Patent Application
20190062373
INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
Any and all applications for which a foreign or domestic priority claim is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 CFR 1.57.
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled PEPT_001A_SUBSTITUTE.TXT, created Nov. 13, 2018, which is 120 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
The present disclosure relates generally to the field of peptide design and protein-protein interactions.
Specific targeting of a protein by a select polypeptide sequence would be extremely useful in many branches of biotechnological sciences including disease prevention, diagnostics, and therapeutics. Animal-sourced antibodies are the present workhorse for detecting target proteins, however, production of these antibodies is tedious, time-consuming, and expensive. It would be highly desirable to develop synthetic antibodies (sAbs) that can be easily synthesized with low cost and time while retaining the favorable molecular recognition characteristics of the animal-sourced antibodies. In pursuit of this end, a number of approaches for predicting or identifying polypeptide sequences for said protein-protein interactions (PPI) have been developed. Computational prediction of PPIs utilizes a diverse database of known protein interactions, primary protein structures, associated physicochemical properties, and appearances of oligopeptide sequences for every protein encoded by the genome of an organism. However, these protein characteristics are not available for all proteins nor all organisms. Although massive library screening methods using the two-hybrid or phage display systems have been broadly accepted as key strategies to identify protein interaction partners, these approaches have been criticized for inaccurate results, and high labor requirements. The protein chip or microarray, another promising method, provides large-scale in vitro PPI data that could be used to identify target binder(s), and chips that expose precisely arranged spots of peptides on a solid support constitute an alternative to the current model. Each of these approaches has unique strengths and weaknesses regarding important factors of PPI such as coverage (library size), binding specificity, identification, experimental bias, post-translational modification, cost, and labor. However, none of these approaches provides a general pairing rule for protein-protein, protein-peptide, or peptide-peptide interaction.
The existence of amino acid complementarity would provide an important insight into protein folding and PPI. There currently are three approaches for formulating amino acid complementarity: 1) The hydropathic complementarity principle (molecular recognition theory); 2) The Root-Bernstein approach, where peptides complementary to a given sequence are encoded by antisense strand read in parallel to the sense strand; and 3) Approaches based on the periodicity of the genetic code.
The hydropathic complementarity principle is closely connected to the concept of sense-antisense peptide interaction, and states that amino acids encoded by the sense strand of DNA are complemented by amino acids with opposite hydropathic scores, coded by the standard 5′→3′ reading of the antisense strand. However, the hydropathic nature of sense and antisense peptides is determined mainly by the central bases of the corresponding codon triplets, and therefore is independent of the direction of the frame reading.
The Root-Bernstein approach suggests that complementary amino acid pairs may result from the parallel reading of complementary DNA strands (i.e. when sense strand is read in 5′-3′ direction, antisense strand is read in 3′→5′ direction). In this approach, it is believed that, of the 210 possible amino acid pairs of the standard 20 amino acids, no more than 26 could meet the physicochemical criteria for probable amino acid pairing. In fact, only 14 of these pairs were found to be genetically encoded pairs using the parallel reading approach. The other 12 pairings were found to be derivatives of the coded pairings in which a single base of the codon triplet had been varied.
In the approaches based on the periodicity of the genetic code, corresponding equivalent codons are categorized into two families of adenine/uracil (A/U) and cytosine/guanine (C/G) based on their central bases. In equivalent codons, the first two nucleotide bases of the triplets are complementary in parallel (3′→5′), with the third being the same. Because of the lack of complementarity with respect to the third base of the codons, peptides designed using this theory cannot be called true “antisense peptides.” The 3′→5′ reading of the complementary DNA strand strongly reduces the impact of the degeneracy of the genetic code on the number of amino acid complements. Thus, there are only minor differences in the assignments of the complementary amino acids according to the various existing approaches. Collectively, it is worth noting that all three approaches share identical complementary amino acid pairing partners for 14 out of 20 standard amino acids.
For all three approaches, successful instances of the complementary peptide-antipeptide interactions have been reported. However, these results have been controversial due to logical contradictions and the inability to repeat some of the studies. These doubts are exacerbated by the low stability of peptide-antipeptide complexes, with most interacting complements possessing dissociation constants (Kd) in the milli- to micromolar range). Furthermore, the sites of many peptide-antipeptide interactions haven't been precisely evaluated with careful attention to important factors including secondary structure, adjacent peptide sequences, amino acid turns in given peptide sequences, protein folding, and composition/spacing of the complementary amino acid pairings. Therefore, it is currently impossible to conclude which of the three approaches outlined above is most effective in predicting peptide-antipeptide interactions. Although various computer programs and publications for designing complementary peptides based on the sense strand of DNA or the resultant amino acid sequence have shown their feasibility, none provides a highly reliable algorithm for designing complementary peptide sequence that can interact with a preselected target peptide sequence with high affinity and specificity, comparable to traditional animal-sourced antibodies. Thus, there is a need for systems and methods that can take advantage of more of the diversity of interactions between amino acids. The present disclosure provides methods of designing binding peptides that go far beyond the limited set of amino acid interactions that could be predicted using previous methods. Further, while methods exist for screening libraries of random peptides for binding to a target protein, none of these methods allows the targeting of a specific region of a target protein, such as a particular region, binding site, or secondary structure element. Therefore, there is a need for methods that can specifically target regions, subsequences, or subdomains of a target protein. Accordingly, there is a need for a method to provide a general amino acid pairing rule for designing polypeptide synthetic antibody (sAb) sequences to interact with a chosen polypeptide sequence in any given target protein.
Disclosed herein is a molecular complex comprising a polypeptide configured to interact with a known binding partner wherein said polypeptide has a polypeptide sequence of between 6 and 20 amino acids in length, wherein said polypeptide sequence is composed by the steps of identifying the sequence of a binding partner; identifying 20% or more of the residues in the sequence of said binding partner; and, for each of the identified residues within the binding partner sequence, selecting the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence as follows: where the identified residue within the binding partner sequence is Phe, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Lys or Glu; where the identified residue within the binding partner sequence is Leu, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Gln, Lys, or Glu; where the identified residue within the binding partner sequence is Ser, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Arg, Gly, Thr, or Ala; where the identified residue within the binding partner sequence is Thr, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Tyr, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Cys, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr or Ala; where the identified residue within the binding partner sequence is Trp, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Pro; where the identified residue within the binding partner sequence is Ile, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Asn, Asp, or Tyr; where the identified residue within the binding partner sequence is Met, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is His; where the identified residue within the binding partner sequence is Asn, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Lys, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Arg, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr, Ala, Ser, or Pro; where the identified residue within the binding partner sequence is Pro, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Arg, Gly, or Trp; where the identified residue within the binding partner sequence is His, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Met or Val; where the identified residue within the binding partner sequence is Gln, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Leu; where the identified residue within the binding partner sequence is Val, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Asn, Asp, Tyr, or His; where the identified residue within the binding partner sequence is Ala, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Asp, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Glu, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Phe or Leu; and where the identified residue within the binding partner sequence is Gly, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr, Ala, Ser, or Pro.
Disclosed herein is a method of making a polypeptide configured to interact with a known binding partner wherein said polypeptide has a polypeptide sequence of between 6 and 20 amino acids in length; wherein said polypeptide sequence is assembled by the steps of: (a) identifying the sequence of said binding partner; (b) identifying 20% or more of the residues in said binding partner sequence; and, (c) for each of the identified residues within the binding partner sequence, selecting the corresponding residue for inclusion in the sequence of said polypeptide sequence according to the relationships disclosed herein.
According to the methods and compositions disclosed herein, the selected residues for inclusion in the polypeptide sequence may occur at one of every two positions in the polypeptide sequence, at every other position in the polypeptide sequence, at one of every three positions in the polypeptide sequence, at every third position in the polypeptide sequence, at two of every three positions in the polypeptide sequence, or at 1, 2, or 3 of every four residues in the polypeptide sequence.
Also disclosed herein are binding peptides made according to the methods described herein, and conjugates and fusions thereof. Such conjugates or fusions may comprise a functional moiety, which may comprise one or more of a polypeptide, a therapeutic molecule, a protein, a nucleic acid, or a diagnostic moiety. Said functional moiety may, for example, comprise one or more of a radiolabel, spin label, affinity tag, or fluorescent label, and may comprise a linker, which may be a peptide, and may have the sequence GSGS (SEQ ID NO: 1), (G)n (SEQ ID NO: 2), (GS)n (SEQ ID NO: 3), (GGSGG)n (SEQ ID NO: 4), (GGGS)n (SEQ ID NO: 5), CYPEN (SEQ ID NO: 6), or KTGEVNN (SEQ ID NO: 7) or the like. Binding peptides designed according to the methods and compositions of the present disclosure may comprise one or more of the sequences LEQIKRLF (SEQ ID NO: 8), LLQVDVILL (SEQ ID NO: 9), LLQVDVILLCYPENLEQIKIRLF (SEQ ID NO: 10), LLQVDVILLCYPENLEQIKIRLFGSGSHHHHHH (SEQ ID NO: 11), EDRLQSYDLD (SEQ ID NO: 12), EDRLQSYDLDGSGSHHHHHH (SEQ ID NO: 13), ELDKAGFIKRQL (SEQ ID NO: 14), LEERGVKDRQLQ (SEQ ID NO: 15), LEILRAKDLALE (SEQ ID NO: 16), LEQIKIRLF (SEQ ID NO: 17), LSGLNEQRTQ (SEQ ID NO: 18), YDVDAIVPQC (SEQ ID NO: 19), CLTYDSHYLQ (SEQ ID NO: 20), LVAHVTSRKC (SEQ ID NO: 21), EYRLYLRALC (SEQ ID NO: 22), IEIVRKKPIF (SEQ ID NO: 23), IEIVRKKPIFC (SEQ ID NO: 24), CEDRLQSYDLD (SEQ ID NO: 25), EKLYLYYLQ (SEQ ID NO: 26), EKLYLYYLQC (SEQ ID NO: 27), LEQIKIRLFGSGSHHHHHH (SEQ ID NO: 28), LSRAYLSYEGSGSHHHHHH (SEQ ID NO: 29), EYRLYLRALCYPENLSRAYLSYEGSGSHHHHHH (SEQ ID NO: 30), DLDYAQLRDKCYPENEDRLQSYDLDGSGSHHHHHH (SEQ ID NO: 31), GKPIPNPLLGLDST (SEQ ID NO: 32), ELDKAGFIKRQLC (SEQ ID NO: 33), LLQVDVILLHHHHHHLEQIKIRLF (SEQ ID NO: 34), and/or CFFDSLVKQ (SEQ ID NO: 35).
