA polynucleotide.
- the biomolecule is a nucleic acid or a fragment thereof of known sequence. In one embodiment, the biomolecule is a nucleic acid or a fragment thereof of unknown sequence.
- nucleic acid sequence of the biomolecule e.g, for hybridization against a nucleic acid sequence of interest in capture assays, for PCR priming and amplification after nucleic acid synthesis processes, for barcoding purposes, etc.
- the biomolecule is an oligonucleotide comprising from 2 to 50 nucleotides. In one embodiment, the biomolecule is an oligonucleotide comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
- the biomolecule is a protein or a fragment thereof, such as, e.g, an amino acid, a peptide or a polypeptide.
- the protein or a fragment thereof is an affinity tag. Examples of affinity tags have been described above and apply here mutatis mutandis.
- the protein or a fragment thereof is an enzyme or a biologically active fragment thereof.
- the enzyme can be any enzyme used in an enzymatic synthesis process of organic molecules.
- the enzyme can be any enzyme used in an enzymatic synthesis process of nucleic acids. Examples of such enzymes include, but are not limited to, DNA-dependent DNA polymerases, DNA- dependent RNA polymerases, DNA primases, terminal deoxynucleotidyltransferases, and DNA ligases.
- the biomolecule is a carbohydrate or a fragment thereof, such as, e.g., a monosaccharide, a di saccharide, an oligosaccharide or a polysaccharide.
- the peptidic scaffold further comprises a linker or spacer bound to the reactive sidechain group of at least one reactive amino acid of the peptidic scaffold, such as, e.g, to the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one of the cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or
- linker or “spacer” are used interchangeably and refer to a chemical moiety that can connect two other moieties (such as, e.g., a peptidic scaffold and a biomolecule) either covalently, or noncovalently, e.g, through ionic or hydrogen bonds or van der Waals interactions.
- the linker or spacer is functionalized.
- the description of the functionalized peptidic scaffold hereinabove applies mutatis mutandis to a functionalized linker or spacer.
- Linkers or spacers may be desirable, to extend the length of the reactive sidechains of the peptidic scaffold and thereby reduce steric hindrance between two reactive amino acids; and/or to modify the nature of the reactive sidechain groups of the peptidic scaffold, e.g., to replace the primary amine of lysine residues with another reactive group such as, without limitation, a thio, a hydroxyl, an imidazole, a guanidino, or a carboxylic acid group, depending on the coupling chemistry chosen by the skilled artisan.
- the linker or spacer has a backbone of 50 atoms or less, preferably of 40 atoms or less, more preferably of 30 atoms or less, even more preferably of 20 atoms or less.
- the linker or spacer is a covalent bond that connects two moieties.
- the linker or spacer is a chain comprising between 1 and 20 atoms in length, for example a chain of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms in length.
- the linker or spacer may be linear, branched, cyclic or a single atom.
- the linker or spacer backbone may comprise or consist of carbon atoms; or alternatively may comprise or consist of a combination of carbon atoms and heteroatoms.
- heteroatom refers to any atom which is not a carbon or a hydrogen. Examples of heteroatoms include, without limitation, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), chlorine (Cl), bromine (Br), iodine (I), lithium (Li) and magnesium (Mg).
- the heteroatom is selected from the group comprising or consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P) and combinations thereof.
- the linker or spacer backbone may comprise or consist of an acyl, acylamino, acyloxy, alkoxy, alkoxy carbonyl, alkoxycarbonylamino, alkyl, trihaloalkyl, alkenyl, alkynyl, amino, ami do, imino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, aryloxy, azido, diazo, biotin, carboxyl, carbonyl, cyano, isocyanate, isothiocyanate, cycloalkyl, guanidyl, halogen, heterocyclyl, heterocyclyloxy, hydroxyl, keto, nitro, nitroso, oxo, thio, thioether, thioalkoxy, thioaryloxy, thioketo, thiol, sulfonate, sulfinate, phosphinate, phosphonate, alkyl, tri
- the number of atoms in the backbone of the linker or spacer is calculated by counting the minimum number of covalently linked atoms between the two moieties connected by the linker or spacer, excluding atoms of the two connected moieties themselves.
- the linker or spacer includes a cyclic moiety, the shortest path around the ring of the cyclic moiety is counted so that a minimum possible number of atoms that connect the two moieties is calculated.
- the linker or spacer may include one or more substituent groups, such as, e.g, an alkyl, aryl or alkenyl group.
- the linker or spacer may include, without limitations, oligo(ethylene glycol), ethers, thioethers, tertiary amines, and/or alkyls, which may be linear or branched, such as, e.g, methyl, ethyl, «-propyl, 1 -methyl ethyl (iso-propyl), «-butyl, «- pentyl, 1,1-dimethylethyl (/-butyl), and the like.
- the backbone of the linker or spacer may include a cyclic group, such as, e.g, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms of the cyclic group are included in the backbone.
- a cyclic group such as, e.g, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms of the cyclic group are included in the backbone.
- the linker or spacer is cleavable. In one embodiment, the linker or spacer is non-cleavable.
- cleavable with reference to a linker or spacer implies that said linker or spacer can be selectively cleaved to produce two products.
- Application of suitable cleavage conditions to a molecule containing a cleavable linker or spacer that is cleaved by said cleavage conditions will produce two “cleavage products”.
- a cleavable linker or spacer is stable to physiological conditions, until it is contacted with a reagent capable of cleaving the cleavable linker or spacer.
- the peptidic scaffold is bound to a solid support. In one embodiment, the peptidic scaffold is covalently bound to a solid support.
- Exemplary materials that can be used as solid supports include, but are not limited to, acrylics, carbon (e.g., graphite, carbon -fiber), cellulose (e.g, cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g, agarose, SEPHAROSETM or alginate), gels, glass (e.g, modified or functionalized glass), gold (e.g, atomically smooth Au(l 11)), graphite, inorganic glasses, inorganic polymers, latex, metal oxides (e.g. , S1O2, T1O2, stainless steel), metalloids, metals (e.g.
- the solid support may comprise a magnetic core, such as a ferrimagnetic or superparamagnetic core.
- a magnetic core such as a ferrimagnetic or superparamagnetic core.
- the present invention also relates to a process for manufacturing the peptidic scaffold according to the present invention.
- the process for manufacturing the peptidic scaffold comprises the steps of: a. producing a peptide comprising a (Xi -Pro-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 2, as described hereinabove; optionally wherein the peptide further comprises at least one aromatic amino acid, as described hereinabove; and b. purifying the synthesized peptide; thereby obtaining a peptidic scaffold.
- the step of producing a peptide can be carried out by solid-phase synthesis; by solution-phase synthesis; by liquid-phase synthesis; or by recombinant expression in bacteria, yeasts, insect cells or mammalian cells.
- peptides shorter than eight amino acids residues are prepared more economically by solution-phase synthesis, and peptides larger than eight amino acid residues are generally produced by solid-phase synthesis.
- Means and methods to purify a peptide are well known to the one skilled in the art. Examples of such methods include, but are not limited to, reverse phase (RP)-high- performance liquid chromatography (HPLC) chromatography, flash chromatography, affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, size exclusion chromatography, hydrophilic interaction chromatography.
