Conductive synthetic peptides for molecular electronics

Abstract

To provide a synthetic peptide, in particular a conductive synthetic peptide with an alpha helix conformation used as a molecular wire in molecular electronics.SOLUTION: In various embodiments, a synthetic peptide used as a molecular wire in a molecular electronic circuit comprises an alpha helical segment further comprising repeating alpha helix motifs. The synthetic peptide may further comprise at least one specific conjugation site between the termini for attachment to 5 molecules such as a binding probe, and may further comprise termini having metal binding functionalities such as repeats of material binding sequences. In various aspects, the synthetic peptide comprises intramolecular hydrogen bonding, salt bridges, and optionally, aromatic rings that provide electrical conductivity through the peptide.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Assignee: Roswell Biotechnologies, Inc. Inventors: Barry Merriman, Tim Geiser, Venkatesh Alagarswamy Govindaraj

[0002] Cross - Reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 62 / 790,828, entitled "Conductive Synthetic Peptides for Molecular Electronics," filed on January 10, 2019, the entire disclosure of which is incorporated herein by reference for all purposes.

[0003] The present disclosure generally relates to synthetic peptides, and more particularly to conductive synthetic peptides having an alpha - helix arrangement that are used as molecular wires in molecular electronics.

Background Art

[0004] The broad field of molecular electronics was introduced by Aviram and Ratner in the 1970s. Their concept was to achieve an extreme scale - down of electrical circuits by using single molecules as circuit components. Figure 1 illustrates the general concept of a molecule attached between nanoscale electrodes, as well as some conjugation groups or mechanisms (small shaded squares) for binding the molecule to the electrodes. In some embodiments, a current i that varies with time can be passed through this molecule, as shown in the insertion plot of current versus time. In various embodiments, this current can be regulated by a molecular device, for example, in a logic processing circuit or a sensing circuit.

[0005] Molecular circuit elements such as those capable of providing various functions depending on the molecule and operating conditions, for example, wires, resistors, switches, rectifiers, actuators or sensors. What is interesting here is the application of such constructs as sensors, which provides the basis for single molecule detection by intermolecular interactions with the molecules in the circuit. What is interesting as a circuit element is the various forms of molecular wires that can serve to provide a conductive connection between two points in a molecular circuit, as shown in FIG. 1.

[0006] To those skilled in the art, the term molecular wire generally refers to a molecule having a relatively long and thin structure and having a regular or repeating structure that is electrically conductive or semiconductive. Examples of such well-studied molecular wires include carbon nanotubes, graphene ribbons, alpha-helical proteins, double-stranded DNA helices, and various synthetic organic polymers formed from aromatic rings, such as well-known examples of polythiophene (polymers formed from thiophene ring components) and PEDOT (poly(3,4-ethylenedioxythiophene)) molecules.

[0007] Despite previous research on molecular wires, there is still a need for new molecules for use in electronic molecular circuits, particularly molecules having precise molecular structures that can be made by established methods of synthetic chemistry. SUMMARY OF THE INVENTION

[0008] In various embodiments of the present disclosure, electrically conductive synthetic peptides that can be used as molecular wires in molecular electronic circuits are described. The synthetic peptides of the present invention can be made by bottom-up synthetic chemistry, and thus the synthetic peptides can (a) include an exact molecular structure, for example, to reduce variability in performance in a molecular electronic circuit, (b) include specific attachment groups located at exact locations on the molecule as needed, and (c) can be efficiently produced in large quantities with high purity and low cost.

[0009] In various embodiments, the synthetic peptide has the formula: [X1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m (wherein each X 1 independently comprises a material-binding peptide containing about 5 to about 15 amino acids, a protease cleavage sequence, or a peptide capture tag, and each X 2 independently comprises a glycine / serine {G, S}-rich linker or a C1-C20 carbon chain molecular linker, and each X 3 independently comprises a covalent bond, a single amino acid, a transition helix-promoting motif, a metal-binding group, or a material-binding peptide containing about 5 to about 15 amino acids, and each X 4 independently comprises an alpha-helix motif containing about 4 to about 40 amino acids, each m is independently 0 to 4, and n is 1 to 40).

[0010] In various embodiments, at least one of the X 4 comprises a conjugation site. In various aspects, the conjugation site can comprise cysteine, lysine, tyrosine, biotin, azide, or a click chemistry group, and thus the synthetic peptide can conjugate with a biomolecule such as a binding probe. In various embodiments, the binding probe can comprise a polymerase enzyme or an antibody.

[0011] In various embodiments, at least one of the X 1 can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 16, 18, or 29.

[0012] In various embodiments, at least one of the X 2 ​​​At least one of them may include glycine, serine, GS, GSG, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 24, SEQ ID NO: 25, or a C1-C20 carbon chain molecular linker.

[0013] In various embodiments, [X 1 X 2 m X 3 For any one grouping of X 3 when m≠0, X 1 X 2 m X 3 in any one grouping of X 3 when m = 0, X 1 X 2 m X 3 customizes the termini of the synthetic peptide represented by X 1 X 2 m for specific utilities such as binding to a metal or conjugation to a biomolecule. In various embodiments, both m's cannot be zero, in which case one terminus of the synthetic peptide has the [X

[0014] In various embodiments, the metal-binding group may include the FLASH-binding motif of C, CC, CCC, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 5, SEQ ID NO: 35, or CCXXCC (wherein X is any amino acid in the sequence).

[0015] In various embodiments, at least one of X 4 may include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, 13, 17, 20, 22, 23, 26, 27, 28, or 31.

[0016] ​​​​ In various embodiments, the synthetic peptides according to the present disclosure have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 14.

[0017] In various embodiments, the synthetic peptides according to the present disclosure have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 15.

[0018] In various embodiments, the synthetic peptides according to the present disclosure have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 19.

[0019] In various embodiments, the synthetic peptides according to the present disclosure have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 21.

[0020] In various embodiments, a molecular electronic circuit is disclosed. The molecular electronic circuit includes a first electrode, a second electrode spaced from the first electrode by a nanogap, and a crosslinking molecular wire including a synthetic peptide according to the general formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m electrically connected to both the first and second electrodes and crosslinked to the nanogap, and a polymerase enzyme conjugated to a conjugation site, wherein the circuit includes a conductive path through the synthetic peptide.

[0021] ​​​In various embodiments, molecular sensors are disclosed. The sensor includes a first electrode, a second electrode spaced from the first electrode by a nanogap, and a crosslinking molecular wire including a synthetic peptide that is electrically connected to both the first and second electrodes and crosslinks the nanogap, having the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m and a molecular electronic circuit including a polymerase enzyme conjugated to a conjugation site, the circuit including a conductive pathway through the synthetic peptide, and a transimpedance amplifier electrically connected to at least one of the first and second electrodes, the transimpedance amplifier providing an output including a measurable electrical parameter.

[0022] In various embodiments, a CMOS chip device includes an array of these sensors.

[0023] In various embodiments, a method of sequencing a DNA molecule is disclosed. The method includes a first electrode, a second electrode spaced from the first electrode by a nanogap, and a crosslinking molecular wire including a synthetic peptide that is electrically connected to both the first and second electrodes and crosslinks the nanogap, having the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m ​​​​​​A molecular electronic circuit comprising a crosslinking molecular wire containing a synthetic peptide according to, and a polymerase enzyme conjugated to a conjugation site, the circuit comprising a conductive pathway through the synthetic peptide, and a transimpedance amplifier electrically connected to at least one of a first electrode and a second electrode, the transimpedance amplifier providing an output comprising a measurable electrical parameter, the method comprising providing a sensor comprising: initiating at least one of a voltage or a current through the circuit; exposing the circuit to a solution containing primed single-stranded DNA and / or dNTPs; measuring an electrical signal through the circuit when the polymerase engages and extends the template; and processing the electrical signal to identify features that provide information regarding the sequence underlying the DNA molecules processed by the polymerase.

[0024] In various embodiments, the molecular electronic circuit comprises a first electrode and a second electrode spaced apart by a nanogap, and a first synthetic peptide according to the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m electrically connected between the first electrode and a first site of the polymerase enzyme, and a second synthetic peptide according to the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m electrically connected between the second electrode and a second site of the polymerase enzyme, the circuit comprising a conductive pathway through a portion of the polymerase enzyme.

[0025] ​​​​​​In various embodiments, the molecular sensor includes a first electrode and a second electrode spaced apart by a nanogap, and a first synthetic peptide electrically connected between the first electrode and a first site of a polymerase enzyme according to the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m and a second synthetic peptide electrically connected between the second electrode and a second site of the polymerase enzyme according to the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m The molecular electronic circuit includes a circuit including a conductive path through a portion of the polymerase enzyme, and a transimpedance amplifier electrically connected to at least one of the first electrode and the second electrode, the transimpedance amplifier providing an output including a measurable electrical parameter.

[0026] In various embodiments, the CMOS chip device includes an array of these sensors.

[0027] In various embodiments, a method for sequencing a DNA molecule includes a first electrode and a second electrode spaced apart by a nanogap, and a first synthetic peptide electrically connected between the first electrode and a first site of a polymerase enzyme according to the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m and a second synthetic peptide electrically connected between the second electrode and a second site of the polymerase enzyme according to the formula [X 1 X 2 m ​​​​​​​​​​X 3 [X 4 n X 3 [X 2 X 1 m A molecular electronic circuit comprising a second synthetic peptide according to the following, the circuit comprising a conductive path through a part of a polymerase enzyme, and a transimpedance amplifier electrically connected to at least one of a first electrode and a second electrode, the transimpedance amplifier providing an output comprising a measurable electrical parameter, the method comprising the steps of providing a sensor comprising the transimpedance amplifier; initiating at least one of a voltage or current through the circuit; exposing the circuit to a solution containing primed single-stranded DNA and / or dNTP; and measuring an electrical signal through the circuit when the polymerase engages and extends the template, and processing the electrical signal to identify features that provide information regarding the sequence underlying the DNA molecule processed by the polymerase.

[0028] The subject matter of the present disclosure is particularly pointed out and distinctly claimed at the end of this specification. However, a more complete understanding of the present disclosure can be obtained by reference to the detailed description in conjunction with the drawings and the claims.

Brief Description of the Drawings

[0029]

Figure 1

[0030]

Figure 2

[0031]

Figure 3

[0032]

Figure 4

[0033]

Figure 5

[0034]

Figure 6

[0035]

Figure 7

[0036]

Figure 8

[0037]

Figure 9

[0038]

Figure 10

[0039]

Figure 11

[0040]

Figure 12

[0041]

Figure 13

[0042]

Figure 14

[0043]

Figure 15A

Figure 15B

Figure 15C

Figure 15D

Figure 15E

[0044]

Figure 16A

Figure 16B

Figure 16C

[0045]

Figure 17A

Figure 17B

Figure 17C

[0046]

Figure 18

[0047]

Figure 19

[0048]

Figure 20

[0049]

Figure 21

[0050] In various embodiments of the present disclosure, synthetic peptides are described. In various embodiments, the synthetic peptides of the present invention exhibit electrical conductivity and are used in molecular electronics, such as single - molecule electronics molecular sensors, and in electrical circuits integrated within CMOS semiconductor chip devices.

[0051] In various embodiments, the synthetic peptide comprises an overall alpha - helix conformation or at least one internal alpha - helix segment. In various embodiments, the synthetic peptide has an amino acid sequence designed to confer an overall alpha - helix conformation to the peptide or an amino acid sequence designed to confer at least one internal alpha - helix segment. In various embodiments, the synthetic peptides of the present invention comprise repetitive helix segments or "helix motifs".

[0052] In various embodiments, the synthetic peptides of the invention include a linear primary structure and thus include two opposing ends (a first end near or at the N-terminus of the peptide and a second end near or at the C-terminus of the peptide) to the sequence, which can be referred to as termini. Either or both of the termini can be used for site-specific conjugation, such as attachment to a metal electrode or attachment to an external chemical group.

[0053] In various embodiments, the synthetic peptides of the invention also include internal sites along the amino acid sequence of the peptide between the termini that can be used to conjugate another molecule to the synthetic peptide in a site-specific, selective manner. The internal sites can include a single cysteine amino acid (abbreviated as C or Cys), or lysine (K or Lys), or tyrosine (Y or Tyr), or an amino acid having a chemical modification used for conjugation, such as the addition of a thiol, biotin, azide, or click chemistry group.