In some embodiments, the methods and compositions disclosed herein comprise a molecular complex comprising a binding polypeptide configured to interact with a known binding partner where the binding polypeptide has a sequence of between 6 and 30 amino acids in length; and, where the binding polypeptide sequence is composed by the steps of identifying the sequence of said binding partner; and, identifying 20% or more of the residues in said binding partner sequence; and, for each of the identified residues within the binding partner sequence, selecting the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence as follows: where the identified residue within the binding partner sequence is Phe, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Lys or Glu; where the identified residue within the binding partner sequence is Leu, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Gln, Lys, or Glu; where the identified residue within the binding partner sequence is Ser, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Arg, Gly, Thr, or Ala; where the identified residue within the binding partner sequence is Thr, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Tyr, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Cys, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr or Ala; where the identified residue within the binding partner sequence is Trp, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Pro; where the identified residue within the binding partner sequence is Ile, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Asn, Asp, or Tyr; where the identified residue within the binding partner sequence is Met, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is His; where the identified residue within the binding partner sequence is Asn, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Lys, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Arg, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr, Ala, Ser, or Pro; where the identified residue within the binding partner sequence is Pro, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Arg, Gly, or Trp; where the identified residue within the binding partner sequence is His, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Met or Val; where the identified residue within the binding partner sequence is Gln, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Leu; where the identified residue within the binding partner sequence is Val, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Asn, Asp, Tyr, or His; where the identified residue within the binding partner sequence is Ala, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Asp, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Glu, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Gly, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr, Ala, Ser, or Pro; and where the binding polypeptide may comprise part of a larger polypeptide.
In some embodiments, the methods and compositions disclosed herein comprise a method of making a polypeptide configured to interact with a known binding partner where the binding polypeptide has a sequence of between 6 and 20 amino acids in length; and, where the binding polypeptide sequence is assembled by the steps of: identifying the sequence of said binding partner; and, identifying 20% or more of the residues in said binding partner sequence; and, for each of the identified residues within the binding partner sequence, selecting the corresponding residue for inclusion in the sequence of said binding polypeptide sequence as follows: where the identified residue within the binding partner sequence is Phe, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Lys or Glu; where the identified residue within the binding partner sequence is Leu, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Gln, Lys, or Glu; where the identified residue within the binding partner sequence is Ser, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Arg, Gly, Thr, or Ala; where the identified residue within the binding partner sequence is Thr, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Tyr, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Cys, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr or Ala; where the identified residue within the binding partner sequence is Trp, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Pro; where the identified residue within the binding partner sequence is Ile, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Asn, Asp, or Tyr; where the identified residue within the binding partner sequence is Met, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is His; where the identified residue within the binding partner sequence is Asn, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Lys, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Arg, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr, Ala, Ser, or Pro; where the identified residue within the binding partner sequence is Pro, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Arg, Gly, or Trp; where the identified residue within the binding partner sequence is His, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Met or Val; where the identified residue within the binding partner sequence is Gln, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Leu; where the identified residue within the binding partner sequence is Val, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Asn, Asp, Tyr, or His; where the identified residue within the binding partner sequence is Ala, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Asp, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Glu, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Gly, the residue at the corresponding position for inclusion in the sequence of said polypeptide sequence is Thr, Ala, Ser, or Pro; and where the binding polypeptide may comprise part of a larger polypeptide.
In some embodiments, the methods and compositions disclosed herein comprise a method as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at one of every two positions in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a method as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at every other position in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a method as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at one of every three positions in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a method as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at every third position in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a method as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at two of every three positions in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a composition as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at one of every two positions in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a composition as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at every other position in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a composition as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at one of every three positions in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a composition as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at every third position in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a composition as described herein, where the selected corresponding residues for inclusion in the binding polypeptide sequence occur at two of every three positions in the binding polypeptide sequence. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide made according to the method as described herein. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide as described herein, which comprises a functional moiety. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide as described herein where the functional moiety comprises one or more of a polypeptide, a therapeutic molecule, a protein, a nucleic acid, or a diagnostic moiety. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide as described herein where the functional moiety comprises one or more of a radiolabel, spin label, affinity tag, or fluorescent label. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide as described herein which comprises a linker. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide as described herein where a linker is a peptide. In some embodiments, the methods and compositions disclosed herein comprise a polypeptide as described herein where the peptide includes the sequence GSGS (SEQ ID NO: 1), (G)n (SEQ ID NO: 2), (GS)n (SEQ ID NO: 3), (GGSGG)n (SEQ ID NO: 4), (GGGS)n (SEQ ID NO: 5), CYPEN (SEQ ID NO: 6), or KTGEVNN (SEQ ID NO: 7),In some embodiments, the methods and compositions disclosed herein comprise a binding polypeptide as described herein, where the binding polypeptide contains residues configured to interact with a second and optionally a third target protein in addition to the first target protein. In some embodiments, the methods and compositions disclosed herein comprise a binding polypeptide generated as described herein, where the binding polypeptide contains residues configured to interact with a second and optionally a third target protein in addition to the first target protein. In some embodiments, the methods and compositions disclosed herein comprise a fusion polypeptide, where the fusion comprises one or more binding polypeptides made according to the methods described herein. In some embodiments, the methods and compositions disclosed herein comprise a fusion polypeptide as described herein, where the fusion comprises 2, 3, 4, 5, or 6 binding polypeptides. In some embodiments, the methods and compositions disclosed herein comprise a molecular complex as disclosed herein, where said binding polypeptide is incorporated within a fusion polypeptide, and where said fusion comprises may further comprise one or more additional binding polypeptides. In some embodiments, the methods and compositions disclosed herein comprise a molecular complex as described herein, where the fusion polypeptide comprises 2, 3, 4, 5, or 6 binding polypeptides. In some embodiments, the methods and compositions disclosed herein comprise a binding polypeptide as described herein, where the sequence of the polypeptide comprises one or more of sequence LEQIKRLF (SEQ ID NO: 8), LLQVDVILL (SEQ ID NO: 9), LLQVDVILLCYPENLEQIKIRLF (SEQ ID NO: 10), LLQVDVILLCYPENLEQIKIRLFGSGSHHHHHH (SEQ ID NO: 11), EDRLQSYDLD (SEQ ID NO: 12), EDRLQSYDLDGSGSHHHHHH (SEQ ID NO: 13), ELDKAGFIKRQL (SEQ ID NO: 14), LEERGVKDRQLQ (SEQ ID NO: 15), LEILRAKDLALE (SEQ ID NO: 16), LEQIKIRLF (SEQ ID NO: 17), LSGLNEQRTQ (SEQ ID NO: 18), YDVDAIVPQC (SEQ ID NO: 19), CLTYDSHYLQ (SEQ ID NO: 20), LVAHVTSRKC (SEQ ID NO: 21), EYRLYLRALC (SEQ ID NO: 22), IEIVRKKPIF (SEQ ID NO: 23), IEIVRKKPIFC (SEQ ID NO: 24), CEDRLQSYDLD (SEQ ID NO: 25), EKLYLYYLQ (SEQ ID NO: 26), EKLYLYYLQC (SEQ ID NO: 27), LEQIKIRLFGSGSHHHHHH (SEQ ID NO: 28), LSRAYLSYEGSGSHHHHHH (SEQ ID NO: 29), EYRLYLRALCYPENLSRAYLSYEGSGSHHHHHH (SEQ ID NO: 30), DLDYAQLRDKCYPENEDRLQSYDLDGSGSHHHHHH (SEQ ID NO: 31), GKPIPNPLLGLDST (SEQ ID NO: 32), ELDKAGFIKRQLC (SEQ ID NO: 33), LLQVDVILLHHHHHHLEQIKIRLF (SEQ ID NO: 34), and/or CFFDSLVKQ (SEQ ID NO: 35). In some embodiments, the methods and compositions disclosed herein comprise a binding polypeptide as described herein, or a nucleic acid encoding said binding peptide, where the sequence of said polypeptide comprises one or more of the sequences provided in Table 6. In some embodiments, the methods and compositions disclosed herein comprise such a binding peptide, or a nucleic acid encoding such a binding peptide, where the sequence of the nucleic acid comprises one or more of the sequences provided in Table 7. In some embodiments, the methods and compositions disclosed herein comprise a method of making a binding polypeptide configured to interact with a known binding partner where the binding polypeptide has a sequence of between 6 and 30 amino acids in length, where the binding polypeptide sequence is composed by the steps of identifying the sequence of said binding partner; and, identifying 20% or more of the residues in said binding partner sequence; and where, for each of the identified residues within the binding partner sequence, selecting the residue at the corresponding position for inclusion in the sequence of the polypeptide sequence according to the corresponding residues given in Table 10.
In one aspect, the present disclosure relates to methods for producing peptides, and especially peptides that can engage in interactions with other peptide sequences. In some embodiments, the present disclosure relates to the making of peptide-peptide or peptide-protein complexes, wherein a peptide is designed to interact with a known protein or a protein of known structure or sequence. In some aspects, the present disclosure relates to small peptides that are capable of interacting with other peptides or with proteins, said peptides being designed according to the methods and compositions described herein.
In some embodiments according to the methods and compositions disclosed herein, peptides can be designed to interact with one or more peptides or proteins of known structure or sequence by identifying the sequence of the target protein and, identifying the sequence of the binding peptide according to the following:
where the identified residue within the binding partner sequence is Phe, the residue at the corresponding position for inclusion in the binding peptide sequence is Lys or Glu; where the identified residue within the binding partner sequence is Leu, the residue at the corresponding position for inclusion in the binding peptide sequence is Gln, Lys, or Glu; where the identified residue within the binding partner sequence is Ser, the residue at the corresponding position for inclusion in the binding peptide sequence is Arg, Gly, Thr, or Ala; where the identified residue within the binding partner sequence is Thr, the residue at the corresponding position for inclusion in the binding peptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Tyr, the residue at the corresponding position for inclusion in the binding peptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Cys, the residue at the corresponding position for inclusion in the binding peptide sequence is Thr or Ala; where the identified residue within the binding partner sequence is Trp, the residue at the corresponding position for inclusion in the binding peptide sequence is Pro; where the identified residue within the binding partner sequence is Ile, the residue at the corresponding position for inclusion in the binding peptide sequence is Asn, Asp, or Tyr; where the identified residue within the binding partner sequence is Met, the residue at the corresponding position for inclusion in the binding peptide sequence is His; where the identified residue within the binding partner sequence is Asn, the residue at the corresponding position for inclusion in the binding peptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Lys, the residue at the corresponding position for inclusion in the binding peptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Arg, the residue at the corresponding position for inclusion in the binding peptide sequence is Thr, Ala, Ser, or Pro; where the identified residue within the binding partner sequence is Pro, the residue at the corresponding position for inclusion in the binding peptide sequence is Arg, Gly, or Trp; where the identified residue within the binding partner sequence is His, the residue at the corresponding position for inclusion in the binding peptide sequence is Met or Val; where the identified residue within the binding partner sequence is Gln, the residue at the corresponding position for inclusion in the binding peptide sequence is Leu; where the identified residue within the binding partner sequence is Val, the residue at the corresponding position for inclusion in the binding peptide sequence is Asn, Asp, Tyr, or His; where the identified residue within the binding partner sequence is Ala, the residue at the corresponding position for inclusion in the binding peptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Asp, the residue at the corresponding position for inclusion in the binding peptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Glu, the residue at the corresponding position for inclusion in the binding peptide sequence is Phe or Leu; and where the identified residue within the binding partner sequence is Gly, the residue at the corresponding position for inclusion in the binding peptide sequence is Thr, Ala, Ser, or Pro. In some embodiments, not all of the residues of the binding peptide will be determined according to the relationships disclosed herein. In some embodiments, for example, every other residue, every third residue, or two of every three residues will be determined according to the disclosed relationships.