- RP reverse phase
- HPLC high-performance liquid chromatography
- HPLC high-performance liquid chromatography
- flash chromatography affinity chromatography
- ion-exchange chromatography hydrophobic interaction chromatography
- hydrophobic interaction chromatography hydrophobic interaction chromatography
- gel filtration chromatography size exclusion chromatography
- hydrophilic interaction chromatography hydrophilic interaction chromatography.
- HPLC of peptides and proteins methods and protocols (Yol. 251). Totowa, NJ: Humana Press.
- the process for manufacturing the peptidic scaffold comprises a step of functionalizing the peptidic scaffold.
- functionalizing or “functionalization”, it is meant coupling at least one reactive sidechain group of the peptidic scaffold with an organic molecule, such as, e.g., a linker or spacer and/or a biomolecule, as defined hereinabove.
- the step of functionalizing the peptidic scaffold comprises coupling the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold with an organic molecule, preferably with a linker or spacer and/or a biomolecule, such as, e.g, coupling the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or coupling the thiol group of at least one of the cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27 with an organic molecule, preferably with a linker or spacer and/or a biomolecule.
- an organic molecule preferably with a linker or spacer and/or a biomolecule
- Functionalization of the peptidic scaffold may therefore comprise or consist of: coupling the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold with a linker or spacer, then coupling said linker or spacer with a biomolecule; or - coupling the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold with a biomolecule; or coupling the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold with a biomolecule comprising a linker or spacer.
- the process for manufacturing the peptidic scaffold comprises a step of coupling the peptidic scaffold with a linker or spacer, as defined hereinabove.
- the step of coupling the peptidic scaffold with a linker or spacer comprises coupling the linker or spacer to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g, to the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one of the cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the linker or spacer comprises coupling the linker or spacer to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g, to the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least
- the step of coupling the peptidic scaffold with a linker or spacer comprises covalently coupling the linker or spacer to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g., to the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one of the cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the linker or spacer comprises a protein-linking group.
- protein-linking group refers to a chemical moiety that is capable of reacting, either spontaneously or after activation through contact with a stimulus, with an accessible reactive sidechain group of a protein or peptide under aqueous conditions to produce a covalent linkage to said protein or peptide.
- Reactive sidechain groups include sidechains of a lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H), arginine (Arg, R), aspartic acid (Asp, D) or glutamic acid (Glu, E) amino acid residue, hence the “protein-linking group” may be amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive, guanidinyl-reactive or carboxy-reactive.
- the protein-linking group can be naturally present on the linker or spacer, or be added to the linker or spacer prior to coupling using techniques well known in the art.
- the protein-linking group is amino-reactive and/or is capable of reacting with the e-amino group of at least one lysine (Lys) residue of the peptidic scaffold, such as the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4, thereby conjugating the linker or spacer with the peptidic scaffold.
- Lys lysine
- amino-reactive protein-linking groups include, but are not limited to, isothiocyanate, isocyanate, acyl azide, /V-hydroxysuccinimide (NHS) ester, sulfonyl chloride, tosyl ester, tresyl ester, aldehyde, amine, epoxide, carbonate, aryl halide, haloacetyl, alkyl halide, imido ester, carboxylate, alkyl phosphate, anhydride, fluorophenyl ester, HOBt ester, hydroxymethyl phosphine, ( -methylisourea, A/A’-disuccinimidyl carbonate (DSC), NHS carbamate, glutaraldehyde, activated double bond, cyclic hemi acetal, NHS carbonate, imidazole carbamate, acyl imidazole, methylpyridinium ether, azlactone
- amino-reactive protein linking groups require, before coupling to the peptidic scaffold, an activation pre-step.
- amino-reactive protein-linking groups which require an activation pre-step prior to coupling include, without limitation, aldehyde, which is activated with sodium cyanoborohydride or aniline; amine, which is activated with formaldehyde; carboxylate, which is activated with carbodiimide; and alkyl phosphate, which is activated with carbodiimide and imidazole.
- the protein-linking group is thiol -reactive and/or is capable of reacting with the thiol group of at least one cysteine (Cys) residue of the peptidic scaffold, such as the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27, thereby conjugating the linker or spacer with the peptidic scaffold.
- Cys cysteine
- thiol -reactive protein-linking groups include, but are not limited to, haloacetyl, alkyl halide, maleimide, aziridine, aryl halide, pyridyl di sulfate, 2,2’-dipyridyl disulfide, 4,4’-dipyridyl disulfide, TNB -thiol, Ellman’s reagent, peroxide, vinyl sulfone, metal surface, phenylthioester, cisplatin, and activated double bond. Section “2. Thiol reactions”, Subsections 2.1 to 2.10. (pp.
- the process for manufacturing the peptidic scaffold further comprises a step of functionalizing the linker or spacer with a biomolecule, as defined hereinabove.
- the biomolecule comprises a chemical moiety that is capable of reacting, either spontaneously or after activation through contact with a stimulus, with an accessible chemical moiety of the linker or spacer under aqueous conditions to produce a covalent linkage to the linker or spacer.
- a chemical moiety can be naturally present on the biomolecule, or be added to the biomolecule prior to coupling using techniques well known in the art.
- the nature of the chemical moiety that is capable of reacting with an accessible chemical moiety of the linker or spacer will depend upon the accessible chemical moiety of the linker or spacer itself.
- the process for manufacturing the peptidic scaffold comprises a step of functionalizing the peptidic scaffold with a biomolecule, as defined hereinabove ( i.e ., without spacer or linker).
- the step of functionalizing the peptidic scaffold with a biomolecule comprises coupling the biomolecule to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g.
- the step of functionalizing the peptidic scaffold with a biomolecule comprises covalently coupling the biomolecule to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g., to the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold such as, e.g., to the e-amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ
- the biomolecule comprises a protein-linking group - as defined hereinabove - to allow coupling to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g, to allow coupling to the e- amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to allow coupling to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the protein-linking group can be naturally present on the biomolecule, or be added to the biomolecule prior to coupling using techniques well known in the art. In particular, custom biomolecules are commercially available, which may include post-synthesis modifications, such as addition of protein-linking groups.
- the process for manufacturing the peptidic scaffold further comprises a step of functionalizing the peptidic scaffold with a biomolecule comprising a linker or spacer, as defined hereinabove.
- a biomolecule comprising a linker or spacer can be produced upstream of the process for manufacturing the peptidic scaffold, by reacting, either spontaneously or after activation through contact with a stimulus, a chemical moiety from the biomolecule with a chemical moiety of the linker or spacer under aqueous conditions to produce a covalent linkage between the two.
- custom biomolecules are commercially available, which may post-synthesis modifications, such as addition of chemical moieties or of a linker or spacer.
- the linker or spacer comprises a protein-linking group - as defined hereinabove - to allow coupling to the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold, such as, e.g., to allow coupling to the e- amino group of at least one of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4; or to allow coupling to the thiol group of at least one cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27.
- the protein-linking group can be naturally present on the linker or spacer, or be added to the linker or spacer prior to coupling using techniques well known in the art.