[0054] Definitions As used herein, the term "peptide" refers to any continuous single chain of amino acids, where the amino acids are standard amino acids, non-standard amino acids or modified amino acids, or amino acid analogs that participate in peptide bonds. In various embodiments, the peptides of the invention can be in the range of 10 to 300 amino acids in length, or 20 to 200 amino acids in length.

[0055] As used herein, the term "motif" refers to a feature within a synthetic peptide according to the present disclosure. In various examples, the motif can be an alpha helix motif, which means a segment of amino acids within a peptide having an alpha helix secondary structure. In other examples, the motif can be one or more amino acids within a peptide having another functional purpose, such as a material-binding peptide that can be used to tether a synthetic peptide to a metal. In other aspects, the motif can include chemical substituents such as functional groups of organic chemistry (e.g., amines, thiols, etc.), or the motif can include chemical linkers (e.g., one or two amino acids, a run of three amino acids, (poly)ethoxylate tethers, 1,4-phenylene linkages, etc.).

[0056] As used herein, the term "alpha helix" refers to the helical secondary structure of a peptide when used in the context of describing protein structural elements in the field of X-ray crystallography. In this specification, a segment of a synthetic peptide can have an alpha helix secondary structure, and this segment can alternatively be referred to as a "helix segment" or a "helix motif". The synthetic peptides of the present invention can include one or more helix motifs such as repetitive alpha helix motifs. In some cases, for a peptide substantially within an alpha helix secondary conformation, when ignoring the peptide bonds at the two ends of the peptide, the term "alpha helix" can be used in place of the term "synthetic peptide". In various examples, "alpha helix" can refer to a synthetic peptide that can be used as a conductive bridging molecule within a molecular electronic circuit.

[0057] As used herein, the term "molecule" refers to an assembly of covalently bonded atoms, or similarly, a well-defined and stably bonded collection of atoms.

[0058] As used herein, the term "molecular electronics" refers to an electrical circuit in which a small molecule complex involving a single molecule or a small number of molecules is incorporated as an element within the electrical circuit. Such small complexes can involve just two or three molecules, less than 10 molecules in various embodiments, or less than 30 molecules. Molecular electronics sensors are examples of molecular electronics.

[0059] As used herein, the term "molecular wire" refers to a relatively long and thin molecule or an aggregate of a small number of molecules that, when incorporated into a circuit, can conduct electricity, for example, by bridging a pair of electrodes or a contact point for charge transfer. Such an aggregate can be a conductor or a semiconductor, and the current-versus-voltage characteristic can be linear or non-linear. Such an aggregate can also exhibit a band gap that suppresses conduction below a threshold voltage. The length of the molecular wire can be on the order of a few nanometers up to a few microns. The synthetic peptides of the present invention that are electrically conductive can themselves function as molecular wires within a molecular electronic circuit. Alternatively, the synthetic peptides according to the present disclosure can be assembled to form molecular wires that make up a larger structure, for example, a bundle of peptides arranged like filaments within a yarn. When the synthetic peptides can be used directly in a molecular electronic circuit, the terms "synthetic peptide" and "molecular wire" can be used interchangeably.

[0060] As used herein, the term "synthetic peptide" refers to a peptide that is not naturally occurring but is manufactured, i.e., a peptide designed, engineered, and / or produced by artificial means. As such, "synthetic" should not be narrowly construed to mean a peptide assembled by linear or convergent chemical synthesis based solely on organic chemical methods. The synthetic peptides of the present invention can also be created by protein expression and genetic engineering. That is, a synthetic peptide can be prepared by expressing a synthetic DNA gene inserted into such a system in a protein expression system. Furthermore, a synthetic peptide produced by protein expression genetic engineering can be further functionalized by organic synthesis, for example, by adding a functional group, removing a protecting group, etc. The synthetic peptides of the present invention are manufactured but can be reminiscent of naturally occurring peptides and can contain naturally occurring amino acid sequences.

[0061] As used herein, the term "terminus" or its plural form "termini" refers to the ends of the amino acid sequence of a synthetic peptide according to the present disclosure, and enables the distinction between the alpha helix of the synthetic peptide and the conductive core, and the end regions that provide other functionalities such as conjugation for attachment to an electrode or tagging. The "end" of the sequence should not be construed as the actual physical end (atom, or one amino acid) of the sequence, but rather, more broadly, as the end region of the synthetic peptide that can potentially be longer than just one amino acid sterically. In other words, the terminus of a synthetic peptide, whether the N-terminus (N-end) or the C-terminus (C-end), can include various combinations of a single amino acid, sequences of amino acids, linkers and functional groups, various combinations of short sequences of linkers and amino acids, etc. In various embodiments, the terminus of the synthetic peptide can include a cysteine at the end of the sequence, and thus a thiol (-SH) functional group is available for conjugation with a metal. In other examples, the terminus of the synthetic peptide can include a single amino acid functionalized to include a non-natural appendage such as an azide group. In other examples, the end of the sequence can include a material-binding peptide having specificity for a particular metal such as gold or palladium, or a double, triple, or quadruple repeat of the material-binding peptide. In other examples, the terminus of the synthetic peptide can include a metal-binding group such as a sulfide-S-atom in a row with an intervening linker that includes one, two, or three amino acids.

[0062] As used herein, the terms "sequence identity" and "percent sequence identity" with respect to two or more peptide sequences refer to the percentage of designated amino acid residues that are the same when two or more sequences or subsequences are compared and aligned for maximum correspondence using one of the sequence comparison algorithms for such amino acid sequence comparison (e.g., BLASTp or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, percent sequence identity can exist over a region of the sequences being compared, e.g., over an alpha-helical segment, or alternatively, over the full length of the two sequences being compared. For sequence comparison, generally one sequence functions as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The percent sequence identity between the test sequence(s) and the reference sequence is then calculated by the sequence comparison algorithm based on the designated program parameters.

[0063] As used herein, the term "polymer" refers to a molecule that is a chain of chemical components obtained from a defined set of finite constitutional unit molecules and linked together through covalent bonds.

[0064] As used herein, the term "aromatic ring" has its general meaning in organic chemistry and is a ring of sp2 hybridized carbon atoms with or without intervening heteroatoms having lone pairs of electrons, and the electrons in the respective orbitals of the atoms within the ring are delocalized. The term aromatic molecule or aromatic amino acid refers to an entity that contains an aromatic ring within its molecular structure.

[0065] It is the convention used herein to write all amino acid or protein sequences from the N-terminus to the C-terminus.

[0066] As used herein, the term "binding probe" refers to a molecule or molecular complex that preferentially binds to a specific target molecule or family of target molecules. Antibodies or antibody fragments are examples of binding probes. Enzymes such as polymerases are also examples of binding probes. In various embodiments herein, a binding probe can be attached to a synthetic peptide used as a molecular wire for crosslinking between electrodes in a molecular electronic circuit.

[0067] As used herein, the term "enzyme" refers to a molecule or molecular complex that catalyzes a chemical reaction and generally comprises one or more proteins. The term "polymerase" refers to an enzyme that synthesizes a DNA or RNA complement to a DNA or RNA single-stranded template. In various embodiments, an enzyme acts as a binding probe within a molecular electronic circuit.

[0068] As used herein, the term "salt bridge" refers to a weak binding phenomenon formed between a positively charged residue and a negatively charged residue of amino acids present within a peptide, and those residues are positioned in proximity within space such that such a binding can form.

[0069] As used herein, the term "bridge" or "bridge molecular" or "bridging" refers to a molecule that can be used to form a conductive bridge between electrodes, which applies to all synthetic peptides contemplated herein having appropriate attachment groups at their termini. Related terms are "arm" or "arm molecular" and refer to the use of a synthetic peptide in a modality that spans between an electrode and a biomolecule or probe molecule as opposed to spanning between electrodes, which also applies to all synthetic peptides contemplated herein having appropriate attachment groups at their termini.

[0070] As used herein, the term "electrode" refers to one of the electrical contact points in an electrical circuit that serves the function of conducting electrons through the completed circuit. Such electrodes are generally composed of metal or doped semiconductors, are relatively highly conductive, and can further derivatize their surfaces to facilitate appropriate electrical and mechanical connections, such as those using molecular wires.

[0071] Synthetic peptides having a structural motif for promoting use as conductive molecular wires in molecular electronic circuits In various embodiments, the synthetic peptides of the present invention include specific structural motifs for enhancing electrical conductivity through the peptide structure and for providing various conjugation sites such as one or both ends and between the ends of the peptide.

[0072] In various embodiments, the synthetic peptides according to the present disclosure can be represented by the following generalized structure: [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m (wherein, [X 4 n represents the repeating alpha helix motif X 4 repeated n times, and the combination of [X 1 X 2 m X 3 at both ends of the sequence represents the ends of the synthetic peptide. There are two important aspects to the generalized structure. First, the motifs X 1 , X 2 , X 3 , and X 4 in the generalized structure are not limited to a single amino acid, but can instead be amino acid sequences. By way of example, X 4 can be EAAAR (SEQ ID NO: 1), which is not a single amino acid. Second, the X 1 that appears within the square brackets in the generalized structure​​​​, X 2 and X 4 It is important to understand that each example of a motif within a repeating substructure such as etc. is independently selected. For example, in the substructure [X 4 n where n = 3, i.e., in the case of [X 4 3, X 4’ X 4’’ X 4’’’ can be represented as, and X 4’ , X 4’’ and X 4’’’ can be different motifs, for example, different amino acid sequences. In various embodiments, at least one of X 4 contains a conjugation site. Also, the two X 3 motifs at either end of the peptide do not have to be identical, and the ends [X 1 X 2 m X 3 of the synthetic peptide do not have to be identical. In other words, the above generalized structure may suggest a certain symmetry by the midpoint of the synthetic peptide fitting the formula, but not all species encompassed by the general structure have to have such symmetry. For example, [X 1 X 2 m X 3 can be designed to bind the N-terminus to gold, while the other end, [X 1 X 2 m X 3 can be designed to bind the C-terminus to palladium, and thus different metal electrodes can be bridged by different synthetic peptide species. In various embodiments, one example of m can be 0, while other examples of m can be 1, 2, or 3. That is, in various embodiments, m is not 0 for both end portions of the synthetic peptide. In various examples, one end [X 1 X 2 m X 3 ​​​​​may include a tag array. In various embodiments, one example of m is 0, while other examples of m are not zero, and thus one end of the synthetic peptide is X 3 ends with, which can be designed, for example, for click chemistry conjugation with a biomolecule, while at the other end, the substructure [X 1 X 2 m (where m = 1, 2, or 3) can be designed to bind to a metal.

[0073] Finally, as a general rule, the above general structure does not imply the motifs within the structure, i.e., the relative lengths of the segments of the synthetic peptide. In other words, the written general structure does not necessarily imply that the length of the internal helix core portion represented by [X 4 n is shorter than the combination of the two ends represented by [X 1 X 2 m X 3 For example, n can be 21, and X 4 can be a 5 - amino - acid peptide, and thus [X 4 n is 105 amino - acids in length, while the entire [X 1 X 2 m X 3 can be only cysteine (C), i.e., m is 0 and X 3 is cysteine.

[0074] The nature and scope of the motifs X 1 , X 2 , X 3 , and X 4 , as well as the ranges of the repeating units m and n, are defined below in this specification.

[0075] ​​​​​In various embodiments, incorporation of specific structural motifs, particularly helical motifs, promotes an alpha-helix structure for at least a portion of the synthetic peptide. Depending on the amino acid sequence within these alpha-helix segments, electrical conductivity through the synthetic peptide is promoted. These motifs can include, for example, (1) amino acids having side chains of opposing charges that can form salt bridges between residues within an alpha-helix conformation at the i and i + 5 positions within the peptide chain, or at the i and i + 4 positions within the peptide chain; (2) amino acids having aromatic ring residues; (3) amino acids having aromatic ring residues at the i and i + 4 positions or at the i and i + 5 positions, such that such rings can be spatially adjacent in an alpha-helix conformation; (4) a hydrocarbon chain staple that stabilizes the alpha-helix structure and is present between the i and i + 4 residues or the i + 5 residues within the chain, and can fix the proximity site in an alpha-helix conformation; or (5) an amino acid sequence motif that mimics or is reminiscent of a biologically conductive protein and that essentially has the biological function of charge transfer and can include.