“Subject” as used herein, has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a human or a non-human animal, for example selected or identified for a diagnosis, treatment, inhibition, amelioration of a disease, disorder, condition, or symptom. “Subject suspected of having” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a subject exhibiting one or more indicators of a disease or condition. In certain embodiments, the disease or condition may comprise one or more of a disease, disorder, condition, or symptom.
“Administering” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to providing a substance, for example a pharmaceutical agent, dietary supplement, or composition, to a subject, and includes, but is not limited to, administering by a medical professional and self-administration. Administration of the compounds disclosed herein or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities such as are consistent with the formulation of said compounds. Oral administrations are customary in administering the compositions that are the subject of the preferred embodiments. In some embodiments, administration of the compounds may occur outside the body, for example, by apheresis or dialysis.
In some embodiments, the methods of the present disclosure contemplate the administration of one or more compositions useful for the amelioration or treatment of one or more disorders, diseases, conditions, or symptoms.
Standard pharmaceutical and/or dietary supplement formulation techniques are used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated herein by reference in its entirety. Accordingly, some embodiments include pharmaceutical and/or dietary supplement compositions comprising, consisting of, or consisting essentially of: (a) a safe and therapeutically effective amount of one or more compounds described herein, or pharmaceutically acceptable salts thereof; and (b) a pharmaceutically acceptable carrier, diluent, excipient or combination thereof.
The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It includes any and all appropriate solvents, diluents, emulsifiers, binders, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, or any other such compound as is known by those of skill in the art to be useful in preparing pharmaceutical formulations of the compounds disclosed herein. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press.
The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the one or more compounds for administration as described herein can be determined by the way the compound is to be administered.
In some embodiments, the methods of the present disclosure contemplate topical or localized administration. In some embodiments, the methods of the present disclosure contemplate systemically or parenterally, such as subcutaneously, intraperitoneally, intravenously, intraarterially, orally, enterically, subdermally, transdermally, sublingually, transbuccally, rectally, or vaginally.
The present disclosure describes binding peptides that interact with proteins or peptides of known structure or sequence. In certain embodiments according to the methods and compositions disclosed herein, said binding peptides may comprise, consist of, or consist essentially of, one or more sequences determined by the steps of: identifying the sequence of the target protein or peptide; and for each residue of the target protein or polypeptide, placing a corresponding residue in the sequence of the binding peptide according to the following relationships: where the identified residue within the binding partner sequence is Phe, the residue at the corresponding position for inclusion in the binding peptide sequence is Lys or Glu; where the identified residue within the binding partner sequence is Leu, the residue at the corresponding position for inclusion in the binding peptide sequence is Gln, Lys, or Glu; where the identified residue within the binding partner sequence is Ser, the residue at the corresponding position for inclusion in the binding peptide sequence is Arg, Gly, Thr, or Ala; where the identified residue within the binding partner sequence is Thr, the residue at the corresponding position for inclusion in the binding peptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Tyr, the residue at the corresponding position for inclusion in the binding peptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Cys, the residue at the corresponding position for inclusion in the binding peptide sequence is Thr or Ala; where the identified residue within the binding partner sequence is Trp, the residue at the corresponding position for inclusion in the binding peptide sequence is Pro; where the identified residue within the binding partner sequence is Ile, the residue at the corresponding position for inclusion in the binding peptide sequence is Asn, Asp, or Tyr; where the identified residue within the binding partner sequence is Met, the residue at the corresponding position for inclusion in the binding peptide sequence is His; where the identified residue within the binding partner sequence is Asn, the residue at the corresponding position for inclusion in the binding peptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Lys, the residue at the corresponding position for inclusion in the binding peptide sequence is Phe or Leu; where the identified residue within the binding partner sequence is Arg, the residue at the corresponding position for inclusion in the binding peptide sequence is Thr, Ala, Ser, or Pro; where the identified residue within the binding partner sequence is Pro, the residue at the corresponding position for inclusion in the binding peptide sequence is Arg, Gly, or Trp; where the identified residue within the binding partner sequence is His, the residue at the corresponding position for inclusion in the binding peptide sequence is Met or Val; where the identified residue within the binding partner sequence is Gln, the residue at the corresponding position for inclusion in the binding peptide sequence is Leu; where the identified residue within the binding partner sequence is Val, the residue at the corresponding position for inclusion in the binding peptide sequence is Asn, Asp, Tyr, or His; where the identified residue within the binding partner sequence is Ala, the residue at the corresponding position for inclusion in the binding peptide sequence is Ser, Gly, Cys, or Arg; where the identified residue within the binding partner sequence is Asp, the residue at the corresponding position for inclusion in the binding peptide sequence is Ile or Val; where the identified residue within the binding partner sequence is Glu, the residue at the corresponding position for inclusion in the binding peptide sequence is Phe or Leu; and where the identified residue within the binding partner sequence is Gly, the residue at the corresponding position for inclusion in the binding peptide sequence is Thr, Ala, Ser, or Pro.
In certain embodiments according to the methods and compositions disclosed herein, said binding peptide sequence may be designed to be parallel to the direction of the target sequence (i.e., with the identified residues in the binding peptide sequence placed from N terminal to C-terminal, corresponding to the residues of the target peptide in their N-terminal to C-terminal orientation) or may be designed to be antiparallel to the direction of the target sequence (i.e., with the identified residues in the binding peptide sequence placed from N terminal to C-terminal, corresponding to the residues of the target peptide in their C-terminal to N-terminal orientation). In some embodiments, a portion, but not all, of the residues of the binding peptide will be determined according to the disclosed relationships. In some embodiments, for example, every other residue, every third residue, one of every three residues, two of every three residues, or one, two, or three out of every four residues will be determined according to the disclosed relationships. In some embodiments, the residues to be determined according to the disclosed relationships will follow a pattern such as [OOXOOOXOO]n, [OOOXOXOOO]n, and [OOOOOXOOOO]n (Where “O” represents a residue determined according to the disclosed relationships, “X” represents any residue, and n represents any integer). In some embodiments, the residues to be determined according to the disclosed relationships will follow a pattern such as [OOO′OOOO′OO]n, [OOOO′OO′OOO]n, and [OOOOOO′OOOO]n (Where “O” represents a residue determined according to the disclosed relationships with respect to a first target protein or peptide, and “O′” a residue determined according to the disclosed relationships with respect to a second target protein or peptide, and n represents any integer).
In some embodiments, without respect to their specific placement within the sequence of the binding peptide, all of the residues of the binding peptide will be selected according to the relationships given herein. In some embodiments, without respect to their specific placement within the sequence of the binding peptide, less than all of the residues of the binding peptide will be selected according to the relationships given herein. In some embodiments, without respect to their specific placement within the sequence of the binding peptide, the percentage of residues within the binding peptide sequence that are selected according to the relationships given herein is 10-30%. In some embodiments, without respect to their specific placement within the sequence of the binding peptide, the percentage of residues within the binding peptide sequence that are selected according to the relationships given herein is between 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 20-90%, 30-90%, or 30-80%. In some embodiments, without respect to their specific placement within the sequence of the binding peptide, the percentage of residues within the binding peptide sequence that are selected according to the relationships given herein is greater than 90%. In some embodiments, without respect to their specific placement within the sequence of the binding peptide, the percentage of residues within the binding peptide sequence that are selected according to the relationships given herein is, or is at least, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, or a range selected from any two of the preceding values.
In some embodiments according to the methods and compositions described herein, a library of binding peptides may be developed according to the relationships and criteria described herein. Said libraries may be screened, such as by surface plasmon resonance spectroscopy, nuclear magnetic resonance spectroscopy, fluorescence resonance energy transfer, fluorescence quenching, Raman spectroscopy, ELISA, western blotting, or dot blot or other methods as are known to those of skill in the art, for binding to the selected target sequence or protein. Sequences identified as having desirable binding properties or other desirable properties may optionally be subjected to another round of design, such as by placing alternate residues still in compliance with the relationships described herein for the design of binding peptides, or by altering the location or register of one or more of the residues selected according to the criteria described herein. Additional rounds of screening and optimization may follow.
In some embodiments, the method is structured according to the steps shown in
The next box illustrates an additional step according to some embodiments of the present method, wherein the length and probable secondary structure of the target sequence can be determined. This may be done according to such criteria as are suitable for the target protein, such as by observing the boundaries of secondary structure elements (e.g. Beta strands, alpha helices, loops, knots, pseudoknots, beta hairpins, 310 helices, and the like) within the three dimensional structure of the target protein or peptide, or by predicting the secondary structures within the target protein using sequence alignments or sequence analysis tools such as are known in the art. Target sequences may be of any length appropriate for the interaction of the binding peptide with the target protein, and as noted herein, exemplary target sequences may be between 2 and 100 amino acids, 2 and 50 amino acids, between 2 and 25 amino acids, between 5 and 20 amino acids, or between 5 and 15 amino acids in length.
The third box depicts a step according to some embodiments of the present method, wherein a binding peptide is designed according to the relationships and design criteria described herein. For example, where the target sequence is primarily alpha helical, CAAP residues corresponding to the residues of the target sequence according to the relationships disclosed herein may be placed at one or two of every three positions within the designed sequence, or when the target sequence comprises significant beta strand character, CAAP residues corresponding to the residues of the target sequence according to the relationships disclosed herein may be placed at every other position within the designed sequence. Likewise, one of skill in the art may determine proper placement of CAAP residues in order to interact with other secondary structure elements, including but not limited to loops, knots, pseudoknots, beta-hairpins, and 310 helices. In some embodiments, the size of the binding peptide may be commensurate with the size of the target sequence, and exemplary binding peptide sequences may be between 2 and 100 amino acids, 2 and 50 amino acids, between 2 and 25 amino acids, between 5 and 20 amino acids, or between 5 and 15 amino acids in length. The contemplated size of the binding peptide, or the binding portion of a protein, is, is about, is at least, or is not more than, 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, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids long, or a range defined by any two of the preceding values.
Optionally, multiple binding sequences may be designed, for example incorporating alternate CAAP residues as disclosed herein and shown in Table 1 or having a different number or placement of the CAAP residues. Exemplary libraries may comprise more than one peptide sequences, between 1 and 5 peptide sequences, between 2 and 10 peptide sequences, 12 or fewer peptide sequences, 24 or fewer peptide sequences, 48 or fewer peptide sequences, 96 or fewer peptide sequences, 192 or fewer peptide sequences, 384 or fewer peptide sequences, 1536 or fewer peptide sequences, or greater than 1536 peptide sequences, or a range between any of the preceding values. Such a library has considerable advantages over conventional library screening methods. For example, while a fully random library of 10-mer peptides would comprise 1013 peptides, an amount which could not reasonably be screened with specificity, by applying the methods described herein, library size and complexity can be reduced by 109-1010-fold, reducing the size of the library to one in which each peptide can reasonably be individually screened.