- the process for manufacturing the peptidic scaffold comprises a step of proline cis-irans isomerization of the peptidic scaffold.
- Peptidyl -prolyl isomerases or PPIase are enzymes capable of catalyzing the isomerization of prolines.
- Proline isomerization can be useful when manufacturing the peptidic scaffold and in particular, when functionalizing the peptidic scaffold, to provide flexibility to the peptidic scaffold and avoid steric hindrance when 2 or more linkers or spacers and/or biomolecules are to be coupled in close proximity on the scaffold.
- the step of proline cis-trans isomerization may be carried out before, during or after the step of functionalizing the peptidic scaffold.
- the step of proline cis-trans isomerization is carried out during (or concomitantly with) the step of functionalizing the peptidic scaffold.
- the peptidyl-prolyl isom erase used to carry out this step of the method shall be compatible with the chemical and/or physical conditions required to carry out the functionalization of the peptidic scaffold.
- the step of proline cis-trans isomerization is carried out before and/or after the step of functionalizing the peptidic scaffold.
- the step of functionalizing the peptidic scaffold may be repeated at least twice, with a step of proline cis-trans isomerization between each repetition, so as to provide flexibility to the scaffold and ensure that all reactive sidechain groups of the peptidic scaffold, such as, e.g, all e-amino groups of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ ID NO: 4 or all thiol groups of the cysteine (Cys) residues of the peptidic scaffold with SEQ ID NO: 26 or SEQ ID NO: 27, are accessible to functionalization at some point during the overall process.
- all reactive sidechain groups of the peptidic scaffold such as, e.g, all e-amino groups of the lysine (Lys) residues of the peptidic scaffold with SEQ ID NO: 3 or SEQ
- the process for manufacturing the peptidic scaffold therefore comprises the steps of: a. producing a peptide comprising a (Xi-Pro-X2-X3-X4-X5)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-X5)n amino acid sequence with
- SEQ ID NO: 2 as described hereinabove; optionally wherein the peptide further comprises at least one aromatic amino acid, as described hereinabove; b. purifying the peptide produced at step a.; thereby obtaining a peptidic scaffold, c. functionalizing the peptidic scaffold, wherein functionalization of the peptidic scaffold comprises:
- the peptide at step a comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, as described hereinabove. In one embodiment, the peptide at step a. comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 5 to SEQ ID NO: 22, as described hereinabove.
- the peptide at step a comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 26 or SEQ ID NO: 27, as described hereinabove. In one embodiment, the peptide at step a. comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 28 to SEQ ID NO: 45, as described hereinabove.
- the peptide at step a comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 24, as described hereinabove.
- the peptide at step a comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 25, as described hereinabove.
- the peptide at step a comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 48, as described hereinabove.
- the peptide at step a comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 49, as described hereinabove.
- the present invention also relates to a use of the peptidic scaffold according to the present invention, as a soluble support.
- the peptidic scaffold comprises a (Xi-Pro-X2-X3-X4-X5)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-Xs)n amino acid sequence with SEQ ID NO: 2, as described hereinabove.
- the peptidic scaffold further comprises at least one aromatic amino acid, as described hereinabove.
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, as described hereinabove. In one embodiment, the peptidic scaffold comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 5 to SEQ ID NO: 22, as described hereinabove.
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 26 or SEQ ID NO: 27, as described hereinabove. In one embodiment, the peptidic scaffold comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 28 to SEQ ID NO: 45, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 24, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 25, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 48, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 49, as described hereinabove. In one embodiment, the peptidic scaffold is functionalized, as described hereinabove.
- the peptidic scaffold is functionalized with at least one biomolecule, as described hereinabove.
- the peptidic scaffold further comprises a linker or spacer bound to the reactive sidechain group of at least one of its reactive amino acids, such as, e.g., to the e-amino group of at least one of its lysine (Lys) residues or to the thiol group of at least one of its cysteine (Cys) residues, as described hereinabove.
- a linker or spacer bound to the reactive sidechain group of at least one of its reactive amino acids such as, e.g., to the e-amino group of at least one of its lysine (Lys) residues or to the thiol group of at least one of its cysteine (Cys) residues, as described hereinabove.
- the peptidic scaffold according to the present invention may be used as a soluble support in any methods which require or else could be carried out using a support, such as, e.g., a solid support.
- a support such as, e.g., a solid support.
- the peptidic scaffold according to the present invention may be used as a soluble support in synthesis processes of organic molecules.
- Examples of synthesis processes of organic molecules in which the peptidic scaffold according to the present invention is useful include, but are not limited to, liquid-phase synthesis.
- liquid-phase synthesis refers to one or a series of chemical or enzymatic reactions carried out to prepare either a single organic molecule, a library of molecularly identical organic molecules or a library of molecularly diverse organic molecules, wherein the chemical or enzymatic reactions are sequentially or cyclically performed in solution, on a soluble support.
- the central feature of liquid-phase synthesis is that it combines the advantages that support-less synthesis offers with those that solid-phase synthesis can provide, through the use of a soluble support such as the peptidic scaffold of the invention.
- Liquid-phase synthesis can be implemented for the production of organic molecules such as biomolecules, including peptides, nucleic acids molecules (including DNA, RNA and mixes thereof), carbohydrates, and conjugates thereof; but could also be applied for synthesis of any organic molecule which would otherwise be synthesized in solution or on a solid support.
- organic molecules such as biomolecules, including peptides, nucleic acids molecules (including DNA, RNA and mixes thereof), carbohydrates, and conjugates thereof; but could also be applied for synthesis of any organic molecule which would otherwise be synthesized in solution or on a solid support.
- liquid-phase synthesis processes include de novo (or template- independent) synthesis, template-dependent synthesis and nucleic acid assembly.
- de novo synthesis refers to a process of synthesizing an organic molecule of any length by iteratively linking organic molecule building blocks until the desired polymeric organic molecule is obtained.
- De novo synthesis processes apply to any type of polymeric organic molecules, including peptides, nucleic acids molecules, carbohydrates, and conjugates thereof. In de novo synthesis, the sequence of the newly synthesized organic molecule is not dictated by any template.
- the process typically involves the following steps - although one or more of these steps can be modified or adapted depending on the organic molecule to be synthesized, as will readily be apparent to the one skilled in the art: (a) providing a functionalized support, optionally functionalized with a biomolecule serving as a synthesis initiator;
- organic molecule building block refers to any organic molecule, which upon sequential or cyclical coupling to other organic molecules (e.g, in liquid-phase synthesis) will form a corresponding polymeric organic molecule.
- organic molecule building blocks in SPS may include natural or synthetic building blocks, such as amino acids, nucleotides, nucleosides, monosaccharides and derivatives or analogs thereof.
- organic molecule building block may further include dimers, trimers and the like.
- the organic molecule building block is selected from the group comprising or consisting of an amino acid, a peptide, a nucleotide, a nucleoside, and a saccharide.
- the protected organic molecule building block is selected from the group comprising or consisting of an iV-protected amino acid, an / ⁇ -protected peptide, an 0-protected nucleotide, an 0-protected nucleoside and an 0-protected saccharide.