[0076] Regarding a general diagram of the alpha-helix peptide concept, FIG. 15 shows five different diagrammatic representations of an alpha-helix peptide. The peptide in this example consists of 40 amino acids, and the primary amino acid sequence (written using the standard AUG amino acid code one-letter designations) consists of eight repeats of the motif EAAAR (SEQ ID NO: 1) (E = glutamic acid, A = alanine, R = arginine), or in shorthand notation 8×(EAAAR) (SEQ ID NO: 4), or explicitly written as EAAAREAAAREAAAREAAAREAAAREAAAREAAAREAAAR (SEQ ID NO: 4) in the standard direction from the N-terminus to the C-terminus.

[0077] Figures 15A - E show various displays of SEQ ID NO: 4. Figure 15A is a protein ribbon diagram of the alpha - helix structure of the peptide, Figure 15B is a ball - and - stick model of the protein peptide - binding backbone, Figure 15C is a space - filling model of the protein peptide - binding backbone, Figure 15D is a ball - and - stick model of the complete molecular structure including amino acid side chains, and Figure 15E is a space - filling model of the complete molecular structure including amino acid side chains.

[0078] The general structure of an alpha - helix consists of 3.6 amino acids per helix turn, a length of 0.54 nm per helix turn, and a helix - core diameter of 1.2 nm. Thus, the example of the peptide of SEQ ID NO: 4 shown in Figures 15A - E has 40 amino acids, or 11.1 helix turns, or a length of 6.0 nm.

[0079] In various embodiments, the synthetic peptides according to the present disclosure include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 4. In various embodiments, the synthetic peptide includes SEQ ID NO: 4. In various embodiments, such synthetic peptides function as conductive molecular wires in molecular electronic circuits. In various embodiments, this alpha - helix structure can be the central core of a larger synthetic peptide.

[0080] In various embodiments of the present disclosure, the alpha - helix peptide can be designed to have an exact length that falls within the range of 3 nm to 100 nm, or in some embodiments 5 nm to 50 nm, or in other embodiments 7 nm to 30 nm, 10 nm to 25 nm, or 10 nm to 15 nm.

[0081] Synthetic peptides can be made by established peptide synthesis methods, such as those known to those skilled in synthetic organic chemistry and biochemistry. These methods can include, but are not limited to, solid-phase or liquid-phase synthesis of amino acids linked to a peptide, and may further include the use of chemical ligation of such synthetic fragments to efficiently produce longer peptides. The amino acid components used can include the standard 22 proteinogenic amino acids found in biology, as well as so-called non-standard amino acids (NSAAs) or unnatural amino acids (UAAs), including well-known examples of chemically modified forms of phenylalanine (F) represented as pAcF (p-acetyl-F), pAzF (p-azido-F), and pBpF (p-benzoyl-dl-F).

[0082] The synthetic peptides according to the present disclosure can also be made by the expression of a synthetic gene representing the desired peptide sequence. Such protein expression methods are well known and generally involve cloning a synthetic DNA segment ("gene") into a bacterial or other expression vector, expressing it, and purifying the resulting protein of interest. For such expression systems, a capture tag peptide (such as a His tag or a FLAG tag or others known in the art) can be added to one of the C-terminus or N-terminus of the target peptide of interest, and in various embodiments, this tag can be removed or not removed after capture. If such a small tag or related peptide residue remains in place, generally, it does not affect the usefulness of the synthetic peptides described herein.

[0083] In the case of expression, when using an expression system created for such a purpose, it is also possible to add NSAA to the peptide, similar to the case of the so-called "extended genetic code". Such expression systems, which rely on the use of a bacterial vector with co-expression of a modified genetic code and a customized transfer RNA charged with the desired NSAA, are well-known to those skilled in the art of extended genetic code. For example, for such systems for protein expression using NSAA, biologists Peter G. Schultz and George M. Church were pioneers. Such systems based on E. coli have been used to date to express proteins containing over 70 different NSAAs.

[0084] A binding group at or near the end of a peptide that may be located at or near the end of an alpha-helix segment and is useful for conjugation of a peptide into a molecular circuit may contain the amino acid cysteine (C), which is useful for thiol-based conjugation such as thiol-metal binding to a metal electrode surface, or is also useful for conjugation based on cysteine-maleimide selective binding to maleimide, or cysteine-cysteine crosslinking or cysteine-sulfide crosslinking as conjugation chemistry. The binding group may also include a cysteine-rich motif, CC, CCC, CCCC (SEQ ID NO: 32), CCCCC (SEQ ID NO: 33), or a "FLASH" tetra-C motif such as CCCGCC (SEQ ID NO: 5) or CCPGCC (SEQ ID NO: 35) CCXXCC (X = any amino acid). The conjugation group at or near the end of the helix segment may also include an amino acid having a group that can bind to a similar derivatized surface, such as an azide or amine group, or a group that can participate in click chemistry binding. The conjugation group at or near the end of the helix segment may also include a material-binding peptide. A material-binding peptide is a peptide generally in the range of 5 to 15 amino acids in length having an amino acid sequence capable of binding to a specific material. Many such material-binding peptides are known to those skilled in the art of bioconjugation. One example, Brown's gold-binding peptide known to selectively bind to gold, has the amino acid sequence MHGKTQATSGTIQS (SEQ ID NO: 6). See, for example, S. Brown, “Metal-recognition by repeating polypeptides,” Nature Biotechnology, 15, 269-272, 1997. Other known metal-binding peptides include, but are not limited to, WAGAKRLVLRRE (SEQ ID NO: 7), VSGSSPDS (SEQ ID NO: 8), TGTSVLIATPYV (SEQ ID NO: 9), LKAHLPPSRLPS (SEQ ID NO: 10), and QQSWPIS (SEQ ID NO: 16). The last example is a palladium-binding peptide.

[0085] Furthermore, the binding group may include such binding peptides arranged as a series of tandem repeats separated by a short spacer peptide. For example, there may be 3 copies of a gold-binding peptide separated by a peptide linker -GSG-, such as the amino acid sequence MHGKTQATSGTIQS-GSG-MHGKTQATSGTIQS-GSG-MHGKTQATSGTIQS (SEQ ID NO: 11). Such groups can be included at either end of the peptide molecular wire, and additional short peptide linkers such as -GSG- or other short peptides composed of G and S, or other common flexible water-soluble peptide linker sequences, may be included to offset it from the primary alpha-helix segment so that the helix conformation is not disrupted. In various embodiments, there may be 2 to 5 such repeats in the binding group.

[0086] In various embodiments, the synthetic peptides according to the present disclosure include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 11. In various embodiments, the synthetic peptides include SEQ ID NO: 11. In various embodiments, such synthetic peptides function as conductive molecular wires in molecular electronic circuits. In various embodiments, this sequence can be part of one or both ends of a larger synthetic peptide.

[0087] In various embodiments, internal binding sites within a peptide, such as near the central position along the amino acid sequence, may include cysteine amino acids to effect cysteine-based conjugations, such as cysteine-maleimide conjugation, at specific sites. The internal binding sites may also include standard amino acids, non-standard amino acids, or modified amino acids that are capable of undergoing specific conjugation reactions at specific positions within a peptide having residues such as azide or amine groups. For example, amino acids containing azide functional groups can be used in click chemistry conjugation reactions. In various embodiments, the internal binding sites can be included within a synthetic peptide that includes an alpha helix motif, such as an alpha helix core segment that further includes a repetitive alpha helix motif, where one or more of the alpha helix repeats includes cysteine.

[0088] One or more isolated amino acids capable of functioning as internal binding sites within a peptide generally do not disrupt the alpha helix structure of the peptide, even if they deviate from the exact repeating motif that defines the helix structure or are present between two such tandem repeats of the motif. In some embodiments, the location of the internal binding site within the peptide chain, and the groups at the ends of the peptide, are spaced in a helical conformation such that the peptide binds to a predetermined position within the circuit without distorting the helical geometry, while conjugating with an auxiliary molecule, and such that the auxiliary molecule is oriented as desired with respect to the electrodes and substrate of the circuit. For example, if the groups at the ends of the peptide bind to the electrodes in a minimum energy configuration and the helix segment is in its standard minimum energy conformation, the internal binding groups can be oriented away from the substrate underlying the circuit and be maximally available for binding other auxiliary molecules in solution.

[0089] The use of salt bridges for both stabilizing the helix structure and providing conduction paths is illustrated by the examples of FIGS. 16, 17, and 18. Salt bridges are formed between spatially close amino acids along the 3D alpha helix structure. These are generally present at positions i and i + 4 or i + 5 within the amino acid sequence. These examples illustrate the principle but do not limit the scope of the present disclosure, which includes other amino acid sequences that achieve a similar effect. Generally, salt cross - bridges can occur between positively charged amino acids and negatively charged amino acids, e.g., between the residues of the standard amino acids R, H, K (positive) and E, D (negative). In various embodiments, charged NSAAs can likewise be used.

[0090] FIGS. 16A - C show a molecular rendering of the molecule of SEQ ID NO: 4. This is based on the repetitive EAAAR (SEQ ID NO: 1) helix peptide motif. The dashed line 1610 within the rendering represents a hydrogen bond within the helix, and the dashed line 1620 represents a salt bridge between the E amino acid side chain and the R amino acid side chain (other side chains are not shown for clarity). Due to the intramolecular hydrogen bond and the salt bridge, a favorable conduction path for electrons across the molecular wire containing the repetition of the motif EAAAR (SEQ ID NO: 1) is provided when the molecular wire is connected within a molecular circuit. The salt bridge shown as the interacting side chains of E and R, which are the i and i + 4 amino acids, further serves to stabilize the helix structure.

[0091] FIGS. 17A - C show a molecular rendering of a synthetic peptide containing repetitions of the EEEERRRR (SEQ ID NO: 2) (E4R4) helix peptide motif. The dashed line 1710 within the rendering represents a hydrogen bond within the helix, and the dashed line 1720 within the rendering represents a salt bridge between the E amino acid side chain and the R amino acid side chain (other side chains are not shown for clarity).

[0092] In various embodiments, enhanced conductivity is achieved by incorporating amino acids containing aromatic rings into synthetic peptides. The aromatic rings are electron-rich and support electron conduction through proteins. By incorporating such aromatic ring side chains into the alpha-helical segments of synthetic peptides, conduction through the peptides can be further enhanced.

[0093] Figure 18 shows such a synthetic peptide based on the repetition of motif EAYAR (SEQ ID NO: 3). Dashed line 1810 indicates a hydrogen bond, and the side chains of E, R, and aromatic Y (1830) are shown. The E and R side chains result in salt bridge 1820, and the Y group (tyrosine) 1830 results in an aromatic ring for enhancing conductivity. Naturally occurring amino acids having an aromatic ring are F, W, Y, P (phenylalanine, tryptophan, tyrosine, proline), and these as well as non-sulfonated aromatic amines (NSAAs) can be used for this effect. In various embodiments, the aromatic amino acids are spaced only between i and i+4 and / or i and i+5 within the peptide chain, and thus, the aromatic rings are spatially close to each other in the helical structure, providing a continuous conduction path for electrons to jump from ring to ring.

[0094] In various embodiments, enhanced conductivity is achieved by including synthetic peptide segments that are highly similar to peptide segments of biologically conductive helical proteins, such as proteins that are known to perform electron transfer or charge transfer in various biological processes or are part of complexes that perform such conductive functions.

[0095] Examples of such families of biological proteins are bacterial pili, such as the pilin protein of Geobacter sulfurreducens, a Gram-negative metal-reducing proteobacterium. This genus was discovered in 1987 and has been well studied with respect to its metal-reducing and electron-conducting properties. See, for example, K. Xiao, et al., “Low Energy Atomic Models Suggesting a Pilus Structure that could Account for Electrical Conductivity of Geobacter sulfurreducens Pili,” Scientific Reports, March 22, 2016, (DOI: 10.1038 / srep23385).