The next box depicts a step according to some embodiments of the present method, wherein a library of designed binding sequences is synthesized or produced, for example by heterologous gene expression. In some embodiments, DNA sequences corresponding to the sequences of the designed binding peptides can be obtained and transformed into appropriate organisms for expression using such methods as are known in the art (see, for example, Green, M. R. and Sambrook, J., Molecular Cloning: A Laboratory Manual, 4th ed. Volume 3, Cold Spring Harbor Laboratory Press (2012); and Greenfield, E.A., ed., which is hereby incorporated by reference for purposes of its description of genetic modification of organisms and heterologuous protein production). Purification of expressed peptides may be carried out by such methods as are known in the art and may optionally include high performance liquid chromatography, precipitation, and/or affinity purification such as, for example, metal affinity purification, glutathione-S-transferase affinity purification, protein A affinity purification, or Ig-Fc affinity purification. Binding peptides may be synthesized using for example solid phase or liquid phase methods, for example, those described in Jensen, K. J. et al., eds. Peptide Synthesis and Applications, 2nd ed., Humana Press (2013), which is hereby incorporated by reference with respect to its disclosure of methods for the synthesis, purification, and characterization of peptides.
The next box in the figure depicts a step according to some embodiments of the present method, wherein and as noted herein, binding peptide libraries are screened for binding to the target protein using such methods as or known in the art and/or are described herein.
The final box depicts a step wherein optionally, sequences screened may be revised, for example by designing new peptides retaining residues shown to be important to binding, and by varying the position and or composition of the remaining CAAP residues utilizing the relationships disclosed herein and in Table 4. A redesigned library may then be produced or synthesized, and screened, as described, in order to identify peptides with optimal binding activity.
In some embodiments, the binding peptide may comprise one part of a larger fusion peptide. Such a fusion polypeptide may comprise, for example, one or more binding peptides and optionally, an effector peptide. In some embodiments, an effector peptide may comprise a therapeutic or diagnostic peptide, an affinity tag, an antibody, a signaling protein, an enzyme, an inhibitor, or any such peptide moiety as may be desired to be bound to the target protein via the binding peptide. In some embodiments, a fusion peptide comprises a linker as described herein or as known to one of skill in the art. In some embodiments, the binding peptide may comprise the full length of a given fusion polypeptide sequence. In some embodiments, the binding peptide may comprise less than the full length of a given fusion polypeptide sequence. In some embodiments, the binding peptide may comprise between 10% and 100% of the length of a given fusion polypeptide sequence. In some embodiments the binding peptide may comprise between 20% and 90% of the length of a given fusion polypeptide sequence. In some embodiments, the binding peptide may comprise less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the length of a given fusion polypeptide sequence. In some embodiments, a fusion polypeptide may comprise one, two, three, four, or more than four binding peptides. In some embodiments, a fusion polypeptide may be from 10 to 600 amino acids in length. In some embodiments, a fusion polypeptide may be from 10 to 500 amino acids in length. In some embodiments, a fusion polypeptide may be from 20 to 400 amino acids, from 30 to 300 amino acids, from 40 to 200 amino acids, from 50 to 100 amino acids, from 10 to 100 amino acids, from 20 to 100 amino acids, from 10 to 200 amino acids, or from 20 to 200 amino acids in length, or a range defined by any two of the preceding values (e.g. 20 to 600 amino acids).
In some embodiments, the binding peptide may be linked to, or may comprise, an affinity tag or an enzyme. Exemplary tags or enzymes include but are not limited to metal affinity tags such as His6, glutathione-S-transferase, protein A, lectins, immunoglobulin constant regions, fluorescent proteins such as the Green Fluorescent Protein and the like, and/or horseradish peroxidase.
In some embodiments, a sequence may be designed to bind to multiple targets. For example, a sequence may have 50% of its residues selected according to the relationships described herein with respect to the sequence of one target sequence, and 50% of its residues selected according to the relationships described herein with respect to the sequence of a second binding target. The second binding target may be a second target protein or may be a second sequence within a single target protein. The division of residues may be more or less than 50%-50%, for example, from 70-90% to from 10-30%, from 60-80% to from 20-40%, from 50-70% to from 30-50%, from 40-60% to from 40-60%, from 30-50% to from 50-70%, from 20-40% to from 60-80%, or from 10-30% to from 70-90%. Likewise, in some embodiments a sequence may be designed to bind to three or more sequences by allocating a percentage of the residues in the binding peptide sequence to interact according to the relationships described herein with the sequences of three or more target sequences.
In certain embodiments, said binding peptides may exist in single copies. In certain other embodiments, said binding peptides may be fused to other binding peptides. In some embodiments, said binding peptides may be present as dimers, trimers, tetramer, pentamers, hexamers, or the like. In some embodiments, said binding peptides may be fused to identical binding peptides. In some embodiments, two or more different binding peptides may be fused together. In some embodiments said binding peptides may be fused in the same orientation (i.e., C terminus to N terminus). In some embodiments, said peptides may be fused in the opposite orientation (i.e., N terminus to N terminus, or C terminus to C terminus). In some embodiments, said binding peptides may be linked together by a peptide linker. In some embodiments, said peptide linker may comprise, consist of, or consist essentially of, one or more sequences such as (G)n (SEQ ID NO: 2), (GS)n (SEQ ID NO: 3), (GGSGG)n (SEQ ID NO: 4), (GGGS)n (SEQ ID NO: 5), CYPEN (SEQ ID NO: 6), or KTGEVNN (SEQ ID NO: 7) or the like. In some embodiments, said binding peptides may be linked together by a nonpeptide linker. Exemplary nonpeptide linkers include but are not limited to polyethylene glycol, polypropylene glycol, polyols, polysaccharides or hydrocarbons. In some embodiments, each binding peptide within the fusion binds to the same target. In some embodiments, the binding peptides within the fusion bind to different targets.
In some embodiments, the present disclosure describes peptides that interact with target proteins. In some embodiments, said target proteins may comprise, consist of, or consist essentially of, one or more of human c-Jun/c-Fos heterodimer; Human Myc/Max heterodimer; Arabidopsis thaliana Hy5/Hy5 homodimer; Yeast GCN4/GCN4 homodimer; Ylan/Ylan homodimer; Drosophila melanogaster DSX/DSX homodimer; human PALS-1-L27N/Mouse PATJ-L27 heterodimer; Staphylococcus pyogenes Cas9; Escherichia coli alkaline phosphatase (AP); and Human Platelet-Derived Growth Factor (PDGF)/PDGF Receptor (PDGFR) complex. In some embodiments, the binding peptides comprise, consist of, or consist essentially of, one or more of the sequences ELDKAGFIKRQL (SEQ ID NO: 14), LEERGVKDRQLQ (SEQ ID NO: 15), LEILRAKDLALE (SEQ ID NO: 16), LEQIKIRLF (SEQ ID NO: 17), LSGLNEQRTQ (SEQ ID NO: 18), YDVDAIVPQC (SEQ ID NO: 19), CLTYDSHYLQ (SEQ ID NO: 20), LVAHVTSRKC (SEQ ID NO: 21), EYRLYLRALC (SEQ ID NO: 22), IEIVRKKPIF (SEQ ID NO: 23), IEIVRKKPIFC (SEQ ID NO: 24), CEDRLQSYDLD (SEQ ID NO: 25), EKLYLYYLQ (SEQ ID NO: 26), EKLYLYYLQC (SEQ ID NO: 27), LEQIKIRLFGSGSHHHHHH (SEQ ID NO: 28), LLQVDVILLCYPENLEQIKIRLFGSGSHHHHHH (SEQ ID NO: 11), LSRAYLSYEGSGSHHHHHH (SEQ ID NO: 29), EYRLYLRALCYPENLSRAYLSYEGSGSHHHHHH (SEQ ID NO: 30), EDRLQSYDLDGSGSHHHHHH (SEQ ID NO: 13), DLDYAQLRDKCYPENEDRLQSYDLDGSGSHHHHHH (SEQ ID NO: 31), GKPIPNPLLGLDST (SEQ ID NO: 32), ELDKAGFIKRQLC (SEQ ID NO: 33), LLQVDVILLHHHHHHLEQIKIRLF (SEQ ID NO: 34), and/or CFFDSLVKQ (SEQ ID NO: 35), or any combination or derivative thereof.
In some embodiments, binding peptides according to the methods and compositions as disclosed herein may be conjugated to a therapeutic moiety. Exemplary therapeutic moieties include but are not limited to, antibacterial agents, antifungal agents, chemotherapeutic agents, and biologics. In some embodiments the binding peptides according to the methods and compositions disclosed herein may be conjugated to a detectable moiety, including, for example, a fluorescent label, a radiolabel, an enzyme, a colorimetric label, a spin label, a metal ion binding moiety, a nucleic acid, a polysaccharide, or a polypeptide. In some embodiments, binding peptides as disclosed herein or made according to the methods described herein bind to or interact with biomarkers of human or animal diseases, disorders, conditions, or symptoms. It is contemplated that such peptides could be attached to a detectable moiety as described herein to provide for diagnosis, prognosis, or identification of said human or animal diseases, disorders, conditions, or symptoms.
Also contemplated herein are methods of treating diseases or disorders in a subject by administering the peptides as disclosed herein, including administering peptides designed and/or made according to the methods described herein, to a subject in need thereof. The present disclosure contemplates the making of peptide-protein complexes wherein said complex may occur in vivo or wherein said complexes are made by contacting the binding peptides disclosed herein or made by the methods as disclosed herein with a target protein or peptide, and wherein said contacting occurs in vivo. The making of said complexes or the contacting of said binding peptides with said target protein or peptide in vitro or ex vivo is also contemplated. Some embodiments according to the methods and compositions of the present disclosure provide for a composition comprising, consisting of, or consisting essentially of, one or more of the binding peptides as disclosed herein or made according to the methods disclosed herein, and optionally one or more excipients as described herein. Said composition may be prepared according to methods known in the art for delivery to the body of a subject, for example by parenteral, topical, subcutaneous, intramuscular, intraocular, intracerebral, intravenous, intraarterial, oral, ocular, intranasal, or transdermal delivery.
Specific targeting of a protein area by pre-selected sequence would be extremely useful for many branches of biotechnological sciences including medical diagnostics, disease prevention/eradication, biomedical engineering, and metabolic engineering. Antibodies are the present workhorse for detecting target proteins because they recognize epitopes with high affinity and specificity. Currently, however, production of antibodies for the pre-selected target sequence is tedious, time-consuming, and expensive. In addition, it is difficult to produce antibodies in very large quantities. As a large protein with disulfide bonds, moreover, antibodies are relatively fragile and unsuitable for certain applications such as delivery into live cells and very small biological environments. Therefore, it is an important goal to develop small biopolymers that retain the favorable molecular recognition characteristics of antibodies but that can be easily synthesized in large amounts. In the present study, we provide a new concept for the protein detection that has a potential to at least in part replace antibodies for protein targeting. Certain embodiments of the methods and compositions described herein are illustrated by the following non-limiting examples.