- polymeric organic molecule refers to any organic molecule, which may be formed upon sequential or cyclical coupling to organic molecule building blocks (e.g. , in liquid-phase synthesis). The nature of the polymeric organic molecule is dependent upon the identities of the organic molecule building blocks.
- polymeric organic molecules prepared in SPS may include biomolecules, such as, e.g, peptides, polypeptides, oligonucleotides, polynucleotides, oligosaccharides and polysaccharides.
- conjugates thereof such as, e.g, peptide-oligonucleotides conjugates, peptide-polysaccharide conjugates, oligonucleotide-oligosaccharide conjugates, and the like.
- template-dependent synthesis applies typically to the synthesis of nucleic acids, and refers to a process that involves the synthesis of a nucleic acid strand that is complementary to a template strand of interest.
- sequence of the newly synthesized nucleic acid strand is dictated by complementary base-pairing with the template strand of interest.
- a functionalized support optionally functionalized with a nucleotide, oligonucleotide or polynucleotide serving either as: a primer for synthesis of a nucleic acid strand which is complementary to at least a portion of a free nucleic acid template hybridized to said primer; or a nucleic acid template for synthesis of its free complementary nucleic acid strand;
- step (b) contacting said functionalized support with at least one organic molecule building block, in particular nucleotides or nucleosides, in conditions suitable to either: couple the organic molecule building blocks with the nucleotide, oligonucleotide or polynucleotide serving as a primer in a complementary fashion to the free nucleic acid template; or couple the organic molecule building blocks to one another in a complementary fashion to the nucleotide, oligonucleotide or polynucleotide serving as a nucleic acid template; (c) repeating step (b) until the desired nucleic acid molecule is obtained.
- organic molecule building block in particular nucleotides or nucleosides
- template-dependent synthesis include enzymatic amplification processes, such as polymerase chain reaction (PCR) which allows to synthetize a DNA (using, e.g, a DNA or RNA-dependent DNA polymerase) or RNA (using, e.g, a DNA or RNA-dependent RNA polymerase) strand from a nucleic acid template (such as, a DNA template or an RNA template).
- PCR polymerase chain reaction
- nucleic acid assembly applies typically to the synthesis of nucleic acids, and refers to a process of assembling a plurality (i.e., two or more) small nucleic acid fragments to produce a longer nucleic acid fragment, using extension-based multiplex assembly reactions, ligation-based multiplex assembly reactions, or a combination thereof, in the presence of a ligase.
- the liquid-phase synthesis processes described above may further include a step of post-synthetic labelling of the polymeric organic molecule, using a modified organic molecule building block.
- modified organic molecule building blocks comprise, e.g. , building blocks bearing a fluorescent dye.
- the peptidic scaffold according to the present invention may be used as a soluble support in molecular biology and biochemistry assays.
- Examples of molecular biology and biochemistry assays in which the peptidic scaffold according to the present invention is useful include, but are not limited to, ligand binding assays, immunoassays, enzyme assays, nucleic acid hybridization assays, affinity purification, and the like.
- ligand binding assay refers to a biochemical test relying on the binding of ligands to a target molecule, such as a receptor, an antibody or any other macromolecule, e.g. , for screening, detecting and/or quantifying a ligand molecule binding to a given target molecule.
- a target molecule such as a receptor, an antibody or any other macromolecule, e.g. , for screening, detecting and/or quantifying a ligand molecule binding to a given target molecule.
- the peptidic scaffold is functionalized with said target molecule.
- ligand/target molecule pairs include, but are not limited to, sub strate/ enzyme; enzyme/ sub strate; antigen/antibody; antibody/antigen; polysaccharide/lectin; lectin/polysaccharide; nucleic aci d/compl ementary base sequence; hormone/receptor; receptor/hormone; poly(A) nucleic acid sequence/RNA comprising a poly(U) nucleic acid sequence; metal ion/poly fusion protein; and poly fusion protein/metal ion.
- the term “immunoassay” refers to a specific type of ligand binding assay in which the presence and/or concentration of a molecule in a solution is measured, through the use of an antibody or an antigen as target molecule.
- the peptidic scaffold is functionalized with said antibody or antigen.
- the molecule detected by the immunoassay is referred to as an “analyte”, and may be, without limitation, a protein or any other type of molecule which may be recognized by or bound by the antibody or antigen.
- immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immune absorbent spot (ELISpot), radioallergosorbent test (RAST), radioimmunoassay, radiobinding assay, and immunofluorescence assays.
- ELISA enzyme-linked immunosorbent assay
- ELISpot enzyme-linked immune absorbent spot
- RAST radioallergosorbent test
- enzyme assay refers to a specific type of ligand binding assay in which the activity of an enzyme is measured, e.g., for studying enzyme kinetics and/or enzyme inhibition. Typically, the consumption of a substrate or the making of a product by said enzyme, acting as target molecule, is measured.
- the peptidic scaffold is functionalized with said enzyme.
- nucleic acid hybridization assay refers to a specific type of ligand binding assay in which the annealing of two complementary strands of nucleic acids is detected.
- the peptidic scaffold is functionalized with an oligonucleotide, of known or unknown sequence.
- affinity purification refers to a variant of a ligand binding assay in which a biochemical mixture is separated, based on a specific interaction between a ligand and its target molecule.
- the molecular biology and biochemistry assay is not or does not comprise a cell delivery assay.
- cell delivery assay refers to a biochemical test relying on the intracellular or cell-surface delivery of an organic molecule.
- the present invention also relates to a kit-of-parts comprising the peptidic scaffold according to the present invention.
- the kit-of-parts comprises the peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-Xs)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3- X4-X5)n amino acid sequence with SEQ ID NO: 2, as described hereinabove.
- the peptidic scaffold further comprises at least one aromatic amino acid, as described hereinabove.
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, as described hereinabove. In one embodiment, the peptidic scaffold comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 5 to SEQ ID NO: 22, as described hereinabove.
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 26 or SEQ ID NO: 27, as described hereinabove.
- the peptidic scaffold comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 28 to SEQ ID NO: 45, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 24, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 25, as described hereinabove. In one embodiment, the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 48, as described hereinabove.
- the peptidic scaffold comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 49, as described hereinabove. In one embodiment, the peptidic scaffold is functionalized, as described hereinabove.
- the peptidic scaffold is functionalized with at least one biomolecule, as described hereinabove.
- the peptidic scaffold further comprises a linker or spacer bound to the reactive sidechain group of at least one of its reactive amino acids, such as, e.g., to the e-amino group of at least one of its lysine (Lys) residues or to the thiol group of at least one of its cysteine (Cys) residues, as described hereinabove.
- a linker or spacer bound to the reactive sidechain group of at least one of its reactive amino acids such as, e.g., to the e-amino group of at least one of its lysine (Lys) residues or to the thiol group of at least one of its cysteine (Cys) residues, as described hereinabove.