[0096] In biological systems, individual pilin protein chains twist into a helical superstructure “filament” formed from 21 chains, which plays an important role in electron transfer in bacteria.

[0097] Xiao, et al. supra, illustrate the basis for the conductivity of biological pilin proteins and pilin filaments. Figure 3 of Xiao, et al. supra, shows the results of the predicted model structure of the G. sulfurreducens pilus: (A) a complete filament superstructure model containing 21 single-stranded helical protein subunits each with a different shading; (B) shows the geometric parameters of the superstructure; and (C) shows an end view of the superstructure.

[0098] In Xiao, et al., FIG. 4 above shows how aromatic rings are organized through the filament superstructure, along with chains of spatially proximate aromatic rings that form continuous conduction paths through the superstructure (A, B, C, and D), and also shows a comparison with a Pseudomonas-derived Geobacter homology model of this aromatic ring structure (E, F): (A) shows the aromatic rings in the pilus model; (B) shows details of adjacent aromatic rings that repeat along the length of the filament. The closest atoms between the rings are 3.5 Å apart. The centers of the rings are 4 - 5 Å apart. "P" indicates the reference protomer. "P+3" and "P+4" indicate the third and fourth protomers further along the helical assembly, respectively; (C) end view of a model containing 21 monomers (model length 275 Å); (D) cross-section of the model filament (thickness 22 Å), with residues from different monomers shown in different shades. The aromatic rings of Phel, Phe24, and Tyr27 are opaque and are off-center in the cross-section; (E) cross-section of the Geobacter homology model based on Pseudomonas; (F) absence of a central channel in the model (left) versus the central channel in the homology model (right).

[0099] FIG. 19 in this specification shows the crystal structure of a specific major pilin chain type IVa derived from this bacterium (Protein Data Base entry PDB ID 2M7G_A). From the X-ray crystal structure visualized using a ribbon plot and a superimposed ball-and-stick model showing all side chains, it is shown that the pilin protein has a visible alpha-helical structure, 13 turns in length, and three aromatic ring side chains.

[0100] The specific amino acid composition of this pilin protein shown in FIG. 19 is the following 61 amino acid sequence obtained from the PDB entry: PDB: 2M7G_A Structure of the type IVa major pilin from the electrically conductive bacterial nanowire of Geobacter sulfurreducens

Chemical formula

[0101] In Xiao, et al., it is shown that the aromatic ring provides the conduction mechanism, and further that the rings are arranged to be in proximity in a 3D structure such that a semi - continuous conductive path of the aromatic ring is created. In other respects, it is known in the field of conductive biological proteins that aromatic rings, often tyrosine, can play some role in conferring conductivity to proteins. The conductivity is, at least hypothetically, due to the presence of aromatic rings that are electron - rich and bring about high conduction around the ring.

[0102] With respect to the peptides of the present disclosure and based on observations of biological conductive proteins, aromatic amino acids can be added to a synthetic peptide sequence to enhance the conductivity of the synthetic peptide alpha - helix. In various embodiments, tyrosine (Y) can be added. In certain aspects, aromatic amino acids are added at locations that are in proximity to each other in a 3D alpha - helix structure to form a conductive path where the aromatic rings are in closer and more continuous spacing. One such example is shown in FIG. 18, where the alpha - helix is based on the repeating motif EAYAR (SEQ ID NO: 3), thereby including tyrosine at positions i and i + 5 as shown in FIG. 18, which leads to a helical pattern of rings around the helix, resulting in helix conduction that enhances the path for electrons to cross the structure.

[0103] In various embodiments, based on the observation of biological conductive proteins, to enhance conductivity, an entire segment derived from an alpha-helix biological conductive protein is incorporated as a tandem repeat into a synthetic alpha-helix peptide. These segments can be identical to the biological sequence or highly similar to the biological sequence, such as having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the biological amino acid sequence. In various embodiments, such as when using a molecular wire in an aqueous medium like a biosensor, a portion of the sequence obtained from or inspired by a biological protein promotes the solubility of the helix and keeps the molecular wire within the molecular circuit in solution rather than moving or precipitating on a substrate, and to prevent precipitation or aggregation during handling in an aqueous medium, for example, during the self-organization of the molecular wire into the circuit or during the assembly with other biomolecular components such as an adhesive enzyme, it should be a soluble or hydrophilic segment, or a segment that does not contain any transmembrane segments.

[0104] In various embodiments, a synthetic helix peptide molecular wire based on the above pyrin sequence PDB:2M7G_A is formed based on the extraction of a 26-amino acid segment (underlined above) from a biological amino acid sequence, namely QFSAYRVKAYNSAASSDLRNLKTALE (SEQ ID NO: 13), and this sequence is used as the repeating motif of the core of the synthetic peptide. This is a water-soluble domain, and a complete synthetic peptide having a molecular wire structure containing three repeats of this domain, one example being the sequence QFSAYRVKAYNSAASSDLRNLKTCLE (SEQ ID NO: 31) and the peptide sequence of SEQ ID NO: 6 which is gold-binding, has the following amino acid sequence:

Chemical formula

[0105] In various embodiments, the synthetic peptides according to the present disclosure include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 14. In various embodiments, the synthetic peptides include SEQ ID NO: 14. In various embodiments, such synthetic peptides function as conductive molecular wires in molecular electronic circuits.

[0106] This construct containing SEQ ID NO: 14 contains a helical segment based on four tandem repeats of a 26 - amino - acid biological segment, where the second repeat contains a cysteine (C) that replaces the A at position 24 of the motif. This internally - located cysteine acts as a specific conjugation site for cysteine - maleimide conjugation reactions, for example, to attach a binding probe to the synthetic peptide. Further, in accordance with the present disclosure, this 104 - amino - acid helical segment is flanked at both the N - terminus and the C - terminus by triple tandem repeats of the gold - binding peptide MHGKTQATSGTIQS (SEQ ID NO: 6), and a flexible soluble linker - GSG - is used to separate each repeat and the primary helix. Overall, the synthetic peptide having SEQ ID NO: 14 is 206 amino acids in length, has a molecular weight of 21.37 kDa, a pI (protein isoelectric point, or the pH at which the charge is neutral) of 10.08, the internal helical segment is 15.5 nm long, and has approximately 28.9 helical turns.

[0107] In various embodiments, the synthetic peptides according to the present disclosure include the following 187 - amino - acid sequence:

Chemical formula

[0108] This synthetic peptide is based on the helix motif EAAAR (SEQ ID NO: 1). This has an alpha-helix segment of 15.6 nm in length, based on an amino acid length of 0.54 nm per turn and 3.6 amino acids per turn and the known alpha-helix pitch. The sequence is further characterized by triple repeats of the palladium-binding peptide QQSWPIS (SEQ ID NO: 16) at each end, separated by a GSG linker. The sequence EACAR (SEQ ID NO: 17) is a helix motif located at the center of the synthetic peptide. This helix motif is EAAAR (SEQ ID NO: 1) modified by using a single cysteine located at the center in place of alanine (A) to provide a conjugation site for use in conjugation reactions based on, for example, maleimide or APN to the synthetic peptide. In SEQ ID NO: 15, the modified helix motif EACAR (SEQ ID NO: 17) is exactly located at the midpoint of the sequence, but this example should not be considered limiting as the site-specific conjugation site, in this case C, can shift in either direction of the sequence. At positions 41 and 147, single alanine (A) residues are present at each end of the primary alpha-helix to avoid the direct attachment of the primary alpha-helix to the linker GSG and the disruption of the secondary structure at the ends of the helix in some cases. Instead, the intervening alanine A provides a transition helix-promoting amino acid and functions somehow as a sacrificial site for the helix structure. SEQ ID NO: 15 also includes the TEV protease cleavage sequence, ENLYFQG (SEQ ID NO: 18), added to provide an option to cleave multiple palladium-binding peptide sequences. Other TEV protease cleavage sequences include substitution of G at the P1’ position by any one of S, A, M or C.

[0109] In various embodiments, the synthetic peptides according to the present disclosure include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 15. In various embodiments, the synthetic peptide includes SEQ ID NO: 15. In various embodiments, such synthetic peptides function as conductive molecular wires in molecular electronic circuits.

[0110] In various embodiments, the synthetic peptides according to the present disclosure include the following 164 - amino - acid sequence:

Chemical formula

[0111] This synthetic peptide is based on the helix motif EEEERRRR (SEQ ID NO: 2). This synthetic peptide has an alpha - helix segment of 15.75 nm in length, based on an amino - acid length of 0.54 nm per turn and 3.6 amino acids per turn, and a known alpha - helix pitch. The sequence is further characterized by a triple repeat of the palladium - binding peptide QQSWPIS (SEQ ID NO: 16) at each end, separated by a GSG linker. The sequence EEEECRRR (SEQ ID NO: 20) is a helix motif located at the center of the synthetic peptide. This helix motif is a modified EEEERRRR (SEQ ID NO: 2) with a single cysteine located approximately at the center used in place of arginine (R) to provide a conjugation site for use in conjugation reactions based on, for example, maleimide or APN within the synthetic peptide.

[0112] In various embodiments, the synthetic peptides according to the present disclosure include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 19. In various embodiments, the synthetic peptide includes SEQ ID NO: 19. In various embodiments, such synthetic peptides function as conductive molecular wires in molecular electronic circuits.

[0113] In various embodiments, the synthetic peptides according to the present disclosure include the following 227 - amino - acid sequence: [Chemical formula]

[0114] This synthetic peptide is based on the helix motif EAAAR (SEQ ID NO: 1). This synthetic peptide has an alpha - helix segment of 25.4 nm in length, based on an amino - acid length of 0.54 nm per turn and 3.6 amino acids per turn, and a known alpha - helix pitch. The sequence is further characterized by a triple repeat of the palladium - binding peptide QQSWPIS (SEQ ID NO: 16) at each end, separated by a GSG linker. The sequence EACAR (SEQ ID NO: 17) is a helix motif located at the center of the synthetic peptide. This helix motif is a modified EAAAR (SEQ ID NO: 1) that uses a single cysteine at the center in place of alanine (A) to provide a conjugation site for use in conjugation reactions, for example, based on maleimide or APN, within the synthetic peptide. In SEQ ID NO: 21, the modified helix motif EACAR (SEQ ID NO: 17) is precisely located at the mid - point of the sequence, but this example should not be considered limiting since the site - specific conjugation site, in this case C, can shift in either direction of the sequence. At positions 31 and 197, a single alanine (A) residue is present at each end of the primary alpha - helix to avoid the secondary structure being disrupted at the ends of the helix when the primary alpha - helix attaches directly to the linker GSG. Instead, the intervening alanine A provides a transitional helix - promoting amino acid and functions in some way as a sacrificial site for the helix structure.

[0115] In various embodiments, the synthetic peptides according to the present disclosure include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 21. In various embodiments, the synthetic peptide includes SEQ ID NO: 21. In various embodiments, such synthetic peptides function as conductive molecular wires in molecular electronic circuits.

[0116] It should be understood that other synthetic peptides capable of functioning as molecular wires can be structurally considered using the same principles as the present invention, and the above examples are merely illustrative of these principles and do not limit the scope. In addition to pyrins based on such synthetic helical peptides, other biologically naturally occurring conductive protein helices such as collagen filaments, dynein, kinesin, components of molecular motors, elements of cytochrome, or chains of immunoglobulins are understood to exist.

[0117] Structural variations of synthetic conductive peptides that can be used as molecular crosslinks in molecular electronic circuits Length of the helical bridge: In various embodiments, the synthetic peptides of the present invention can be designed to have a specific length by shortening or lengthening one or more helical segments and / or by expanding or reducing the repetition of the helical motif. All helical motifs present can be considered to define helices of any length. For example, in the synthetic peptide, EAAAR....EAAAR, the motif EAAAR (SEQ ID NO: 1) can be repeated indefinitely, and any desired length of crosslinked helix can be provided by any resulting array segment. For peptide bridges used to bridge between spaced electrodes, such as those shown in FIG. 1 or FIG. 12, the preferred length is from 5 nm to 100 nm, more preferably from 10 nm to 50 nm, and most preferably from 15 nm to 40 nm. For synthetic peptides used as connecting arms between probe molecules and electrodes, such as those shown in FIG. 13, the preferred length is from 2 nm to 50 nm, more preferably from 5 nm to 30 nm, and most preferably from 7 nm to 20 nm. For naturally occurring helical sequences, i.e., alpha-helical sequences present within proteins of biological origin, subsegments obtained therefrom can be used as shorter bridges, and sequences formed by repeating all or subsegments or segments of the helical sequence allow for longer bridges compared to naturally occurring alpha-helices.