We summarized pairings of amino acids in Table 1. This pairing is named “complementary amino acid pairing (CAAP)”. Using the hydrophobicity grouping of amino acids [Kyte J, and Doolittle RF (1982) J Mol Biol 157: 105-132], we found that there are four different types of pairing relationships between the CAAP residues: hydrophilic-hydrophobic (44%), hydrophilic-neutral (20%), neutral-hydrophobic (13%), and neutral-neutral (23%). There are no hydrophilic-hydrophilic and hydrophobic-hydrophobic relationships. Interestingly, 38% of the CAAP interactions (shaded in Table 1) belong to the acceptable amino acid pairings [Root-Bernstein, R. S. J Theor Biol. 1982 Feb. 21; 94(4):885-94]. In addition, the most CAAP interactions have a good stereochemical arrangement: the high molecular weight (bulky) side chains are pairing with the low molecular weight (small) side chains, and vice versa. These observations led us to postulate that the physicochemical and stereochemical natures of the CAAP relationships between two polypeptide chains may provide an attractive environment for protein-protein interaction.
We first focused on finding the CAAP interactions in the protein-protein interaction structure database from the protein data bank (PDB). We first examined the well-known leucine zipper proteins: human c-Jun/c-Fos heterodimer [PDB_1FOS]; Human Myc/Max heterodimer [PDB_1NKP]; Arabidopsis thaliana Hy5/Hy5 homodimer [PDB_20QQ]; and Yeast GCN4/GCN4 homodimer [PDB_2DGC]. As shown in
Next, we expanded the search for the CAAP boxes into some non-leucine-zipper proteins: Staphylococcus aureus Ylan/Ylan homodimer [PDB_20DM]; Drosophila melanogaster DSX/DSX homodimer [PDB_1ZV1]; and human PALS-1-L27N/Mouse PATJ-L27 hetero dimer [PDB_1VF6]. The CAAP boxes are also found in all protein-protein interaction domains of the non-leucine-zipper proteins (
We assessed the composition of all amino acid pairings in the CAAP boxes to obtain information on pairing preference and how the CAAPs were spaced out. First, we wrote a simple computational program to count all amino acid pairings in two different sets, parallel alignment and antiparallel alignment.
The numbers are shown in
To test our CAAP design system, we selected target sequences in the three different proteins: Streptococcus pyogenes Cas9 [PDB_5B2R]; Escherichia coli alkaline phosphatase (AP) [PDB_3TG0]; Human Platelet-Derived Growth Factor (PDGF)/PDGF Receptor (PDGFR) complex [PDB_3MJG], and Horseradish Peroxidase plus V5 epitope (
First, we performed a dot blot experiment to detect a Cas9 target sequence (PTD12 (SEQ ID NO: 27)) using the His-tagged CAAP oligopeptides, PTD13 (SEQ ID NO: 28) and PTD14 (SEQ ID NO: 11), (
Dual detection using a purified polypeptide V5C2-L-HRPC2 with two CAAP box dimer arms designed to interact with V5 epitope and HRP was also achieved. The V5C2-L-HRPC2 was designed with dual CAAP dimers to detect V5 epitope and HRP. Dot blot analysis using synthetic polypeptides, PTD1 (SEQ ID NO: 14) (unrelated, control) and immobilized PTD19 (SEQ ID NO: 32) (part of V5 epitope), as target molecules in presence or absence of V5C2-L-HRPC2 showed that the first interaction between immobilized V5 epitope and V5C2-L-HRPC2 was required for the second interaction between V5C2-L-HRPC2 and purified HRP protein. The interactions were visualized using a HRP chromogenic substrate (
To verify these results, we produced three recombinant fusion proteins, C9-813-92P (monomer, parallel), C9-813-93P (monomer, antiparallel), and C9-813-CAA2 (dimer, antiparallel and parallel), that consist of the N-terminal His-tag (for purification), CAAP oligopeptide, and alkaline phosphatase (AP). Then the same amount of the purified proteins (
Finally, we further examined the performance of the CAAP oligopeptides to detect the whole Cas9 protein in both non-denatured (dot blot) and denatured (western blot) conditions. We used two different forms of the Cas9 protein: the Cas9 protein without any tag (no tag) as an actual target and the His-tagged Cas9 protein as a positive control. The purified Cas9 proteins are shown in
To evaluate the specificity of the synthetic CAAP oligopeptides, PTD13 (SEQ ID NO: 28) and PTD14 (SEQ ID NO: 11), we used them to detect any potential target in the whole proteome of E. coli BL21 Star DE3 (
To investigate whether the CAAP-base protein interaction might be applicable for detecting the β-sheet structure, we designed CAAP oligopeptides to interact with two more target oligopeptide sequences: n_LVAHVTSRKC_c (SEQ ID NO: 21) (PTD8 (SEQ ID NO: 21), coil-beta sheet-coil) in the AP and n_IEIVRKKPIF_c (SEQ ID NO: 23) (PTD10 (SEQ ID NO: 24), beta sheet) in the PDGF-β. We first tested two synthetic His-tagged CAAP oligopeptides, PTD15 (SEQ ID NO: 29) (monomer, antiparallel) and PTD16 (SEQ ID NO: 30) (dimer, parallel and antiparallel), to detect the synthetic oligopeptide PTD8 (SEQ ID NO: 21) (
The CAAP oligopeptide PTD14 induces non-specific DNA binding activity of the Cas9 nuclease
The PTD14 (SEQ ID NO: 11) target site [E813 to Q821] in the Cas9 protein is located in the HNH domain, which is important for DNA binding and DNA cleavage by conformational change. Thus we first tested the effect of the PTD14-Cas9 (SEQ ID NO: 11) interaction on the RNA-guided DNA cleavage by Cas9 nuclease. The PTD16 (SEQ ID NO: 30) was used as negative control. We used a 510 bp human AAV1 region as a target DNA and in vitro transcribed gRNA. We designed a gRNA specific for the AAVS1 to produce 191bp and 319 bp DNA cleavage products (
Oligonucleotides were obtained from Integrated DNA Technologies (IDT) and Thermo Fisher Scientific, and listed in Table 1. Synthetic DNA fragments were obtained from IDT DNA, and listed in Table 1. Synthetic peptides were purchased from Peptide 2.0 and listed in Table 1. Restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs (NEB) and Thermo Fisher Scientific. The purified horseradish peroxidase (HRP) was obtained from PROSPEC.
The bacterial expression vector, pET-21b, was obtained from EMD Millipore (catalog # 69741-3). All plasmids were constructed by assembling two linear DNA fragments, vector and insert, with overlapping ends using a seamless DNA assembly method following the manufacturer's protocol [Thermo Fisher Scientific, GeneArt™ Seamless Cloning and Assembly Enzyme Mix, catalog # A14606]. Briefly, the pET-21b vector was digested with SwaI/XhoI, and assembled with a 143 bp DNA fragment, 92_6HNLS to produce vector pC9-813-92 or 93_6HNLS to produce vector pC9-813-93. The DNA fragments correspond to the parallel CAAP box and antiparallel CAAP box used to detect the Cas9 protein, respectively. The pC9-813-92 and pC9-813-93 vectors were digested with BamHI, and assembled with a 1501 bp DNA fragment 92P or 93P, corresponding to the E. coli alkaline phosphatase (AP) fusion, to generate pC9-813-92P and pC9-813-93P, respectively. The pC9-813-92P vector was digested with BgIII, assembled with a 204 bp synthetic DNA fragment Sp-C9_813-821_CAA, corresponding to the CAAP box tetramer used to detect Cas9, to generate pC9-813-CAA4. The pC9-813-CAA4 vector was digested with BgIII, and self-ligated (to remove 117 bp DNA fragment encoding two CAAP boxes), producing pC9-813-CAA2 which corresponds to the CAAP box dimer to used detect Cas9. A 258 bp synthetic DNA fragment V5C2-L-HRPC2, corresponding to the dual CAAP box dimer arms used to detect both V5 epitope and HRP, was assembled with the SwaI/XhoI-digested pET-21b to generate pV5C2-L-HRPC2.
For production of the recombinant Cas9 proteins, the pET-Spy-Cas9_6His and pET-Spy-Cas9_d6H vectors were constructed by assembling five parts with overlapping DNA ends using the seamless DNA assembly kit. Briefly, four insert parts [a 1000 bp Spy-Cas9_1, a 1030 bp Spy-Cas9_2, a 1030 bp Spy-Cas9_3, and a 1300 bp Spy-Cas9_4, corresponding to the His-tagged Cas9] and the SwaI/XhoI-digested pET-21b were assembled, to create pET-Spy-Cas9_6His. Similarly, four insert parts [a 1000 bp Spy-Cas9_1, a 1030 bp Spy-Cas9_2, a 1030 bp Spy-Cas9_3, and a 1303 bp Spy-Cas9_5, corresponding to the tagless Cas9] and the SwaI/XhoI-digested pET-21b were assembled, to create pET-Spy-Cas9_d6H.
The E. coli strain DH10B T1 [Thermo Fisher Scientific, catalog # 12331013] was used as a cloning host. The E. coli strain BL21 Star (DE3) [Thermo Fisher Scientific, catalog # C601003] was used for production of the recombinant proteins.
For the recombinant protein production, the BL21 Star (DE3) cells harboring an expression vector were grown to mid-log phase (optical density at 600 nm [0D600] of 0.6) in LB medium [ampicillin (Amp), 100 μg/ml] at 28° C. and induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 5 h. Cells were harvested by centrifugation at 3000 rpm for 10 min. The harvested cells were disrupted by using a chemical lysis method following the manufacturer's protocol [Thermo Fisher Scientific, B-PER™ Complete Bacterial Protein Extraction Reagent, catalog # 89821]. Cell debris and insoluble proteins in the lysate were separated by centrifugation at 16,000×g for 5 minutes. The His-tagged recombinant proteins were purified by a metal-affinity chromatography using the Dynabeads™ His-Tag Isolation and Pulldown beads following the manufacturer's protocol [Thermo Fisher Scientific, catalog # 10103D].
The recombinant Cas9 proteins were purified using the HiTrap heparin HP column [GE Healthcare, catalog # 17-0406-01] as previously described (Karvelis et al., 2015).
The sgRNA targeting human AAVS1 region (target sequence GGCTACTGGCCTTATCTCACAGG (SEQ ID NO: 36), PAM sequence underlined) was synthesized by in vitro transcription using a 118 bp PCR-assembled DNA fragment AAVS1_T23826 as template, following the manufacturer's protocol [Thermo Fisher Scientific, TranscriptAid T7 High Yield Transcription Kit, catalog # K0441]. The sgRNA product was purified using the GeneJET RNA Purification Micro Column [Thermo Fisher Scientific, catalog # K0841].