- the kit-of-parts may comprise: the peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-Xs)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-X5)n amino acid sequence with SEQ ID NO: 2, as described hereinabove; optionally the peptidic scaffold further comprises at least one aromatic amino acid, as described hereinabove; a linker or spacer comprising a protein-linking group, as described hereinabove; optionally the linker or spacer is functionalized, as described hereinabove; optionally the linker or spacer is functionalized with at least one biomolecule, as described hereinabove; and instructions to couple the linker or spacer to the peptidic scaffold.
- the kit-of-parts may comprise: the peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-Xs)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X 3 -X4-X5)n amino acid sequence with SEQ ID NO: 2, as described hereinabove; optionally the peptidic scaffold further comprises at least one aromatic amino acid, as described hereinabove; a biomolecule comprising a protein-linking group, as described hereinabove; and instructions to couple the biomolecule to the peptidic scaffold.
- the kit-of-parts may comprise: the peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-Xs)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-Xs)n amino acid sequence with SEQ ID NO: 2, as described hereinabove; optionally the peptidic scaffold further comprises at least one aromatic amino acid, as described hereinabove; a linker or spacer comprising a protein-linking group, as described hereinabove; a biomolecule, as described hereinabove; and instructions to couple the linker or spacer and the biomolecule to the peptidic scaffold.
- the kit-of-parts may further comprise any reagents suitable to couple the linker or spacer and/or the biomolecule to the peptidic scaffold.
- the kit-of-parts may further comprise any reagents suitable to use the peptidic scaffold as a soluble support. In any of the previous embodiments, the kit-of-parts may further comprise any reagents suitable to perform synthesis processes of organic molecules and/or molecular biology and biochemistry assays using the peptidic scaffold as a soluble support, as described hereinabove.
- kit-of-parts may further comprise instructions for use.
- a peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 2, with:
- n is an integral number ranging from 2 to 20, and
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H) and arginine (Arg, R); preferably XI is a reactive amino acid selected from the group consisting of lysine (Lys, K) and cysteine (Cys, C); more preferably XI is lysine (Lys, K); and X2, X3, X4 and X5, independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid, preferably selected from the group consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (lie, I), leucine (Leu, L), valine (Val, V), and proline (Pro, P); wherein said peptidic scaffold
- said at least one organic molecule is a biomolecule selected from the group consisting of a nucleotide, an oligonucleotide, a polynucleotide, an amino acid residue, a peptide, a polypeptide, a protein, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide.
- said at least one organic molecule is covalently bound to the e-amino group of the at least one lysine residue through a linker or spacer.
- the peptidic scaffold further comprises at least one aromatic amino acid selected from the group consisting of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), and histidine (His, H), preferably selected from the group consisting of tryptophan (Trp, W), and tyrosine (Tyr, Y).
- the peptidic scaffold comprises a (Lys-Pro-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 3, or a (Pro-Lys-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 4, with “n”, X2, X3, X4 and X5 being as defined above.
- the peptidic scaffold comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5 to 22; preferably comprises the amino acid sequence of
- the peptidic scaffold comprises a (Cy s-Pro-X2-X 3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 26, or a (Pro-Cys-X2-X 3 -X4-Xs-)n amino acid sequence with SEQ ID NO: 27, with “n”, X2, X3, X4 and X5 being as defined above.
- Also disclosed herein is a process for manufacturing a peptidic scaffold according to the present invention, comprising the steps of: a. producing a peptide comprising a (Xi -Pro-X2-X3 -X4-X5 -)n amino acid sequence with SEQ ID NO: 1, or a (Pro-Xi-X2-X3-X4-Xs-)n amino acid sequence with SEQ ID NO: 2, with
- n being an integral number ranging from 2 to 20;
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys,
- XI is a reactive amino acid selected from the group consisting of lysine (Lys, K) and cysteine (Cys, C); more preferably XI is lysine (Lys, K); and X2, X3, X4 and X5, independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid, preferably selected from the group consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (He, I), leucine (Leu, L), valine (Yal, V), and proline (Pro, P); b. purifying the peptide produced at step a.; thereby obtaining a peptidic scaffold, c. functionalizing
- the biomolecule or the linker or spacer comprises a protein-linking group capable of reacting directly or indirectly with the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold.
- a peptidic scaffold according to the present invention as a soluble support.
- the peptidic scaffold is used as a soluble support in synthesis processes of organic molecules.
- the synthesis process of organic molecules is a synthesis process of nucleic acids, peptides, carbohydrates, or conjugates thereof; preferably the synthesis process of organic molecules is an enzymatic nucleic acid synthesis process.
- the peptidic scaffold is used as a soluble support in molecular biology and biochemistry assays.
- the molecular biology and biochemistry assay is a ligand binding assay, immunoassay, enzyme assay, nucleic acid hybridization assay, or affinity purification.
- kit-of-parts comprising: a peptidic scaffold comprising a (Xi-Pro-X2-X3-X4-X5-)n amino acid sequence with
- SEQ ID NO: 1 or a (Pro-Xi-X2-X3-X4-X5-)n amino acid sequence with SEQ ID NO: 2, with
- n being an integral number ranging from 2 to 20,
- Xi being a reactive amino acid selected from the group consisting of lysine (Lys, K), cysteine (Cys, C), serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), histidine (His, H) and arginine (Arg, R); preferably XI is a reactive amino acid selected from the group consisting of lysine (Lys, K) and cysteine (Cys, C); more preferably XI is lysine (Lys, K); and
- X2, X3, X4 and Xs independently from each other and independently for each “n” repeat, being absent or being an aliphatic amino acid, preferably selected from the group consisting of alanine (Ala, A), glycine (Gly, G), isoleucine (lie, I), leucine (Leu, L), valine (Val, V), and proline (Pro, P); a biomolecule and/or a linker or spacer, optionally the linker or spacer is functionalized with at least one biomolecule; wherein the biomolecule, or the linker or spacer, comprises a protein-linking group capable of reacting directly or indirectly with the reactive sidechain group of at least one of the reactive amino acids of the peptidic scaffold; instructions to couple the biomolecule and/or the linker or spacer to the peptidic scaffold.
- Figure 1 is a MALDI-TOF/T OF spectrum of a peptide with SEQ ID NO: 24.
- y-axis intensity in %.
- Figure 2 is a spectrophotometry spectrum showing the absorbance of the peptide with SEQ ID NO: 24 (y-axis) peaking at 280 nm (x-axis), at three different concentrations (1, 5 and 10 mg/mL).
- Figure 3 is a spectrophotometry spectrum at 600 nm, showing the change of absorbance at 600 nm (y-axis) over time (x-axis, in minutes).
- Figure 4 depicts a 33-mer oligonucleotide [33-mer] comprising a 5’ NHS-carboxy-CIO moiety.
- Figure 5 shows a peptidic scaffold with SEQ ID NO: 24 functionalized with 33-mer oligonucleotides using the NHS coupling strategy.
- Figure 6 depicts an oxidized 5’ C6 di thiol 33-mer oligonucleotide [33-mer]
- Figure 7 depicts a reduced 5’ C6 thiol-modified 33-mer oligonucleotide [33-mer]
- Figure 8 depicts a 5 , -C2-aldehyde 33-mer oligonucleotide [33-mer]
- Figure 9 shows the reaction process of a peptidic scaffold according to the invention, with a /V-Boc-aminooxy acetic acid /V-hydroxysuccinimide ester.