[0118] Amino acid sequence similarity: For the above helical amino acid sequences, other preferred sequences that may have similar properties and utilities include sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the provided sequence. Similarly, for any naturally occurring alpha helix sequence, at least 5 amino acid segments obtained therefrom and having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence similarity to the natural form can result in a preferred crosslinked helix, or a segment that creates a repeatedly crosslinked helix.

[0119] Functionally similar amino acid substitutions: For any of the helix sequences described herein, certain individual amino acid substitutions can be made that are predicted to retain the helix structure and other functionality. For example, by replacing one or more amino acids within a helix with amino acids that promote helix formation, it can be predicted that a helix of similar structure and utility will result. In particular, helix formation is promoted by the amino acids A, M, L, E, and K, and in particular, it is known that the amino acids within the helix can be replaced with these, and there is a high likelihood that the helix structure will be retained. More generally, for each amino acid, a score has been developed that ranks their tendency to form or conform to a helix structure, or to occur more frequently in naturally occurring helices. Substitution with amino acids that score equally well or better is predicted to conserve or even enhance the helix structure. One such score is the helix propensity (see Pace, C. N., & Scholtz, J. M. (1998). A helix propensity scale based on experimental studies of peptides and proteins. Biophysical journal, 75(1), 422-427), where amino acids are numerically ranked from highest (low score) to lowest (high score) compatibility with a helix as follows: Ala = 0, Leu = 0.21, Arg = 0.21, Met = 0.24, Lys = 0.26, Gln = 0.39, Glu = 0.40, Ile = 0.41, Trp = 0.49, Ser = 0.50, Tyr = 0.53, Phe = 0.54, Val = 0.61, His = 0.61, Asn = 0.65, Thr = 0.66, Cys = 0.68, Asp = 0.69, and Gly = 1.

[0120] Furthermore, salt bridges formed between oppositely charged amino acids located at positions i and i+4 or i+5 that are spatially proximal in a helix, such as those exemplified by the EAAAR motif (SEQ ID NO: 1) and the EEEERRRR motif (SEQ ID NO: 2), can generally be formed by replacing pairs of amino acids that form any salt bridge, including negatively charged amino acids {E, D} that form a salt bridge with positively charged amino acids {R, K, H}. Thus, starting from any of the sequences described herein, by replacing one of {R, K, H} at position i and one of {E, D} at i+4 or i+5 within the sequence, it is predicted that either the one already present will be maintained or perhaps one with enhanced stability or conductivity will be added to form a salt bridge. For example, the described motifs EAAAR (SEQ ID NO: 1) and EEEERRRR (SEQ ID NO: 2) suggest functionally substituted motifs such as DAAAK (SEQ ID NO: 22), and EEDDRKHK (SEQ ID NO: 23). In the case of aromatic ring amino acids present within a helix, or for such introductions to potentially increase conductivity, any of the aromatic ring amino acids such as F, W, V, or P can be considered.

[0121] More generally, the classification of amino acids into similar groups such as charged (positive, negative), polar, hydrophobic or hydrophilic, or aromatic, also serves as a guide for a number of substitutions that replace a given amino acid with an amino acid of the same type that is predicted to have similar functionality. All such substitutions fall within the scope of modifications that can be applied to the exemplary sequences described herein.

[0122] Non-standard amino acid substitution. Non-standard amino acids (NAAs), also known as unnatural amino acids (UAAs), are amino acids other than the 22 that are present in biological proteins. In particular, there are many such NAAs that are chemically modified forms of standard amino acids and can be used as alternatives to standard amino acids. These can be incorporated into proteins using peptide chemical synthesis, or expression in an expression system in which a non-standard genetic code is implemented, or through chemical modifications made to standard amino acids present in a protein. Such NAAs can still embody important design principles such as helix propensity, salt bridges, aromatic rings, spacers / linkers, or binding to electrodes - in particular, when these are modified forms of the analogous standard amino acids that embody these properties. Thus, substitution of standard amino acids with NAAs of a similar form results in a large class of changes that can be made to the sequences described herein, which are predicted to result in similar structures and utility. These NAAs can be incorporated into proteins using peptide chemical synthesis, or by expression in an expression system in which a non-standard genetic code is implemented, or through chemical modifications made to standard amino acids present in a protein.

[0123] Linker Variation: In the examples presented, the amino acid sequence GSG is used as a linker / spacer at the ends of crosslinks between primary helices and between one or more linking groups at the ends. Such a large variety of linkers is known to those skilled in protein engineering and has been used to space apart functional domains of interest within a protein or to reduce steric interference, and many of them can be used as alternative linkers / spacers in the sequences described herein. For example, other common linkers are G / S sequences such as G, GS, SG, GSGS (SEQ ID NO: 24), GSSSGSSSG (SEQ ID NO: 25), etc. More generally, {G, S}-rich sequences result in conveniently commonly used linkers. More generally, a large variety of linkers / spacers are used in the literature, any of which may be suitable as alternative linkers in the sequences described herein. In general, these linkers tend to be sequences that form flexible hydrophilic chains, which can be conveniently done by short sequences with a high {S, G} content. Such linkers are preferably short, preferably 1 to 10 amino acids, but longer amino acids can also function similarly. Compilation of various linkers / spacers used in various applications can be found at http: / / parts.igem.org / Protein_domains / Linker. In addition to peptide linkers / spacers such as GSG, other flexible molecular linkers can be used, and in the case of peptides made by chemical synthesis, the addition of non-amino acid elements to the peptide chain becomes possible. One common such family is short carbon chain linkers such as C3, C6, or C12 (3, 6, or 12 chains of hydrocarbons). In such cases, these or other short molecular linkers can be used in place of peptide linkers such as GSG.

[0124] Stapled helix: Known techniques for stabilizing an alpha helix are the use of hydrocarbon staples between the turns of the helix to chemically link them together. Any of the helices described herein can also be modified to include one or more staples to further stabilize the helix. Such methods are described, for example, in Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress, Loren D. Walensky and Gregory H. Bird, Journal of Medicinal Chemistry, 57 (15), 6275-6288 (2014).

[0125] Functionally similar motif variations: The helix motifs described herein have many direct extensions that embody the same design principle. For example, from the motif EAAAR (SEQ ID NO:1), functionally similar motifs such as EAAARRAAAE (SEQ ID NO:26) or EAAARAARAAAR (SEQ ID NO:27) are suggested, which use the helix-forming amino acid A and have E-R salt bridges that can form between the i and i+4 positions. Or, for example, from the motif EEEERRRR (SEQ ID NO:2), a functionally similar motif in a longer form, EEEERRRRRRRREEEE (SEQ ID NO:28), is suggested, which still has each E / R pairing with i+4 R / E. The presented motifs, EAAAR (SEQ ID NO:1) and EEEERRRR (SEQ ID NO:2), are simply the simplest and / or shortest of these families, while the same principle is embodied by many longer and / or more complex motif patterns.

[0126] General cross-bridge architecture: The scope of synthetic peptide structures beyond the specific examples of sequences presented herein, such as SEQ ID NOs: 14, 15, 19, and 21, includes synthetic peptides having the following general formula: [X 1 X 2 ​m X 3 [X 4 n X 3 [X 2 X 1 m (wherein each X 1 is independently a material-binding peptide containing about 5 to about 15 amino acids, a protease cleavage sequence, or a peptide capture tag, each X 2 is independently a glycine / serine {G, S}-rich linker or a C1-C20 carbon chain molecular linker, each X 3 is independently a covalent bond, a single amino acid, a transition helix-promoting motif, a metal-binding group, or a material-binding peptide containing about 5 to about 15 amino acids, each X 4 is independently an alpha helix motif containing about 4 to about 40 amino acids, each m is independently 0 to 4, n is 1 to 40.

[0127] The types of synthetic peptides each belong to [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m are included therein, and the motifs defined herein, and the respective ranges of n and m, are used, for example, as conductive molecular wires bridging spaced electrodes as illustrated in FIG. 2, or, for example, as synthetic peptide arm molecules conjugating between an electrode and a specific site on a binding probe as illustrated in FIG. 13. The differences in the usefulness of conductive synthetic peptides are, at least in part, for example, whether a conjugation site for attaching a probe molecule to the synthetic peptide is provided along the sequence of the synthetic peptide, and [X 1 X 2 m and X​​​​​​3 The motif composition, e.g., whether these motifs are designed to bind to biomolecules such as metal electrodes or binding probes, and whether the ends of the synthetic peptide are the same or different, is determined. As described above, the synthetic cross-linked peptide can be characterized as having a cysteine (C) or other conjugation site near the midpoint within the peptide sequence, e.g., along the length of the synthetic peptide. That is, X 4 At least one of the alpha helix motifs can include a conjugation site such as a cysteine residue. In various embodiments, X 1 The motif includes a material-binding peptide, which can be arranged in a triplet with a glycine / serine-rich linker such as -GSG-X 2 intervening. In these cases, when m is not 0, X 3 can be used as a spacer between the material-binding sequence of the triplet arrangement and the core alpha helix segment composed of the repetitive alpha helix motif. In a particular example, when m is not 0, X 3 can be a transitional helix-promoting amino acid such as alanine (A). If a transitional helix-promoting amino acid is not desired, X 3 can simply be a covalent bond.

[0128] In other examples above, [X 1 X 2 m It is possible that one or both of the terminal motifs are not present in their entirety, i.e., m = 0. In various embodiments where m = 0, [X 1 X 2 m means that the segment is absent, and X 3 can serve as the binding motif in the terminal region of the synthetic peptide, either one end or both ends. For example, when m = 0, X 3 ​​may contain cysteine (to provide a single - SH group at the peptide terminus), or may contain a run of two or more cysteine amino acids, or a more complex arrangement for specific conjugation, such as a material - binding peptide. In other cases, when m = 0, X 3 may contain a non - natural amino acid having an azide or other functional group for specific binding.

[0129] The above general considerations provide some perspectives on how to select motifs to impart various physical properties to synthetic peptides. However, with the following motif options, the general formula of the synthetic peptide [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m is further defined as follows:

[0130] In various embodiments, each X 1 may include any one of a material - binding peptide containing about 5 to about 15 amino acids, a protease cleavage sequence, or a peptide capture tag. In certain examples, X 1 includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NO: 6, 7, 8, 9, 10, 11, 16, 18, or 29. In certain examples, X 1 includes any one of SEQ ID NO: 6, 7, 8, 9, 10, 11, 16, 18, or 29. In various embodiments, X 1 includes a sequence configured to bind to a specific metal. Each instance of X 1 can be selected independently, and thus, it is important to recall that, if necessary, the N - terminus and C - terminus of the synthetic peptide can be made different. When configured as a binding domain, X 1 is a material - binding peptide, or an intervening linker X 2 ​​​It may consist of a series of such material-binding peptide sequences separated by , or may contain amino acids that bind / conjugate to a particular material or functional group, such as cysteine that can bind to various metals or conjugation group maleimides. The binding domain may also include other known conjugation groups such as thiol, biotin, maleimide, APN, lysine, azide, or amine, or many others known to those skilled in conjugation chemistry. The peptide target tag may contain a peptide target of an antibody, such as a FLAG epitope tag, HIS tag (poly-histidine), Myc tag, HA, GST, or other epitope tags, or a peptide group such as an avitag or an aldehyde tag that is a conjugation target. In other examples, X 1 may contain a capture tag at one end of the peptide for synthesis, or one or both ends may contain a protease cleavage sequence, so that the synthetic peptide can be digested before or after coupling to other elements, such as a pre-bound electrode or other circuit element, or other biomolecules that can be conjugated.