For dot blot analysis, 1 μl (2.5 μg) or 2 μl (5 μg) of samples were spotted onto the nitrocellulose (NC) membrane and dried completely. Then, non-specific sites were blocked by soaking the membrane in the blocking solution made for NC membranes [Thermo Fisher Scientific, WesternBreeze™ Blocker/Diluent (Part A and B), catalog # WB7050]. The membrane was washed twice with water (1 ml per cm2 membrane), and incubated with the 1st antibody (Ab) in a binding/wash (BW) buffer [50 mM sodiumphosphate, pH 8.0, 300 mM NaCl, and 0.01% Tween 20] for 1 h. The membrane was washed 4 times (for 2 minutes per wash) with the wash buffer [Thermo Fisher Scientific, WesternBreeze™ Wash Solution, catalog # WB7003]. If the 1st oligopeptide was Anti-Cas9 Ab-HRP conjugate [Thermo Fisher Scientific, catalog # MAC133P] or the peptide-AP fusions, the membrane was washed twice with water, and incubated with the chromogenic substrates, Chromogenic Substrate (TMB) [Thermo Fisher Scientific, catalog # WP20004] for HRP and NBT/BCIP substrate solution for AP [Thermo Fisher Scientific, catalog # 34042]. Otherwise, the membrane was incubated with in the blocking solution for 1 h. To detect His-tagged peptide and proteins, the Anti-6His Ab-HRP conjugate [Thermo Fisher Scientific, catalog 46-0707] was used. Then the membrane was washed four times with the wash buffer and two times with water. Finally, the blot was incubated with the chromogenic substrates.
For the western blot analysis, the protein samples were resolved in 4-20% gradient SDS-PAGE gel, transferred to NC membrane, and subjected to the western blot analysis using the same method for the dot blot analysis.
A 510 bp human AAVS1 region was amplified from HEK293 genomic DNA by PCR using a primer set (CH1161 and CH1162) and used as a target DNA for the in vitro CRISPR/Cas9 assay. Performance of the Cas9 protein was assessed in various concentrations of Cas9 [100, 50, 25, 12.5, and 0 ng] in presence or absence of sgRNA and peptides (PTD14 (SEQ ID NO: 11) and PTD16 (SEQ ID NO: 30)) in the 1×buffer K [20 mM Tris-HCl, pH 8.5, 10 mM MgCl2, 1 mM Dithiothreitol (DTT), and 100 mM KCl]. The PTD16 (SEQ ID NO: 30) was used as an unrelated peptide control. The reaction mixture was incubated at 37° C. for 15 minutes. The reaction was stopped by adding a stop buffer [1 mM Tris-HCl (pH 7.5), 10 mM EDTA, 6.5% (w/v) Sucrose, 0.03% (w/v) Bromophenol Blue] and heat inactivated at 75° C. for 5 minutes. The reaction samples were resolved in 4% agarose gel.
gagcaccaccaccaccaccactgagatccggct (SEQ ID NO: 49)
gagcaccaccaccaccaccactgagatccggct (SEQ ID NO: 50)
AGATCTGTGAAACAAAGCACTATT (SEQ ID NO: 51)
KAKERLEA (SEQ ID
Homo
sapiens
FHKLTHQR (SEQ ID
ERQQLVET (SEQ ID
Homo
sapiens
LSLSQNMR (SEQ ID
ELSAATHL (SEQ ID
Homo
sapiens
LHTAASLE (SEQ ID
TSVQNVRR (SEQ ID
Homo
sapiens
RSTYVDET (SEQ ID
FEMLIKEILK (SEQ
Enterobacter
KLIEKILMEF (SEQ
IGGTASLITASQ
Helicobacter
pylori 26695
YQRKSQELSREL
LEELDALERSLEQS
Helicobacter
pylori 26695
KLSEVLTQSATILSA
T
LKKKVRKL (SEQ ID
Cyanobacterium
Cyanothece
KKKLQDLE (SEQ ID
Mycobacterium
tuberculosis
AGLSPEEQ (SEQ ID
Allochromatium
vinosum
GAQRTEIQ (SEQ ID
Allochromatium
vinosum
LREHFEKLEK (SEQ
Homo
sapiens
KELKEFHERL (SEQ
KRCSCSSL (SEQ ID
Homo
sapiens
LSSCSCRK (SEQ ID
SYGVGRQG (SEQ ID
Shewanella
RRSIETFA (SEQ ID
LEKEKSEFKLEL
Homo
sapiens
KLEKEKSEFKLE
KLEELERDLRKL
LKRLDRELEELK
ASNLLTTS (SEQ ID
Haemophilus
influenzae
STTLLNSA (SEQ ID
SLINAVKT (SEQ ID
Haemophilus
influenzae
TKVANILS (SEQ ID
Helicobacter
pylori
DKFSEVLDNLKSTF
Helicobacter
pylori
IREKLWAIQEQAAE
QSIKKLKQS (SEQ ID
Bos taurus
LAALQEKAR (SEQ
LSGEQEVLRGELEA
Homo
AK
sapiens
KAAELEGRLVEQE
GSL
Bacteriophage
Lambda
RLIKFLYQS (SEQ ID
SQYLFKILR (SEQ ID
SERIRSTYLGR (SEQ
RGLYTSRIRES (SEQ
FIRSQTLT (SEQ ID
Escherichia
coli
ELLTLTQS (SEQ ID
ESLHDHADEL (SEQ
Escherichia
coli
FRALCSRYLE (SEQ
SLSQASADL (SEQ ID
Homo
sapiens
RKTLSQEIE (SEQ ID
QSTIDLKN (SEQ ID
Homo
sapiens
LRGICQKL (SEQ ID
KSYVHSALKIFKTA
Homo
sapiens
LRCEEATKFIKLAS
YVLYMKYV (SEQ ID
Homo
sapiens
VYKMYLVY (SEQ ID
Homo
sapiens
GSMSVTGI (SEQ ID
Homo
sapiens
PKFTYSII (SEQ ID
QRILELMEHV (SEQ
Caenorhabditis
elegans
LIRKLEKADN (SEQ
Homo
sapiens
ASLQQVLQ (SEQ ID
Caenorhabditis
elegans
SIEELVEK (SEQ ID
Homo
sapiens
IQELRKLL (SEQ ID
Saccharomyces
cerevisiae
DILKNIQR (SEQ ID
LQKRLLALDP (SEQ
Homo
sapiens
ERLAEELKQR (SEQ
VLDRLKMK (SEQ ID
Homo
sapiens
NQVLQLLL (SEQ ID
Mus
musculus
LSMFYETL (SEQ ID
Homo
sapiens
QIHKLSSF (SEQ ID
Mus
musculus
LFSKELRC (SEQ ID
Homo
sapiens
EYRNLQEE (SEQ ID
LEDLVIEFITEMTH
Homo
sapiens
EVVEGVFVKSIGSM
Pseudopleuro
nectes
americanus
VQKHIDLLHTYNEI
Schizosaccharomyces
pombe
HLLDIHKQVTQKA
D
EQQKEQLESSLQ
Schizosaccharomyces
pombe
LKALADQLSSEL
Arenicola
marina
EALEKSEARRKELE
Homo
E
sapiens
LKEALEKSEARRKE
L
EKNDLQLQVQ (SEQ
Homo
sapiens
LLQEKNDLQL (SEQ
ELKRDIDDLE (SEQ
Homo
sapiens
LKRDIDDLEL (SEQ
LKEKLEES (SEQ ID
Mus
musculus
ELKEKLEE (SEQ ID
LEDLKQQLQ (SEQ
Homo
sapiens
QLEDLKQQL (SEQ
LLQEQLEQLQ (SEQ
Homo
sapiens
ELLQEQLEQL (SEQ
AYFAMVKR (SEQ ID
Homo
sapiens
GEAMAYFA (SEQ ID
HLEHDLVH (SEQ ID
Homo
sapiens
VQSHILHL (SEQ ID
LEKRLSEK (SEQ ID
Homo
sapiens
KELEKRLS (SEQ ID
EEGQYVVNEYSR
Drosophila
melanogaster
LMPLMYVILKDA
VEAAVNRL (SEQ ID
Mus
musculus
HFFRELAE (SEQ ID
Homo
sapiens
GVGRQGEQ (SEQ ID
Shewanella
AGLADAFA (SEQ ID
RLERLEQL (SEQ ID
Saccharomyces
cerevisiae
SRLERLEQ (SEQ ID
RRSRARKLQRMKQ
Saccharomyces
LE
cerevisiae
ARRSRARKLQRMK
QL
Caenorhabditis
elegans
Caenorhabditis
elegans
SDDARIAL (SEQ ID
Methanobacterium
fervidus
RIIKNAGA (SEQ ID
LSQLQTEL (SEQ ID
Mus
musculus
KLSQLQTE (SEQ ID
LSQLQTEL (SEQ ID
Mus
musculus
EALIQALG (SEQ ID
LNKLLKQN (SEQ ID
Mus
musculus
ERLNKLLK (SEQ ID
SAYLSELE (SEQ ID
Arabidopsis
thaliana
GSAYLSEL (SEQ ID
ALSEMIQF (SEQ ID
Homo
sapiens
SKAVEQVK (SEQ ID
LARERDTSRRLLAE
Homo
KEREMA
sapiens
EDSLARERDTSRRL
LAEKER
DSFHSLRD (SEQ ID
Homo
sapiens
IQYMRRKV (SEQ ID
Homo
sapiens
KQVEEILR (SEQ ID
Bos taurus
LQQLRDEE (SEQ ID
LQKLQQLRD (SEQ
Bos taurus
EILRLEKEI (SEQ ID
LRQQLQQA (SEQ ID
Mus
musculus
EDLRQQLQ (SEQ ID
QEQLEQLQREF
Mus
musculus
LQEQLEQLQRE
LQVYNNKLE (SEQ
ELQVYNNKL (SEQ
ELEVARLKKL (SEQ
LELEVARLKK (SEQ
LKRKLHKLQ (SEQ
Homo
sapiens
ELKRKLHKL (SEQ
DELELELDQKDELI
Homo
sapiens
IDELELELDQKDELI
QLQNE
LQQLQKDL (SEQ ID
Saccharomyces
cerevisiae
KYLQQLQK (SEQ ID
LDEEISRVRKD (SEQ
Mus
musculus
ERLLDEEISRV (SEQ
GADSLNVAMDCISE
Saccharomyces
A
cerevisiae
ASKEEIAALIVNYFS
Salmonella
enterica
serovar
Typhimurium
EVLDTQFGLQKEVD
Staphylococcus
FAVK
aureus
LYEEVLDTQFGLQK
EVDF
Homo
sapiens
LEKKNEALKERA
Mus
musculus
ERLQKKVEQLSR
LEDEKSALQ (SEQ
Mus
musculus
QLIQQVEQL (SEQ
LKAQNSEL (SEQ ID
Homo
sapiens
EDEKSALQ (SEQ ID
Homo
sapiens
AQECQNLE (SEQ ID
Homo
sapiens
RLEGLTQD (SEQ ID
LILQQAVQVI (SEQ
Mus
musculus
KIETLRLAKN (SEQ
GCPAEQRA (SEQ ID
Homo
sapiens
TNGPKIPS (SEQ ID
EERVSELRHQLQ
Homo
sapiens
LDKDLEEVTMQL
REVYETVY (SEQ ID
Caenorhabditis
elegans
THDVVAHE (SEQ ID
Homo
sapiens
LLEEQLEY (SEQ ID
Saccharomyces
cerevisiae
QKKLVEVE (SEQ ID
LRKRREQL (SEQ ID
Homo
sapiens
KRQNALLE (SEQ ID
LSKNEILR (SEQ ID
Homo
sapiens
KLLILQQA (SEQ ID
Escherichia
coli
Rattus
norvegicus
Rattus
norvegicus
KAQNSELAST (SEQ
Homo
sapiens
LKAQNSELAS (SEQ
KLTVEDLE (SEQ ID
Homo
sapiens
LKLTVEDL (SEQ ID
Homo
sapiens
EALKENEKLHK
Homo
sapiens
LYEALKENEKL
KDDFARFNQR (SEQ
Escherichia
coli
FNAFRSDFQA (SEQ
EIRAAFLE (SEQ ID
Homo
sapiens
LEIRAAFL (SEQ ID
Synthetic peptides were purchased from Peptide 2.0 and are listed in Table 6. Synthetic DNA fragments are listed in Table 7. E. coli strain DH10B T1 [Thermo Fisher Scientific, catalog # 12331013] was used as a cloning host. E. coli strain BL21 Star (DE3) [Thermo Fisher Scientific, catalog # C601003] was used for the production of the recombinant proteins.