- Figure 10 shows the reaction process of the functionalized, deprotected peptidic scaffold with 5’-C2-aldehyde 33-mer oligonucleotides.
- Figure 11 is a photograph of a urea-polyacrylamide gel (15%) upon electrophoresis of neosynthesized oligonucleotide by enzymatic nucleic acid synthesis using a peptidic scaffold functionalized with a 33-mer oligonucleotide as synthesis initiator.
- First lane 33-mer oligonucleotide initiator (control);
- second lane 33-mer +1 after one cycle of synthesis;
- third lane 33-mer +2 after two cycles of synthesis;
- fourth lane 33-mer +3 after three cycles of synthesis;
- fifth lane 33-mer +5 after five cycle of synthesis.
- Figure 12 is a photograph of a 3 % agarose gel, showing the efficacy of the functionalized peptidic scaffold over classically used solid supports (magnetic beads) or free ssDNA primer in a terminal transferase assay.
- Lane 1 MW ladder
- lane 2 unpurified functionalized peptidic scaffold
- lanes 3&4 free ssDNA primer ⁇ enzyme
- lanes 5&6 peptidic scaffold with 4 Oligo 1 ⁇ enzyme
- lanes 7&8 peptidic scaffold with 6 Oligo 1 ⁇ enzyme
- lanes 9&10 solid beads with Oligo 1 ⁇ enzyme.
- Figures 13A-F are a set of six diagrams illustrating exemplary steps of an RNA synthesis process using the peptide frame of the invention.
- Figures 14A-C are a set of three diagrams illustrating exemplary steps of an RNA synthesis process using the peptide frame of the invention.
- Figure 15 is a photograph of a 3 % agarose gel, showing the efficacy of PCR amplification of a gene of interest using the peptidic scaffold functionalized with a T7 terminator primer.
- Lane 1 free single stranded Oligo 2 (/. ⁇ ? ., T7 terminator primer); lane 2: template plasmid, no primer; lane 3: free single stranded Oligo 2 + template plasmid; lane 4: peptidic scaffold with 4 Oligo 2 + template plasmid; lane 5: peptidic scaffold with 5 Oligo 2 + template plasmid; lane 6: peptidic scaffold with 6 Oligo 2 + template plasmid.
- PKPKPKPKPKPKPKPKPKPYPWP A peptide with SEQ ID NO: 24 (PKPKPKPKPKPKPKPKPKPKPYPWP) was synthetized by the “Plateforme de Proteomique de l’lnstitut de Biologie Paris-Seine” (Paris, France).
- the synthesis was carried out using an automated peptide synthesizer, on a polystyrene-based solid support, from C-term to N-term, using Fmoc chemistry.
- the peptide was purified by reverse phase HPLC, lyophilized, and weighted.
- TOF/TOF MALDI tandem time-of-flight
- the peptide at 3 mg/mL was analyzed by absorption spectrophotometry at 600 nm for four minutes. The results were compared to those of a suspension of Praesto ® CNBr resin (Purolite Life Sciences), a highly crosslinked pre activated agarose-based matrix typically used as solid support in molecular biology and biochemistry assays (protein purification, production of customized affinity chromatography media, etc.) and suitable for coupling biomolecules including oligos, peptides, and the like.
- Aromatic amino acid residues tyrosine (Tyr, Y) and tryptophan (Trp, W) were incorporated for spectrophotometry monitoring, their absorption profile allowing direct A280 measurement of peptide concentration (Fig. 2).
- PKPKPKPKPKPKPKPKPKPKPKPYPWP peptide with SEQ ID NO: 24
- Another peptide with SEQ ID NO: 25 was also successfully synthesized and purified using the same routine methods.
- peptidic scaffolds with SEQ ID NO: 24 (PKPKPKPKPKPKPKPKPKPKPKPYPWP) and SEQ ID NO: 25
- AKPGKPLKPAKPGKPLKPGKPYPWP were functionalized, on the e-amino group of its lysine (Lys) residues, with a biomolecule being an oligonucleotide.
- Lys lysine residues
- any other biomolecule can be readily coupled to the peptidic scaffold, depending on the intended use in synthesis processes or molecular biology and biochemistry assays.
- a 33-mer oligonucleotide comprising a 5’ NHS-carboxy-C 10 was purchased at GeneLink (Fig. 4).
- the NHS moiety serves as an amino-reactive protein linking group
- the carboxy-CIO moiety serves as a linker or spacer.
- Coupling was performed according to GeneLink’ s protocol, with a ratio of 4 mg of e- amines for 200 nmoles of oligonucleotide (the peptidic scaffold with SEQ ID NO: 24 comprises 8 lysine residues, hence 8 e-amines).
- a first step is to reduce the disulfide bond of the di thiol oligonucleotide, to obtain a 5’ C6 thiol-modified 33-mer oligonucleotide
- One suitable protocol is divided into two main sub steps: (1) sulfhydryl formation and (2) byproduct removal.
- the oxidized 5’ C6 di thiol 33-mer oligonucleotide is dissolved in 100 mM DTT/100 mM sodium phosphate buffer (pFI 8.3-8.5) and incubated for one hour.
- TCEP can be used in place of DTT.
- reaction mixture is eluted on a NAP- 10 column in 100 mM sodium phosphate buffer (pH 6.0).
- the 5’ C6 thiol-modified 33-mer oligonucleotide (Fig. 7) can be coupled with the e-amino group of the lysine (Lys) residues of the peptidic scaffold using EMCS (6-maleimidohexanoic acid A-hydroxysuccinimide ester), as described in Ghosh et al. , 1990 Bioconjug Chem. l(l):71-6.
- the C6 moiety serves as a linker or spacer.
- a 5’-C2-aldehyde 33-mer oligonucleotide was purchased at Kaneka Eurogentec (Fig. 8).
- the C2 moiety serves as a linker or spacer.
- the functionalized peptidic scaffold was purified, then deprotected in presence of TFA/H2O/TIS (80/16/4) to remove the Boc moiety.
- the deprotected peptidic scaffold comprises thus oxy amine groups.
- PKPKPKPKPKPKPKPKPKPYPWP The peptide with SEQ ID NO: 24 (PKPKPKPKPKPKPKPKPKPKPYPWP) could be successfully functionalized with a 33-mer oligonucleotide, using various coupling strategies well known in the art.
- the functionalized peptidic scaffold can hence serve as a soluble support for enzymatic nucleic acid synthesis.
- a support such as, e.g, a solid support.
- the soluble, functionalized peptidic scaffold produced in Example 2 was used in a process of enzymatic nucleic acid synthesis, the peptidic scaffold replacing the commonly used solid support.
- the 33-mer oligonucleotide serves as a synthesis initiator.
- the process was carried out using 3’-0-NH2-dNTPs as organic molecule building block and a terminal deoxynucleotidyl transferase (TdT) as reacting enzyme.
- TdT terminal deoxynucleotidyl transferase
- the protecting 3’-( -NH2 group was removed using 700 mM sodium nitrite (NaNCh) in 1 M sodium acetate buffered at pH 4.5-5.0 with acetic acid.