[0131] In various embodiments, X 2 may include a glycine / serine-rich linker or a hydrocarbon-type linker. Regarding the former, as non-limiting options, among other glycine / serine-rich sequences, G, S, -GS-, and -GSG- are envisioned up to at least 10 or more amino acids. The linker X 2 may also include a "tether" referred to as a "C1-C20 carbon chain molecular linker". The only limitation on the nature of the C1-C20 carbon chain molecular linker is that it is divalent so that one part of the synthetic peptide can be tethered to another part by the linker. In various embodiments, such linkers include, but are not limited to, methylene -CH2- and its homologs, -(CF2) p-(where p = 1 to about 20), ethoxylates from 1 EO to about 10 EO, 1,4-phenylene, -CO2-, -C(O)-NH, etc. are included. The C1-C20 linker may also include any combination of carbon atoms and heteroatoms, and may be acyclic, cyclic, aliphatic, or aromatic, or a combination thereof. In various embodiments, X 2 may include a combination of an amino acid sequence and C1-C20 non-amino acid species. X 2 The length can be customized for a particular device, for example, according to the contact area of the metal electrode, or to achieve a desired separation distance between the probe molecule bound to the synthetic peptide and the metal electrode. For example, the material-binding peptide X 1 can be deliberately lengthened to space it apart from the binding probe attached near the approximate midpoint of the synthetic peptide (i.e., near the center of the [X 4 n segment). 2

[0132] In various embodiments, the motif X 3 may include a covalent bond, a single amino acid, a transitional helix-promoting motif, a metal-binding group, or a material-binding peptide containing about 5 to about 15 amino acids. As discussed, the choice of X 3 depends at least in part on the choice of X 1 when m = 0 in particular, and the ends of the synthetic peptide are completely defined by the nature of X 3 . When X 3 includes a "covalent bond", X 3 is not present in the synthetic peptide, and the ends of the internal alpha-helix segment defined by [X 4 n bind directly to X 2 . In other words, stating that X 3 can be a covalent bond is equivalent to stating that the presence of X 3 is optional. In various embodiments, X 3 may include a single amino acid such as alanine (A), which is [X 4 n ​​​​Functions as a spacer that does not impair the alpha - helix segment defined thereby. In various embodiments, X 3 may include a spacer longer than just one amino acid and may include a transition helical promoting motif that is a spacer that promotes an alpha - helix secondary conformation. Such a transition helical promoting motif may contain up to about 5 amino acids and may be simple, such as a poly - alanine sequence.

[0133] In various embodiments, for example, when m = 0, X 3 may include a metal - binding group. The selection of such metal - binding groups has been discussed in detail herein and includes species such as thiol groups, carbenes, amine groups, diazonium groups, or any other functional group that can bind to a metal such as Au, Pt, or Pd to at least some extent. In various embodiments, the metal - binding group may be derivatized to include an amino acid that provides a thiol group (i.e., cysteine), or a functional group not native to the amino acid and may include an amino acid that can bind to a metal. In other words, X 3 may include a single amino acid, but the single amino acid selected is not for the purpose of functioning as a spacer that promotes an alpha - helix conformation and can be selected because the single amino acid provides a functional group that can bind to a metal. In various embodiments, X 3 may include two or more cysteine residues, for example, a run of 6 or more cysteines. In various embodiments, X 3 may include a tetra - cysteine FLASH - binding motif CCXXCC, such as CCCGCC (SEQ ID NO: 5) as described above, (X = any amino acid). In various embodiments, X 3 may include the FLASH - binding motif CCXXCC, where XX is proline - serine, i.e., CCPSCC (SEQ ID NO: 34).

[0134] In various embodiments, the helical core portion of the synthetic peptide, i.e., [X4 n may include repeats of any amino acid sequence that promotes an alpha-helical secondary structure for that portion, such as the repeat motif QFSAYRVKAYNSAASSDLRNLKTALE (SEQ ID NO: 13) or SEQ ID NO: 1 or 2. As is apparent from the examples, the repeating integer n, and the repeating alpha-helical motif X 4 The length of the sequence of may have a substantial impact on the overall length of the synthetic peptide, and both of these variables, along with the choice of terminal portions, can be manipulated to define a very precise length of the synthetic peptide. As mentioned, a predictable and accurate length of the synthetic peptide is important when using the synthetic peptide as a molecular wire that crosslinks spaced electrodes or connects a biomolecule to an electrode within a molecular electronic circuit.

[0135] Within the helix, for example, X that is repeated to define the alpha-helical core of the synthetic peptide 4 The conjugation site within at least one of the alpha-helical motifs can be an amino acid such as cysteine or lysine, or a modified amino acid such as lysine with a free azide group attached, or lysine with biotin attached, or a modified amino acid or non-amino acid site positioned within the peptide by chemical modification or synthesis. Further, and such a primary conjugation site can be chemically functionalized or converted to another group for conjugation, for example, converting cysteine to azide by using an azide-maleimide bifunctional linker that reacts with cysteine. Many other such conversions from the primary conjugation site to the desired conjugation group are possible and are well known to those skilled in the art of conjugation chemistry.

[0136] In various embodiments, the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m ​​​​The synthetic peptide includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 14. In certain examples, the synthetic peptide includes SEQ ID NO: 14.

[0137] In various embodiments, the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m The synthetic peptide includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 15. In certain examples, the synthetic peptide includes SEQ ID NO: 15.

[0138] In various embodiments, the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m The synthetic peptide includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 19. In certain examples, the synthetic peptide includes SEQ ID NO: 19.

[0139] In various embodiments, the formula [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m ​​​​​​​​​The synthetic peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 21. In certain examples, the synthetic peptide comprises SEQ ID NO: 21.

[0140] Production of crosslinked molecules: The crosslinked molecules described herein can be made using known methods of chemical peptide synthesis and protein expression. In the case of chemical peptide synthesis where amino acids are added stepwise in a chemical coupling step to synthesize the desired chain, it is also possible to add modified / unnatural amino acids, as well as internal or terminal non-amino acid groups such as chemical linkers or conjugation groups. In the case of protein expression, the target crosslinking has to be done across the entire amino acid, creating the corresponding gene, introducing it into a biological expression system such as E. coli bacteria, expressing the gene to produce the resulting protein, and then extracting and purifying it from the cultured cells. Such proteins can be further chemically modified to create certain desired groups, for example, reacting cysteine with maleimide to conjugate it with other groups at that site, or reacting lysine with NHS, or reacting with terminal amino or carboxy groups. Crosslinks created by any of these methods can generally be ordered from commercial vendors that perform these processes as a service. It is also possible to insert such NAAs into protein products using a genetically modified expression system for using non-standard amino acids.

[0141] The principles of the present invention are extended to molecular wires organized as bundles of the single-stranded helical peptides described above. For example, the triple helix formed by the three helical strands outlined above is a further extension of this concept. For one embodiment of such a class, such examples can be based on synthetic forms of collagen that form triple helices naturally. Similarly, pyrin is naturally organized into a multi-stranded superstructure and thus, similar multi-stranded constructs can be formed from synthetic molecular wires promoted by pyrin.

[0142] Use of synthetic peptides as molecular wires in molecular electronics There are at least two notable applications for the conductive synthetic peptides according to the present disclosure. The conductive synthetic peptides according to the present disclosure can be used as molecular wires for forming conductive connections between other extremely important molecular circuit elements, as shown, for example, in FIGS. 3, 4, 5, and 6. In these illustrated devices, the main function of the peptide is to provide a conductive path that does not overly impede the transport of electrons or other charge carriers through the circuit. In another notable application, the conductivity of the synthetic peptide varies due to its interaction with its immediate environment, in which case it can function as part of a sensor construct.

[0143] In some embodiments, the wire directly interacts with molecules in the environment, e.g., gas molecules in a gaseous environment or molecules in solution in a liquid environment, and as a result of these interactions, the conductivity or resistivity changes (see FIG. 1). In other embodiments, such as those shown in FIG. 2, a secondary molecule is conjugated to the primary molecular wire spanning the electrodes, and the interaction of this secondary molecule with its binding target can create a local environmental change that regulates the conductivity of the wire, thereby providing a sensor for these interactions by monitoring the current through this complex under an applied voltage as shown in FIG. 2. In such a sensor system, the charge transport through the primary wire is disrupted by local charge perturbations resulting from the target substrate engaged with the enzyme, and is thus recorded as a change in conductivity or current over time, as indicated by the increasing change in the i vs. t current in the inset plot. The informative current changes can be increases or decreases, pulses, or other temporal variations.

[0144] For illustrative purposes, and not to limit the scope of the disclosure, FIGS. 2-10 and 12-14 illustrate various such molecular sensor constructs that include one or more of the synthetic peptides disclosed herein that function as molecular wires in a molecular electronic circuit, with the synthetic peptides schematically shown as ball-and-stick representations (FIGS. 1-8) or as long, thin rods (FIGS. 9-10, and 12-14) in the various figures. The conjugation systems used to connect the synthetic peptides to electrodes and / or other molecules are schematically shown in the drawings by small shaded squares at the intersections of the synthetic peptide / biomolecule and synthetic peptide / electrode or substrate. Such conjugation points can represent any known molecular conjugation for creating a specific connection between two molecular / atomic (metal) components, such as click chemistry coupling, thiol-metal bonding, biotin-avidin bonding, material-binding peptides, diazonium-metal bonding, carbene-metal bonding, cysteine-maleimide coupling, amine-reactive crosslinking, or many others known to those skilled in the art of chemical conjugation, bioconjugation, or material surface conjugation. The analogous elements necessary for this conjugation are generally incorporated into the structure and attachment sites of the synthetic peptides designed and manufactured in the present invention. Briefly stated, electronic constructs that include synthetic peptides functioning as molecular wires are illustrated in the drawings as follows:

[0145] FIG. 2 shows a binding probe molecule conjugated with a synthetic peptide spanning two electrodes. This circuit functions as a sensor, and under an applied voltage, when the probe molecule binds to its cognate target molecule, the conductivity of the wire is adjusted, resulting in a current increase signal (increase in conductivity). In various embodiments, the binding probe molecule includes an enzyme such as polymerase, and the sensor can be used for DNA sequencing.

[0146] In various embodiments, the sensor of FIG. 2 includes an advanced enzyme molecular electronics sensor. In various examples, the sensor includes a first electrode, a second electrode spaced from the first electrode by an electrode gap, and a sensor complex including an advanced enzyme conjugated with a synthetic peptide according to the present disclosure that is electrically connected to the first and second electrodes and bridges the gap therebetween. The advanced enzyme can include a native or genetically engineered polymerase, reverse transcriptase, helicase, exonuclease, or molecular motor for packaging viral DNA. For a functioning sensor, a transimpedance amplifier is electrically connected to at least one of the first and second electrodes, and an output including an electrical parameter measurable by the transimpedance amplifier is provided, and the measurable electrical parameter includes a distinguishable signal corresponding to the enzyme activity of the advanced enzyme.

[0147] FIG. 3 shows a configuration in which a binding probe molecule is directly electrically connected to a pair of spaced electrodes by two synthetic peptides functioning as arm molecules. The synthetic peptides here mainly function as conductive connectors for the probe molecule, and the conductivity of the probe molecule itself changes when it binds to its homologous target. Since conjugation along the sequence of the peptide from the central point is not necessary, the synthetic peptide of the present invention for use as an arm molecule only needs to have functionalized ends. Any configuration of FIG. 2 or FIG. 3 incorporating the synthetic peptide according to the present disclosure can be used as a sensor for DNA sequencing.

[0148] In various embodiments, the molecular electronic circuit includes first and second electrodes spaced apart by a nanogap on a substrate, and a synthetic peptide that is electrically connected to both the first and second electrodes and thus bridges the nanogap. This embodiment is represented, for example, in FIG. 2. In various embodiments, the underlying substrate includes SiO2. In various embodiments, the electrodes include a metal such as Au, Pt, or Pd. In various embodiments, the first and second electrodes are the positive and negative electrodes in the circuit. In various embodiments, the crosslinkable synthetic peptide is conjugated to a binding probe such as a polymerase enzyme. The conjugation site may be located substantially in the center of the synthetic peptide sequence such that the attached polymerase does not contact either electrode of the pair of electrodes. In various embodiments, a gate electrode can be placed between the electrodes and under the crosslinkable peptide, such as, for example, a gate electrode embedded in the substrate. In various embodiments, a transimpedance amplifier is electrically connected to at least one of the first and second electrodes, and this transimpedance amplifier provides an output that includes measurable electrical parameters. The measurable electrical parameters include distinguishable signals corresponding to binding events involving the binding probe conjugated to the crosslinkable peptide.