The bacterial expression vector, pET-21b, was obtained from EMD Millipore (catalog # 69741-3). The pET-21b vector was digested with SwaI/XhoI, and assembled with a linear 143 bp synthetic DNA fragment, 92_6HNLS or 93_6HNLS, using a seamless DNA assembly method following the manufacturer's protocol [Thermo Fisher Scientific, GeneArt™ Seamless Cloning and Assembly Enzyme Mix, catalog # A14606] to produce vector pC9-813-92 and vector pC9-813-93, respectively. The pC9-813-92 and pC9-813-93 vectors were digested with BamHI, and assembled with a PCR-amplified 1501 bp DNA fragment 92P [primer set: AGCGTTGAAGTTCAGCAGCTGAGATCTGTGAAACAAAGCACTATTG (CH1424) and GGACTTTGCGTTTCTTTTTCGGATCCGCAGATGAACCGTGATGGTGATGGTGATG GCTAGAGCCGGAAGCTTTCAGCCCCAGAGCGGCTTTC (CH1425ART-R)] or 93P [primer set: CAGATTAAAATCCGTCTGTTTAGATCTGTGAAACAAAGCACTATTG (CH1425) and GGACTTTGCGTTTCTTTTTCGGATCCGCAGATGAACCGTGATGGTGATGGTGATG GCTAGAGCCGGAAGCTTTCAGCCCCAGAGCGGCTTTC (CH1425ART-R)] from the E. coli MG1655 genome, corresponding to the E. coli alkaline phosphatase (AP) fusion, to generate pC9-813-92P and pC9-813-93P, respectively. The pC9-813-92P vector was digested with BgIII, assembled with a 204 bp synthetic DNA fragment Sp-C9_813-821_CAA, corresponding to the CCAAP box tetramer recombinant antibody (rAb) against Cas9, to generate vector pC9-813-CAA4. The pC9-813-CAA4 vector was digested with BgIII, and self-ligated to remove 117 bp DNA fragment encoding two CCAAP boxes, producing pC9-813-CAA2 which corresponds to the CCAAP box dimer antibody used to detect Cas9. To introduce two mutations, D153G and D330N, into the E. coli AP protein, we PCR-amplified three DNA fragments, P957-1 [primer set: GAATACCTGTTTATTGAAAAATTAAGATCCGGTGGTGGAGGATCAGGATCCGGT GGTGGAGGATCAGGATCTGTGAAACAAAGCACTATTG (CH1483ART-F) and CAGCGCAGCGGGCGTGGCACCCTGCAACTCTGCGGTAG (CH1486)], P957-2 [primer set: CTACCGCAGAGTTGCAGGGTGCCACGCCCGCTGCGCTG (CH1487) and CAAGGATTCGCAGCATGATTCTGTTTATCGATTGACGCAC (CH1492)], and P957-3 [primer set: GTGCGTCAATCGATAAACAGAATCATGCTGCGAATCCTTG (CH1493) and GTGCTCGAGTTTCAGCCCCAGAGCGGCTTTCATG (CH1494)] and assembled to produce a 1,473-bp DNA fragment corresponding to the mutant AP (or P957). This PCR product was digested with BamHI and XhoI, and ligated into BgIII/XhoI digested pC9-813-CAA2, to generate p813C2-P957dB. For the production of the recombinant antibodies (rAbs), two synthetic DNA fragments, Anti-Bace1 (130 bp) and Anti-PDGFR (130 bp) (Table 7), were digested with SwaI/BgIII and ligated into the same enzyme site of the pC9-813-CAA2, to generate pAnti-Bace1-P and pAnti-PDGFR-P, respectively. Four synthetic DNA fragments, Anti-Brca1 (124 bp), Anti-Hsp90 (124 bp), Anti-EstR (124 bp), and Anti-Xiap (124 bp) (Table 7), were digested with SwaI/BgIII and ligated into the SwaI/BamHI sites of the p813C2-P957dB, to generate pAnti-Brca1-P957, pAnti-Hsp90-P957, pAnti-EstR-P957, and pAnti-Xiap-P957, respectively. To produce the recombinant Cas9 protein, pET-Spy-Cas9_d6H vectors were constructed by assembling five parts with overlapping DNA ends using the seamless DNA assembly kit. Briefly, four insert parts [a 1000 bp Spy-Cas9_1, a 1030 bp Spy-Cas9_2, a 1030 bp Spy-Cas9_3, and a 1303 bp Spy-Cas9_5, corresponding to the tagless Cas9] (Table 7) and the SwaI/XhoI-digested pET-21b were assembled, to create pET-Spy-Cas9_d6H.
For recombinant protein production, BL21 Star (DE3) cells harboring an expression vector were grown to mid-log phase (optical density at 600 nm [OD600] of 0.6) in LB medium [ampicillin (Amp), 100 μg/ml] at 28° C. and induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 5 h. Cells were harvested by centrifugation at 3000×g for 10 min. Harvested cells were disrupted using a chemical lysis method following the manufacturer's protocol [Thermo Fisher Scientific, BPER™ Complete Bacterial Protein Extraction Reagent, catalog # 89821]. Cell debris and insoluble proteins in the lysate were separated by centrifugation at 16,000×g for 5 minutes. His-tagged recombinant proteins were purified via metal-affinity chromatography using Dynabeads™ His-Tag Isolation and Pulldown beads following the manufacturer's protocol [Thermo Fisher Scientific, catalog # 10103D]. Recombinant Cas9 proteins were purified using the HiTrap Heparin HP column [GE Healthcare, catalog # 17-0406-01] as previously described (Karvelis et al. 2015).
For dot blot analysis, 2 μl (5 μg) of samples were spotted onto a nitrocellulose (NC) membrane and dried completely. Then, non-specific sites were blocked by soaking the membrane in blocking solution [Thermo Fisher Scientific, WesternBreeze™ Blocker/Diluent (Part A and B), catalog # WB7050] for 1 hr at room temperature (or up to 72 hr at 4° C.). The membrane was washed twice with water (1 ml per cm2 of membrane), and incubated with the 1st antibody (Ab) in a binding/wash (BW) buffer [50 mM sodium phosphate, pH 8.0, 300 mM NaCl, and 0.01% Tween 20] for 1 hr at room temperature. The membrane was washed 4 times (2 minutes per wash) with wash buffer [Thermo Fisher Scientific, WesternBreeze™ Wash Solution, catalog # WB7003]. If the 1st Ab was Anti-Cas9 Ab-HRP conjugate [Thermo Fisher Scientific, catalog # MAC133P] or the peptide-AP fusions (2nd Ab not required), the membrane was washed twice with water, and incubated with a chromogenic substrate: Chromogenic Substrate (TMB) [Thermo Fisher Scientific, catalog # WP20004] for HRP and NBT/BCIP substrate solution for AP [Thermo Fisher Scientific, catalog # 34042]. Otherwise, the membrane was incubated with 2nd Ab in the blocking solution for 1 hr. To detect His-tagged peptide and proteins, the Anti-6His Ab-HRP conjugate [Thermo Fisher Scientific, catalog # 46-0707] was used as 2nd Ab. Then the membrane was washed four times with the wash buffer and two times with water. Finally, the blot was incubated with the chromogenic substrates. For the western blot analysis, the protein samples were resolved in 4-20% gradient SDS-PAGE gel, transferred to an NC membrane, and analyzed using the same method for the dot blot analysis [note: we have obtained the best result with a long blocking time (72 hr at 4° C.)].
For the image processing, we used Adobe Photoshop 7.0. Quantitative image analysis of the digital images was carried out using measuring tools of imaging software ImageJ (Schneider et al. 2012). Image analysis results were calculated by averaging data from three independent experiments.
Statistical analyses were performed using a one-way analysis of variance (ANOVA) and confirmed by Student's t-test [two tails, two-sample equal variance (homoscedastic)]. p values<0.05 considered statistically significant, and scored with five different levels: ♦, p<0.05; ♦♦, p<0.01; ♦♦♦, p<0.001; ♦♦♦♦, p<0.0001; and; ♦♦♦♦♦, p<0.00001. All graphs display mean±SD.
In the present study, we demonstrate that the pairing between two amino acids encoded by a codon and the reverse complementary codon (c-codon) is favored in PPI. We name this pairing the “Complementary Amino Acid Pairing (CAAP).” We summarize all possible CAAPs in
To address the CAAP hypothesis for PPI, we first focused on finding the CAAP interactions in the PPI structure database from the Protein Data Bank (PDB). We examined the well-known leucine zipper proteins: Saccharomyces cerevisiae GCN4/GCN4 homodimer [PDB_2ZTA], Mus musculus NF-k-B essential modulator (NEMO) homodimer [PDB_4OWF], and Homo sapiens c-Jun/c-Fos heterodimer [PDB_1FOS], and Rattus norvegicus C/EBPA homodimer [PDB_1NWQ] (
We also investigated 75 additional PPI structures for CCAAP interactions (Table 8). A total of 84 protein structures were selected for their relatively simple PPI structures, which limit the effect of any other potential parameters. Protein structures were also categorized according to parallel or antiparallel alignment. We found CCAAP boxes in all PPI sites in the 82 structure data from PDB (Table 8). However, we could not find any CCAAP box from PPI sites of two dimers: Homo sapiens ERBB2-EGFR heterodimer [PDB_2KS1] and Bos taurus If1 homodimer [PDB_1GMJ]. Interestingly, the PPI sites of these two dimers have a high content of either charged amino acids [PDB_2KS1] or hydrophobic amino acids [PDB_1GMJ]. We found 79 CCAAP boxes in the parallel (↓↓) interactions (76 helix/helix, 2 β-sheet/coil, and 1 β-sheet/β-sheet interactions) and 81 CCAAP boxes in antiparallel (↓↓) interactions (67 helix/helix and 14 β-sheet/β-sheet interactions) (Table 8). Notably, 93% of the β-sheet/β-sheet interactions are antiparallel interactions.