- the process was carried out as known in the art, including steps of:
- the soluble peptidic scaffold because of its size (2.461 kDa for the peptidic support itself, plus 8 times ⁇ 10 kDa for the 33-mer oligonucleotide), allows to vacuum filter the sample, for easily washing and eluting reagents and by-products with no risks of eluting the support and/or growing oligonucleotide.
- Example 4
- a first strategy involves the digestion of the peptidic support itself, to release synthetized oligonucleotides comprising: a Lys residue or Lys-Pro dipeptide; - optionally, when present, the linker or spacer; the synthesis initiator (e.g, the 33-mer oligonucleotide); and the neosynthesized oligonucleotide sequence upstream of the synthesis initiator.
- a prolyl -endopeptidase which cleaves peptide bonds in the peptidic scaffold at the C -terminal side of each proline residues, thereby releasing the synthetized oligonucleotides with a Lys-Pro dipeptide;
- Lys-C and/or Lys-N endopeptidases which cleave peptide bonds in the peptidic scaffold at the C -terminal side and N-terminal side, respectively, of each lysine residues, thereby releasing the synthetized oligonucleotides with a Lys residue or Lys-Pro dipeptide.
- This first strategy has the advantage, among others, to maintain a fixed sequence (the synthesis initiator) which can serve, e.g, a priming site for probe hybridization or PCR amplification.
- a second strategy involves the use of endonucleases.
- the one skilled in the art can readily choose any endonuclease, in particular, in accordance with the sequence of the synthesis initiator.
- the peptidic scaffold remaining functionalized with the synthesis initiator, can be recycled for performing further processes of enzymatic nucleic acid synthesis, or any other process which would require such functionalized support.
- the peptidic scaffold with SEQ ID NO: 25 was functionalized, on the e-amino group of its lysine (Lys) residues, with a biomolecule being an oligonucleotide, prior to nucleic acid synthesis assays.
- a peptidic scaffold with SEQ ID NO: 25 (AKPGKPLKPAKPGKPLKPGKPYPWP) was synthetized as described in Example 1.
- ESI-ITMS Electrospray ionization ion trap mass spectrometry
- Oxime ether coupling was carried out as described in Example 2, using two different oligonucleotides: - Oligo 1 consisting of a 5’ -C3 -aldehyde 30-mer oligonucleotide with
- SEQ ID NO: 46 comprising a 6-FAM label on the thymidine at position 6
- Oligo 2 consisting of a 5’ -C3 -aldehyde 22-mer oligonucleotide with SEQ ID NO: 47, comprising a 6-FAM label on the thymidine at position 6.
- the band of interest corresponding to the peptidic scaffold bearing 4 ( ⁇ 120-mer) or 6 ( ⁇ 180-mer) oligos were excised from the gel. These bands were dry-grinded and mixed with 150 pL of ultrapure H O. The sample was left incubating for 45 minutes at 50°C while shaking at 2000 rpm, before ethanol precipitation.
- thermostable archaeal enzyme 5.1 pM (final) of a proprietary thermostable archaeal enzyme. 36 pL of this reaction mix were then added to 4 pL (for a final volume of 40 pL) of either of:
- the enzymatic assay was carried out as follows: for the functionalized peptidic scaffold bearing single stranded Oligo 1 :
- ⁇ elongation 30 minutes at 70°C
- ⁇ reaction stop twice 40 pL of water to wash the enzymatic mix on a magnetic separation rack (DynaTMMag 2 Magnet from ThermoFisher); and
- the samples were then loaded onto a 3 % agarose gel with 2.5 % SybrGreen II, and migrated for 1 hour at 135 V in TBE IX buffer.
- Fig. 12 shows the results of this assay.
- 20 times more genetic material could be coupled before the enzymatic reaction on the peptidic support compared to the beads; moreover, the configuration of the peptidic scaffold-Oligo 1 complex does not interfere with terminal transferase activity compared to synthesis on free ssDNA.
- the fixation of SYBRgreen II and the excess of fluorescence intensity using the peptidic support also corroborated the length of ssDNA fragment being elongated.
- messenger RNA molecules on a large scale represents a major industrial challenge mainly due to the low production yields of conventional methods, as well as the intrinsic instability of these molecules at room temperature.
- conventional methods require a first step of grafting the template gene of interest onto a functionalized chromatographic resin or of cloning the template gene in a plasmid vector, in the form of double-stranded DNA; followed by an in vitro transcription step.
- RNA polymerase of phage T7 requires the use of synthetic enzymes or enzymatic complexes, such as the RNA polymerase of phage T7, but also of maturation enzymes specialized in the addition of a cap at the 5’ end and of the polyadenylation at the 3 ’ end.
- the produced messenger RNA molecules must be purified by chromatography in order to be separated from synthetic enzymes and template DNA.
- synthesis processes are mastered at the laboratory scale, none of these steps is optimized for industrialization and mass production, the reaction yields being directly linked to the quantity of starting material and dependent from one stage to another.
- RNA polymerases produce a large number of abortive strands that very often result in the synthesis of truncated RNA strands.
- the peptidic scaffold of the invention can overcome these issues by increasing the quantity of starting template DNA.
- a first step one or more copies, in a single-stranded DNA form, of the complementary strand of a gene to be transcribed, are coupled to the peptidic scaffold of the invention.
- the full length of this complementary strand can be coupled to the peptidic scaffold; or alternatively, only a complementary fragment of the gene to be transcribed is coupled to the peptidic scaffold, serving as a primer for a first step of amplification in presence of the template gene in ssDNA form, thereby yielding the full complementary strand of the gene being coupled to the peptidic scaffold (Fig. 13A).
- synthesis cycles can be carried out, comprising three successive steps and repeated between 1 and 50 times: a.
- thermostable RNA polymerase e.g ., the mutated Tgo DNA polymerase from Thermococcus gorgonarius described by Cozens etal, 2012.
- RNA primer on the complementary strand at a temperature ranging from about 50°C to about 70°C
- a third step successive ultrafiltrations or filtrations of the reaction medium are carried out with a buffer or water, in order to remove salts, nucleotides and the polymerase.
- This washing step allows to retains only the RNA strands and the template strand attached to the peptidic scaffold. Filtration can take place in a bioreactor capable of stirring, controlling the temperature and filtering, or directly in ultrafiltration units.
- the sample can be incubated in the presence of an enzyme mixture able to perform a 5’ capping and a 3’ poly-adenylation to the newly produced messenger RNA (Fig. 13D), before additional rounds of washing and optionally, of DNAse/protease treatment (Fig. 13E), and ultimately, recovery of the messenger RNA strands (Fig. 13F).
- an enzyme mixture able to perform a 5’ capping and a 3’ poly-adenylation to the newly produced messenger RNA (Fig. 13D), before additional rounds of washing and optionally, of DNAse/protease treatment (Fig. 13E), and ultimately, recovery of the messenger RNA strands (Fig. 13F).
- a first step one or more copies, in a single-stranded DNA form, of the complementary strand of a gene to be transcribed, are coupled to the peptidic scaffold.