[0149] In various embodiments, the molecular electronic circuit includes first and second electrodes spaced by a nanogap on a substrate, and two synthetic peptides that electrically connect a binding probe, such as a polymerase enzyme, to both the first and second electrodes, and thus function as arm molecules that crosslink across the nanogap and force an electrically conductive pathway through a portion of the binding probe. This embodiment is represented, for example, in FIG. 3. In various embodiments, the underlying substrate comprises SiO2. In various embodiments, the electrodes comprise a metal such as Au, Pt, or Pd. In various embodiments, the first and second electrodes are the positive and negative electrodes within the circuit. The lengths of the two synthetic peptide arm molecules determine how the binding probe is positioned centrally between the electrodes, and these lengths can be precisely manipulated so that the attached polymerase does not contact either electrode of the pair of electrodes. In various embodiments, a gate electrode can be placed between the electrodes and under the crosslinkable peptide, such as a gate electrode embedded in the substrate. In various embodiments, a transimpedance amplifier is electrically connected to at least one of the first and second electrodes, and this transimpedance amplifier provides an output that includes measurable electrical parameters. The measurable electrical parameters include distinguishable signals corresponding to binding events involving binding probes electrically wired into the circuit by the peptide arm molecules.

[0150] Described with respect to FIGS. 2 and 3, sensor circuits, such as those including one or more synthetic peptides according to the present disclosure, can be arranged as an array on a chip, and thus multiple such sensors can be operated as a system for DNA sequencing. In various aspects of the system, the system includes an array of sensors and further includes a CMOS sensor array chip that supports pixel circuits for performing measurements of measurable electrical parameters.

[0151] In various aspects of the system, the system includes at least two of: a CMOS sensor array chip; an electronic hardware system for controlling and managing the electrical inputs and data outputs of the chip; a fluid system for introducing synthetic DNA molecules in a buffer into the chip; and a signal processing and data recording system for capturing distinguishable signals and converting the distinguishable signals back into information.

[0152] Figures 4 - 10 and 12 - 14 illustrate other configurations of molecular electronic circuits that include one or more synthetic peptides according to various embodiments of the present disclosure. These circuits can be part of the biosensors described above with respect to Figures 2 and 3 and can be arranged in an array on a chip as discussed for these sensors.

[0153] Figure 4 shows a particular case of a molecular electronic circuit that is a protein having two synthetic peptides where the probe molecule has an internal alpha helix as part of its structure and the ends of the internal alpha helix of the protein are connected to a positive electrode and a negative electrode to provide a conductive path through a portion of the probe molecule.

[0154] Figure 5 shows another version that instead connects synthetic peptide arm molecules to an internal beta sheet within a protein probe molecule, again creating a preferred conduction path.

[0155] Figure 6 shows a sensor configuration that uses three synthetic peptide arm molecules to wire a probe to an electrode using multiple connections to the electrode.

[0156] Figure 7 shows using one synthetic peptide in a regulated manner of Figure 2 using two other synthetic peptides that connect the probe molecule directly to the first and second electrodes.

[0157] Figure 8 shows a somewhat more complex configuration where a synthetic peptide arm molecule connects a probe to a multiple - electrode system, thereby enabling multiple simultaneous measurements.

[0158] FIG. 9 shows a specific case of FIG. 3 where the probe molecule is an IgG antibody protein and the homologous binding target is the corresponding antigen, and such a system can be used to detect antibody-antigen binding.

[0159] FIG. 10 shows a specific case of FIG. 3 where the probe molecule is a single-stranded DNA oligonucleotide and its binding target is a complementary oligo, and such a system can be used to detect hybridization probe binding.

[0160] FIG. 11 shows the detailed protein structure of the Klenow polymerase enzyme that can be used as a binding probe in a situation where the DNA sequence is detected when the template is copied by the polymerase.

[0161] FIGS. 12, 13, and 14 show the use of the synthetic peptides according to the present disclosure for wiring polymerase enzymes, which are specific examples of FIGS. 2, 4, and 6 respectively, into various configurations, and the resulting circuits can be used as sensors for the sequences of templates processed by the polymerase.

[0162] The conductive synthetic peptides used as molecular wires according to the present disclosure can be part of a molecular sensor complex used in molecular electronics sensors. In any of the embodiments disclosed in FIGS. 2-14, the crosslinkable synthetic peptides and / or synthetic peptide arm molecules used in these circuits can include any one of the synthetic peptides disclosed herein. Molecular sensors that can utilize the synthetic peptides of the present invention are also disclosed in U.S. Pat. Nos. 9,829,456; 10,036,064; 10,125,420; 10,151,722; and 10,508,296; and U.S. Patent Application Publication No. 20180340220, each of which is incorporated herein by reference in its entirety. As incorporated herein by reference, such sensors can be arranged in an array of sensors on a chip fabricated by a CMOS process. The disclosed sensors can be used for DNA sequencing.

[0163] In various embodiments, methods for sequencing DNA molecules are disclosed. The method includes providing a circuit that further includes a positive electrode; a negative electrode spaced from the positive electrode; a conductive synthetic peptide electrically connected to the positive and negative electrodes; and a polymerase enzyme conjugated to the synthetic peptide at a conjugation site located along the synthetic peptide sequence, initiating at least one of a voltage or current through the circuit, exposing the circuit to a solution containing primed single-stranded DNA and / or dNTPs, and measuring an electrical signal through the circuit when the polymerase engages and extends the template, and processing the electrical signal to identify features that provide information regarding the sequence underlying the DNA molecule processed by the polymerase. In various embodiments, the synthetic peptide connecting the positive and negative electrodes has the formula: [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m , (wherein each X 1 is independently a material-binding peptide, protease cleavage sequence, or peptide capture tag containing about 5 to about 15 amino acids, each X 2 is independently a glycine / serine {G, S}-rich linker or a C1-C20 carbon chain molecular linker, each X 3 is independently a covalent bond, a single amino acid, a transition helix-promoting motif, a metal-binding group, or a material-binding peptide containing about 5 to about 15 amino acids, each X 4 is independently an alpha helix motif containing about 4 to about 40 amino acids, each m is independently 0 to 4, n is 1 to 40, X 4 and at least one of which contains a conjugation site)​​​ comprises

[0164] In various embodiments, the conjugation site includes a thiol, biotin, azide, amine, click chemistry group, cysteine, lysine or tyrosine, or any functional group capable of conjugating a synthetic peptide to a biomolecule such as a polymerase enzyme.

[0165] In various embodiments, another method for sequencing a DNA molecule is disclosed. The method includes a positive electrode; a negative electrode spaced from the positive electrode; a first conductive synthetic peptide arm molecule electrically connected to the positive electrode and a first site on the polymerase enzyme and a second conductive synthetic peptide arm molecule electrically connected to the negative electrode and a second site on the polymerase enzyme, thus providing a step of providing a circuit that results in a conductive path through the polymerase enzyme, a step of initiating at least one of a voltage or current through the circuit, a step of exposing the circuit to a solution containing a primed single-stranded DNA and / or dNTP, and a step of measuring an electrical signal through the circuit when the polymerase engages the template and extends it, and processing the electrical signal to identify features that provide information regarding the sequence underlying the DNA molecule processed by the polymerase. In various embodiments, the first and second synthetic peptide arm molecules each have the formula: [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m (wherein each X 1 is independently a material-binding peptide containing about 5 to about 15 amino acids, a protease cleavage sequence, or a peptide capture tag, each X 2 is independently a glycine / serine {G, S}-rich linker or a C1-C20 carbon chain molecule linker, each X 3 ​​​is, independently, a covalent bond, a single amino acid, a transitional helix promoting motif, a metal-binding group, or a material-binding peptide containing about 5 to about 15 amino acids, each X 4 is, independently, an alpha helix motif containing about 4 to about 40 amino acids, each m is, independently, 0 to 4, n is 1 to 40) comprises.

[0166] In various embodiments, the [X 1 X 2 m or X 3 at least one of the motifs is configured to bind the end of the synthetic peptide to any one of a positive electrode, a negative electrode, a first site on a polymerase enzyme, or a second site on a polymerase enzyme.

Example

[0167] Experimental verification of the usefulness of cross-linking: Versions of synthetic peptides containing repeats of the alpha helix motifs EAAAR (SEQ ID NO: 1) and EEEERRRR (SEQ ID NO: 2) were produced, which have a palladium-binding peptide at the end, a central cysteine residue available for conjugation, and lengths of approximately 15.6 nm, 15.75 nm, and 25.4 nm (for the alpha helix portion of the cross-linking, not including the linker and the binding peptide), close to an integer number of helical turns, and thus the binding group can contact the electrode surface without causing excessive torsional stress on the helix. The following three synthetic peptides were used in these experiments:

Chemical formula

[0168] ​These synthetic peptides were produced using the standard commercial protein expression service of Genscript, Inc. Briefly, they were expressed in E. coli, contained a FLAG tag added to the N-terminus, and enabled FLAG column purification. The materials were purified to over 95% and suspended in standard PBS buffer.

[0169] Furthermore, for the purification in the production of these synthetic peptides by the expression means, a FLAG epitope tag (7-mer peptide DYKDDDK (SEQ ID NO: 29)) was added as DYKDDDK-GSG- (SEQ ID NO: 30) using a linker to the N-terminus of this sequence, thus enabling the purification of the expression product using a FLAG tag affinity column. This added epitope tag was left in place for experimental work, thus providing a site for anti-FLAG antibody binding that could be used for antibody-based labeling in experimental applications.

[0170] Cross-bridge conductivity experiment: In the first experiment, a comparison of cross-bridge conductivity was carried out between SEQ ID NO: 15 and SEQ ID NO: 19. Nanoelectrodes were fabricated by e-beam lithography. Briefly, a resist was spin-coated onto a silicon wafer substrate, and using e-beam lithography, an electrode pattern was exposed in the resist to define a nanoelectrode pattern with a width of 50 nm and a tip-to-tip gap of 20 nm. The resist was developed, and a 5-nm-thick titanium adhesion layer and a 20-nm-thick palladium metal electrode layer were deposited using sputtering deposition. A lift-off process was used to fabricate the finished palladium nanoelectrodes.

[0171] Using photolithography and the lift-off method, palladium microelectrodes extending to visible pads for electrical connection were added, and using additional photolithography, a 100-nm-thick sputtered SiO2 passivation layer was added, leaving only a 4-micron-wide channel concentrated near the electrode gap exposed, and only a 2-micron-long portion of the nanowire exposed for contact with the solution applied to the device.

[0172] The crosslinking peptide was placed in solution on these devices, which were small cube-shaped (4 mm × 8 mm) and had eight pairs of nanoelectrodes cut into them. A custom flow cell and current measurement station were used to apply the solution to these devices and measure the electrode current under a DC applied voltage. The devices were exposed to a solution containing the crosslinking molecule at a concentration of 10 nM, and the time for crosslinking to occur with the electrodes was taken. The current passing through the resulting electrodes was measured at an applied DC voltage of 1 V. The difference between the current at 1 V and the current at 0 V was defined as the "delta maximum" current.

[0173] Figure 20 shows the current distribution of this delta maximum current obtained based on the observation of 29 electrodes from the crosslinking of SEQ ID NO: 19 and 71 electrodes from the crosslinking of SEQ ID NO: 15. Figure 20 shows the distribution of the observed current in picoamperes (pA), indicating that the conductivity of the SEQ ID NO: 15 peptide is higher.