TYNVT
Rattus norvegicus
GREWR
LEELERDLRKLK
KLKRLDRELEEL
LEPSKKIVVSTKYLQQLQ
Saccharomyces
EPSKKIVVSTKYLQQLQK
cerevisiae
LSPEEQIE
Allochromatium
KGMNWGMF
vinosum
LFSKELRC
Homo sapiens
EYRNLQEE
LEDLVIEFITEMTH
Homo sapiens
EVVEGVFVKSIGSM
LTGECKELEK ETQHKVLELT
Mus musculus
Mus musculus
QYLKDLIE
Mus musculus
LSTLRNLF
Homo sapiens
LKAQNSEL
Homo sapiens
EDEKSALQ
Homo sapiens
PEELAALESE GKLAQLKSKL
LEKKLAAL
KKELAQLE
RLERLEQL
Saccharomyces
SRLERLEQ
cerevisiae
Homo sapiens
Escherichia coli
KAQNSELAST
Homo sapiens
LKAQNSELAS
MHSLQNVI
HSLQNVIP
ELQVYNNKLERDLQNKIGSLT
LQVYNNKLERDLQNKIGSLTS
IDDLEDELYAQKL
Rattus norvegicus
DDLEDELYAQKLK
LRKRREQL
Homo sapiens
KRQNALLE
Rattus norvegicus
Rattus norvegicus
LSCCE
Laticauda
ECCSL
semifasciata
Homo sapiens
VHTLQQDIDDLK
Homo sapiens
HTLQQDIDDLKR
LEQQVRAL
Homo sapiens
EQQVRALE
DNEIARLK
Homo sapiens
NEIARLKK
RCSCS
Homo sapiens
SCSCR
GEAMAYFA
Homo sapiens
AFYAMAEG
FPIDDRVQ
Homo sapiens
KRTVHVLD
Homo sapiens
Mus musculus
ELTSTWDLMLQTRINLSRSAARM
Salmonella
enterica serovar
LTSTWDLMLQTRINLSRSAARMM
Typhimurium
SELTSTWDLM GLAEGLANQM
Salmonella
enterica serovar
Typhimurium
Homo sapiens
Homo sapiens
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
QRILELMEHVQ LIRKLEKADNN
Caenorhabditis
elegans Homo
sapiens
Caenorhabditis
elegans Homo
sapiens
Drosophila
melanogaster
Homo sapiens
Homo sapiens
Homo sapiens
QNLEVERQ
Homo sapiens
LEGLTQDL
Homo sapiens
FIIVN
Homo sapiens
KIVMV
EFTRLKEALEKSEARRKEL
Homo sapiens
FTRLKEALEKSEARRKELE
LQEKNDLQL
Homo sapiens
QEKNDLQLQ
KLEDECSELKRDIDDLE
Homo sapiens
LEDECSELKRDIDDLEL
KDDFARFNQR FNAFRSDFQA
Escherichia coli
ADEQADICE
Escherichia coli
RALCSRYLE
LEKHKAPVDLS ELVAIMDNVIA
SLIALGNMA
AMNGLAILS
EALAQAFSNSL LSNSFAQALAE
Arenicola marina
ARTPLIAA
Homo sapiens
RTPLIAAG
Homo sapiens
CVSLT
Homo sapiens
TLSVC
Homo sapiens
VKHLKILN
Homo sapiens
NLIKLHKV
IQEYLEKALN NLAKELYEQI
Homo sapiens
EVLDTQMFGLQKEVDFAVK
Staphylococcus
LYEEVLDTQMFGLQKEVDF
aureus subsp.
aureus MW2
QLTKDADE
Staphylococcus
LKVAFDVE
aureus subsp.
aureus MW2
GSAYLSEL
Arabidopsis
SAYLSELE
thaliana
LENKNSEL
Arabidopsis
ENKNSE LE
thaliana
LEERLSTL
Arabidopsis
EERLSTLQ
thaliana
Mus musculus
EAFRELGR
Mus musculus
LAKNYIWA
ILQQAVQV
Mus musculu
NAALDNLR
LEDEKSALQ
Mus musculus
QLIQQVEQL
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
QLEDKVEE
Saccharomyces
LEDKVEEL
cerevisiae
LENEVARLKK ENEVARLKKL
Saccharomyces
cerevisiae
LKQMNVQL
Homo sapiens
KQMNVQLA
KVRKLNSK
Cyanobacterium
LTEEWINL
Cyanothece
LHDLAEGL
Cyanobacterium
ERFIEYTK
Cyanothece
EVREFVGHLERF
Helicobacter
LNHFHNSLSNVE
pylori
RDKFSEVLDNL AIQEQAAEDFE
Helicobacter
pylori
Enterobacter sp.
ELMQQVNVLKLTVEDL
Homo sapiens
LMQQVNVLKLTVEDLE
Homo sapiens
EYLADLVK
Caenorhabditis
LREVNSFM
elegans
DEDSLKAVRLIKFLY
YLFKILRVAKLSDED
Helicobacter
Pylori
IDELELELDQKDELIQML
Homo sapiens
DELELELDQKDELIQMLQ
QDALAKLKNRDAKQTV
Schizosaccharomyces
LAIDRIENYTHLLDIH
pombe
Schizosaccharomyces
pombe
VQKHIDLLHTYNE
Schizosaccharomyces
HLLEQQKEQLESS
pombe
LKQINIQL
Mus musculus
KQINIQLA
EIAALIVNYF
Saccharomyces
FYNVILAAIE
cerevisiae
ADSLNVAMDCISEAFG
Saccharomyces
GFAESICDMAVNLSDA
cerevisiae
QLEDLKQQL
Homo sapiens
LEDLKQQLQ
ELLQEQLEQLQREYSKL
Homo sapiens
LLQEQLEQLQREYSKLK
TPDYLXQL
Mus musculus
RSIEEDLL
KFLREHFEKL LKEFHERLKK
Homo sapiens
Homo sapiens
Homo sapiens
LRHKLTVMYSQIN
Homo sapiens
KASKLRGISTKPV
VRNRQQDV
Homo sapiens
HKLIKGIH
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
QLEDLRQQL
Mus musculus
LEDLRQQLQ
Mus musculus
LKAQADIY
Mus musculus
KAQADIYK
AREKLVEKKEY
Mus musculus
LQEQLEQLQREFNKL
REKLVEKKEYL
QEQLEQLQREFNKLK
Homo sapiens
EERVSELRHQLQ
Homo sapiens
LDKDLEEVTMQL
DLIAKVDQ
Homo sapiens
IRNELKVK
LKRKLHKLQ
Homo sapiens
ELKRKLHKL
EIRAAFLE
Homo sapiens
LEIRAAFL
GQCFS
Homo sapiens
SFCQG
KLEKEKSEFKLELDDVT
Homo sapiens
LEKEKSEFKLELDDVTS
ELGEQIDNL
Homo sapiens
LGEQIDNLQ
LQQLRVNYG QQLRVNYGS
Homo sapiens
Homo sapiens
Ixodes scapularis
ASRSE
Homo sapiens
GECRA
LKEKLEESEKLIKEL
Mus musculus
ELKEKLEESEKLIKE
LESMGISLETSG QLESMGISLETS
Mus musculus
LKEKLEES
Mus musculus
ELKEKLEE
Homo sapiens
Acinetobacter
Bos taurus
LEKEIEDLQ
Bos taurus
QLDEIEKEL
Pelecanus crispus
Drosophila
melanogaster
aCAAP interactions underlined
We assessed the composition of all amino acid pairings in the CCAAP boxes (Table 8) to obtain information on pairing preference and how the CAAPs were spaced out in the CCAAP box, which may be important factors for binding affinity, specificity, and stability. The raw abundance numbers are shown in Table 9 and summarized in
Saccharomyces cerevisiae GCN4
Mus musculus NF-k-B essential modulator
Homo sapiens c-Jun/c-Fos Heterodimer [PDB_1FOS]
Rattus norvegicus C/EBPA Homodimer
Saccharomyces cerevisiae Put3 Homodimer
Salmonella enterica serovar Typhimurium
Mus musculus E47-NeuroD1 Heterodimer
Arenicola marina (lugworm) Arenicin-2
Laticauda semifasciata Erabutoxin
To test the sAb design tool based on the CCAAP principle, we selected a target sequence in the HNH domain of the Staphylococcus pyogenes Cas9 protein [PDB_5B2R]. S. pyogenes CRISPR-Cas9 system has been broadly applied to edit the genome of bacterial and eukaryotic cells. The target sequence for the Cas9 is nEKLYLYYLQc (Helix: E813 to Q821). We designed two different types of synthetic antibody (sAb) molecules, sAb monomer (PTD13, Table 6) and sAb dimer (PTD14, Table 6), to detect the target protein sequences. As shown in the dot blot experiment (
To verify these results, we first produced three recombinant antibody (rAb) constructs, C9-813-92P (monomer, parallel), C9-813-93P (monomer, antiparallel), and C9-813-CAA2 (dimer, antiparallel and parallel). As shown in
Finally, we further examined the performance of the CCAAP oligopeptides to detect the whole Cas9 protein in both non-denatured (dot blot) and denatured (western blot) conditions (
To generalize the CCAAP principle for protein targeting, we have designed a synthetic antibody (sAb) construct and 6 recombinant antibody (rAb) constructs to detect 7 additional clinically important proteins: Anti-PDGF sAb (PTD18, Table 1) for Human Platelet-Derived Growth Factor B (PDGF-B) [PDB_3MJG]; Anti-Bace1 rAb for Human Bace1 [PDB_4B05]; Anti-Brca1 rAb for Human Brca1 [PDB_3PXE]; Anti-Hsp90 rAb for Human Hsp90 [PDB_2VCI]; Anti-EstR rAb for Human Estrogen Receptor [PDB_1A52]; Anti-Xiap rAb for Human Xiap [PDB_2KNA]; and Anti-PDGFR rAb for PDGF Receptor (PDGFR) [PDB_3MJG] (
In the present study, we have developed a novel CCAAP principle and obtained experimental evidence that CCAAP box is a critical driving force for PPI. Therefore, we conclude that the CCAAP concept can be applied to design sAb or rAb that can specifically interact with a preselected oligopeptide sequence (8-10 amino acids) in the target protein.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to plural as is appropriate to the context and/or application. The various singular/plural permutations can be expressly set forth herein for sake of clarity.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims can contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (for example, “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed herein. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62552272 | Aug 2017 | US | |
| 62553757 | Sep 2017 | US |