- the attached strand should have a plurality of deoxythymidine at their 5’ end, preferably contiguous, preferably between 200 and 300. Coupling to the peptidic scaffold of the invention can be made via the 5’ end, the 3’ end or both indiscriminately.
- the full length of this complementary strand can be coupled to the peptidic scaffold; or alternatively, only a complementary fragment of the gene to be transcribed is coupled to the peptidic scaffold (with its plurality of deoxythymidine at the 5’ end), serving as a primer for a first step of amplification in presence of the template gene in ssDNA form, thereby yielding the full complementary strand of the gene being coupled to the peptidic scaffold (Fig. 14A).
- synthesis cycles can be carried out, comprising three successive steps and repeated between 1 and 50 times: a. denaturation at 95°C of the complementary strand attached to the peptidic scaffold, in the presence of a 5’ capped RNA primer specific to the 3’ end of this complementary strand and of a thermostable RNA polymerase (e.g ., the mutated Tgo DNA polymerase from Thermococcus gorgonarius described by Cozens et al. , 2012. Proc Natl Acad Sci USA. 109(21):8067-72) (Fig. 14B). Denaturation takes place in a bioreactor capable of stirring, controlling the temperature and filtering, or directly in a thermal cycler; b.
- a thermostable RNA polymerase e.g ., the mutated Tgo DNA polymerase from Thermococcus gorgonarius described by Cozens et al. , 2012. Proc Natl Acad Sci USA. 109(21):
- RNA primer hybridization of the RNA primer on the complementary strand at a temperature ranging from about 50°C to about 70°C; c. synthesis of the messenger RNA strand, complementary to the template ssDNA strand, by the polymerase, at temperatures ranging from about 60°C to about 75°C
- a third step successive ultrafiltrations or filtrations of the reaction medium are carried out with a buffer or water, in order to remove salts, nucleotides and the polymerase.
- This washing step allows to retains only the RNA strands and the template strand attached to the peptidic scaffold. Filtration can take place in a bioreactor capable of stirring, controlling the temperature and filtering, or directly in ultrafiltration units.
- sample can optionally be incubated in the presence of DNAse/proteases, before additional rounds of washing and ultimately, recovery of the messenger RNA strands.
- a PCR was performed using the Q5 polymerase Mix 2X (NEB) and following manufacturer’ s protocol.
- Example 7 a peptidic scaffold with SEQ ID NO: 48 (APKAPKAPKAPKAPKAPKAPKWPW) was functionalized, on the e-amino group of its lysine (Lys) residues, with a biomolecule being an oligonucleotide.
- a peptidic scaffold with SEQ ID NO: 48 (APKAPKAPKAPKAPKAPKAPKAPKAPKWPW) was synthetized as described in Example 1.
- this peptidic scaffold with SEQ ID NO: 48 was modified post synthesis as follows: the A-terminal end of the peptide was protected with an acetyl group (CO-CFE;
- the acid moieties thereby exposed from the succinyl linker on the peptidic scaffold act similarly to a peptidic scaffold of SEQ ID NO: 1 or SEQ ID NO: 2 wherein Xi would be an aspartic acid or glutamic acid residue.
- Amine-carboxylic acid coupling was carried out using “Oligo 3”, consisting of a 5’-C6- amine 30-mer oligonucleotide with SEQ ID NO: 46, comprising a 6-FAM label on the thymidine at position 6.
- the reaction was carried out in 25 mM MES pH 4.5, in presence of EDC (ethyl -3 (3- dimethylaminopropoyl)carbodiimide hydrochloride), for amide bond formation from carboxylic acid and amine groups.
- the amount of Oligo 3 was kept constant at 1.25 nmol, while the amount of peptidic scaffold varied from 0.3 to 3.7 nmol, to screen the best ratio giving the best coupling efficacy.
- an oligo: active site molar ratio of 0.3:1 i.e., 1.25 nmol of Oligo 3 and 3.7 nmol of peptidic scaffold
- 100 % of the Oligos were coupled to the scaffold, with a high amount of +4 and +5 coupled sites.
- a peptidic scaffold with SEQ ID NO: 49 (APKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKWPW) was synthetized as described in Example 1.
- TOF/TOF MALDI tandem time-of-flight
- this peptidic scaffold with SEQ ID NO: 49 was modified post synthesis as follows: either a succinyl linker (-CO-CH2-CH2-COOH) was added to the reactive sidechain group of each -amino group of the lysine (Lys) by reaction with succinic anhydride at room temperature for 5 hours in DMF, thereby exposing a carboxylic acid group at the end of each reactive sidechain; or a thiol linker was added to the reactive sidechain group of each e-amino group of the lysine (Lys), thereby exposing a thiol group at the end of each reactive sidechain.
- a succinyl linker -CO-CH2-CH2-COOH
- the acid moieties thereby exposed from the succinyl linker on the peptidic scaffold act similarly to a peptidic scaffold of SEQ ID NO: 1 or SEQ ID NO: 2 wherein Xi would be an aspartic acid or glutamic acid residue.
- SEQ ID NO: 49 with thiol linker
- APKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKAPKWPW was synthetized as described in Example 1. Prior to coupling, this peptidic scaffold with SEQ ID NO: 49 was modified post synthesis by addition of a succinyl linker (-CO-CH2-CH2-COOH) to the reactive sidechain group of each e-amino group of the lysine (Lys), as described in Example 8.
- a succinyl linker -CO-CH2-CH2-COOH
- the acid moieties thereby exposed from the succinyl linker on the peptidic scaffold act similarly to a peptidic scaffold of SEQ ID NO: 1 or SEQ ID NO: 2 wherein Xi would be an aspartic acid or glutamic acid residue.
- Amine-carboxylic acid coupling was carried out using bovine serum albumin (BSA) or a Elis-tagged recombinant protein previously expressed in A. coli and purified successively by Ni-NTA affinity chromatography and heparin column.
- the reaction was carried out by: a. mixing 19 pmol of l-ethyl-3-(3-dimethylaminopropyI)carbodiimide (EDC) and 75 pL of DMSO or alternatively 75 pL of EbO in a tube; b. adding 1.5 nmol of succinyl acid-modified peptidic scaffold; c. pre-activating the reaction mix for 1 hour at 30°C; d. adding 11 pL of BSA or His-tagged recombinant protein (c°: 138 pmol) to the mix, and incubating overnight at 30°C with tube shaking.
- BSA bovine serum albumin
- EDC l-ethyl-3-(3-dimethyl
- the coupling is visualized after agarose gel (1-5%) migration and Coomassie blue staining.
- the peptidic scaffold according to the invention has a theoretical binding capacity of 4000 pmol of oligo/g of support.
- a solid support such as the one described in US20190264017 has a theoretical coupling capacity of 15-350 pmol of oligo/g of support, depending on the length of the oligo (Table 3 of US20190264017).
- the advantage of the peptidic scaffold according to the invention lies in its low molar mass ( ⁇ 3 kD) and its negligible mass in the reaction bulk medium. This scaffold being soluble, it allows to consider any dimensioning of the bioreactor (from miniaturization (microfluidics) to a several liters tanks.
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