[0174] Figure 20 shows that the first population of electrodes for SEQ ID NO: 19 and SEQ ID NO: 15 is concentrated below 4 pA, which is presumably interpreted as no crosslinking across the electrodes being formed. The other major population of electrodes was found to have currents concentrated at 16 pA (synthetic peptide having SEQ ID NO: 19) and 20 pA (synthetic peptide having SEQ ID NO: 15), which are interpreted as the conductivity being observed with a single crosslink, and thus SEQ ID NO: 15 shows a higher conductivity than SEQ ID NO: 19. The small number of populations with higher-order currents around 30 pA and 45 pA are interpreted as being due to multiple crosslinks spanning the electrode gaps.

[0175] Crosslinking sensor experiment: In these experiments, a 25.4 nm long synthetic peptide having SEQ ID NO: 21 was constructed to create sensors that detect binding events and enzyme activity events. The central cysteine C of the synthetic peptide was conjugated to the 5’ end of a linear single-stranded DNA oligonucleotide using standard cysteine conjugation chemistry. The attached oligonucleotide functioned as a probe molecule for binding to a primer DNA oligonucleotide. An assembly of the resulting synthetic peptide and DNA oligonucleotide probe molecule was fabricated and deployed for experiments similar to the above cross-bridge conductivity experiments and cross-bridged across the palladium electrodes. Once established in solution, this cross-bridge / probe assembly was first bound to a complementary primer oligonucleotide, resulting in a first type of binding event to be detected, thereby establishing a 3’ end priming site where 5 bases were removed from the conjugation junction site, which is the location where the cysteine of the cross-linkable peptide contacts the 5’ end of the template oligonucleotide. Next, polymerase was introduced into the solution, bound to the primer site, resulting in a second type of binding event to be detected. The polymerase was maintained in a non-catalytic buffer (using strontium as the divalent cation), thus unable to incorporate nucleotides, and thus reversibly bound to and exited from the pocket, thereby resulting in a third type of binding event to be detected by the sensor.

[0176] Figure 21 shows the current trace over time for the entire experiment over a 5000 second period, indicating that the sensor detects a strong current spike when polymerase is added and a series of strong spikes after nucleotides are added. These spikes are interpreted as the binding of signal-transducing polymerase to the primer complex on the cross-bridge, followed by the non-productive and transient binding and release of nucleotides into the polymerase pocket. This experiment exemplifies the use of such cross-bridged peptides in the context of a sensor.

[0177] Synthetic peptides are provided that are used as conductive bridging molecules or arm molecules in molecular electronic circuits of various arrangements. In the detailed description herein, references to "various embodiments", "one embodiment", "an embodiment", "example embodiments", etc., indicate that the described embodiments may include certain features, structures, or characteristics, but that not all embodiments necessarily include those particular features, structures, or characteristics. Further, such phrases do not necessarily refer to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is proposed that it is within the knowledge of one of ordinary skill in the relevant art(s) to envision such feature, structure, or characteristic in connection with other embodiments, whether or not explicitly described. After reading the description, one of ordinary skill in the relevant art(s) will be apparent as to how to implement the present disclosure in alternative embodiments.

[0178] Benefits, other advantages, and solutions to problems are described herein with respect to particular embodiments. However, benefits, advantages, solutions to problems, and any element that may give rise to or make more prominent any benefit, advantage, or solution are not to be construed as critical, required, or essential features or elements of the present disclosure. Accordingly, the scope of the present disclosure is defined only by the appended claims, and references to singular elements are not to be construed as meaning "only" unless explicitly so recited, but rather "one or more". Further, when phrases such as "at least one of A, B, and C" or "at least one of A, B, or C" are used in the claims or specification, such phrases are to be construed to mean that in one embodiment A may exist alone, in one embodiment B may exist alone, in one embodiment C may exist alone, or any combination of elements A, B, and C may exist in a single embodiment; for example, it is to be construed to mean A and B, A and C, B and C, or A and B and C.

[0179] All structural, chemical, and functional equivalents known to those of ordinary skill in the art of the elements of the various embodiments described above are hereby expressly incorporated by reference and are intended to be included within the scope of the claims. Further, with respect to any chemical entity, molecular electronics structure, or method for addressing any and all problems attempted to be solved by the present disclosure, it is not necessary that they be included within the scope of the claims. Additionally, none of the elements, components, or method steps of the present disclosure are intended to be dedicated to the public, whether or not such elements, components, or method steps are expressly recited in the claims. None of the elements of the claims are intended to invoke 35 U.S.C. 112(f), except where such element is expressly recited using the phrase "means for." As used herein, the terms "comprise," "comprising," or any other variation thereof are intended to cover a non-exclusive inclusion, such that a chemical substance, chemical composition, process, method, article, or apparatus that includes a list of elements does not include only those elements but may also include other elements not expressly listed or inherent to such chemical substance, chemical composition, process, method, article, or apparatus.

[0180] [Item 1] Formula: [X 1 X 2 m X 3 [X 4 n X 3 [X 2 X 1 m A synthetic peptide comprising, wherein, each X 1 independently comprises a material-binding peptide, a protease cleavage sequence, or a peptide capture tag comprising from about 5 to about 15 amino acids, each X 2 independently comprises a glycine / serine {G, S}-rich linker or a C1-C20 carbon chain molecular linker, each X 3 ​​​independently includes a covalent bond, a single amino acid, a transitional helix promoting motif, a metal-binding group, or a material-binding peptide containing about 5 to about 15 amino acids, each X 4 independently includes an alpha helix motif containing about 4 to about 40 amino acids, each m is independently 0 to 4, n is 1 to 40, a synthetic peptide. [Item 2] X 4 at least one of which includes a conjugation site, the synthetic peptide according to Item 1. [Item 3] The conjugation site includes cysteine, lysine, tyrosine, biotin, azide, or a click chemistry group, the synthetic peptide according to Item 2. [Item 4] X 1 at least one of which includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 16, 18 or 29, the synthetic peptide according to Item 1. [Item 5] X 2 at least one of which includes glycine, serine, GS, GSG, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 25, or a C1-C20 carbon chain molecule linker, the synthetic peptide according to Item 1. [Item 6] [X 1 X 2 m X 3 In any one grouping of, when m≠0, X 3 includes a covalent bond, a single amino acid, or a transitional helix promoting motif, the synthetic peptide according to Item 1. [Item 7] [X 1 X 2 m X 3 ​​In any one of the groupings, when m = 0, X 3 is the synthetic peptide according to item 1, comprising a metal-binding group or a material-binding peptide containing about 5 to about 15 amino acids. [Item 8] The synthetic peptide according to item 1, wherein the metal-binding group comprises a FLASH-binding motif of C, CC, CCC, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 5, SEQ ID NO: 35, or CCXXCC (wherein X is any amino acid in the sequence). [Item 9] X 4 At least one of which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, 13, 17, 20, 22, 23, 26, 27, 28 or 31, the synthetic peptide according to item 1. [Item 10] The synthetic peptide according to item 1, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 14. [Item 11] The synthetic peptide according to item 1, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 15. [Item 12] The synthetic peptide according to item 1, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 19. [Item 13] The synthetic peptide according to item 1, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 21. [Item 14] A first electrode, A second electrode spaced from the first electrode by a nanogap, A crosslinking molecular wire comprising the synthetic peptide according to item 2, electrically connected to both the first electrode and the second electrode and crosslinked to the nanogap, A polymerase enzyme conjugated to a conjugation site A molecular electronic circuit comprising The circuit is a molecular electronic circuit comprising a conductive path through the synthetic peptide. [Item 15] The molecular electronic circuit according to item 14, A transimpedance amplifier electrically connected to at least one of the first electrode and the second electrode, the transimpedance amplifier providing an output comprising measurable electrical parameters A sensor comprising [Item 16] A CMOS chip device comprising an array of sensors according to item 15. [Item 17] A method for sequencing a DNA molecule, comprising Providing the sensor according to item 15, Initiating at least one of a voltage or a current through the circuit, Exposing the circuit to a solution containing primed single-stranded DNA and / or dNTPs, Measuring an electrical signal through the circuit when the polymerase engages the template and extends it, Comprising Processing the electrical signal to identify features that provide information about the sequence underlying the DNA molecule processed by the polymerase. A method. [Item 18] A first electrode and a second electrode spaced apart by a nanogap, The first synthetic peptide according to item 1, electrically connected between the first electrode and the first site of the polymerase enzyme, The second synthetic peptide according to item 1, electrically connected between the second electrode and the second site of the polymerase enzyme, A molecular electronic circuit comprising The circuit is a molecular electronic circuit including a conductive path passing through a part of a polymerase enzyme. [Item 19] The molecular electronic circuit according to item 18, and a transimpedance amplifier electrically connected to at least one of the first electrode and the second electrode, the transimpedance amplifier providing an output including measurable electrical parameters A sensor comprising. [Item 20] A CMOS chip device including an array of the sensors according to item 19. [Item 21] A method for sequencing a DNA molecule, comprising: providing the sensor according to item 19; initiating at least one of a voltage or a current through the circuit; exposing the circuit to a solution containing primed single-stranded DNA and / or dNTPs; measuring an electrical signal through the circuit when the polymerase engages the template and extends it; and processing the electrical signal to identify features that provide information about the sequence underlying the DNA molecule processed by the polymerase.

Claims

1. A synthetic peptide comprising the formula: [X １ X ２ ｍ X ３ [X ４ ｎ X ３ [X ２ X １ ｍ ​​​ wherein each m is independently 0 to 4; Each X １ independently contains a material-binding peptide containing 5 to 15 amino acids, a protease cleavage sequence, or a peptide capture tag, Each X ２ independently includes a glycine / serine {G, S}-rich linker or a C1-C20 carbon chain molecular linker, Each X ３ independently contains a metal-binding group or a material-binding peptide containing 5 to 15 amino acids, Each X ４ independently includes an alpha-helix motif containing 4 to 40 amino acids, n is 1 to 40; the conjugation site comprises cysteine, lysine, tyrosine, biotin, azide, or a click chemistry group;

2. X ４ is a synthetic peptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 15, 16, and 19.

3. X １ At least one example of X comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 16, 18, or 29 ２ At least one example of X comprises glycine, serine, GS, GSG, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 24 or SEQ ID NO: 25, or a C1-C20 carbon chain molecular linker, the synthetic peptide according to claim 1

4. [X １ X ２ ｍ X ３ In any one grouping of X, when m≠0, X ３ is a synthetic peptide according to claim 1, comprising a covalent bond, a single amino acid, or a transitional helix promoting motif.​

5. [X １ X ２ ｍ X ３ In any one grouping of X, when m = 0, X ３ is the synthetic peptide according to claim 1, which comprises a metal-binding group or a material-binding peptide containing 5 to 15 amino acids.​

6. The synthetic peptide according to claim 1, wherein the metal-binding group comprises a FLASH-binding motif of C, CC, CCC, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 5, SEQ ID NO: 35, or the sequence CCXXCC (wherein X is any amino acid) in the sequence.

7. X ４ At least one example of is an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 13, 17, 20, 22, 23, 26, 27, 28, or 31, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 15, the synthetic peptide according to claim 1.

8. An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 21, the synthetic peptide according to claim 1.

9. A first electrode; A second electrode spaced from the first electrode by a nanogap; A cross-linking molecular wire comprising the synthetic peptide according to claim 1, electrically connected to both the first electrode and the second electrode and cross-linked to the nanogap; A polymerase enzyme conjugated to the conjugation site; A molecular electronic circuit comprising: The circuit comprises a conductive path through the synthetic peptide.

10. A sensor comprising the molecular electronic circuit according to claim 8, and A transimpedance amplifier electrically connected to at least one of the first electrode and the second electrode, the transimpedance amplifier providing an output comprising a measurable electrical parameter.

11. A method for sequencing a DNA molecule, comprising: Providing the sensor according to claim 9; Initiating at least one of a voltage or a current through the circuit; Exposing the circuit to a solution containing primed single-stranded DNA and / or dNTPs; Measuring an electrical signal through the circuit when the polymerase engages the template and extends it; And Processing the electrical signal to identify features that provide information about the sequence underlying the DNA molecule processed by the polymerase.

12. ​ A first electrode and a second electrode spaced apart by a nanogap, The synthetic peptide according to claim 1, electrically connected between the first electrode and the first site of the polymerase enzyme, The synthetic peptide according to claim 1, electrically connected between the second electrode and the second site of the polymerase enzyme, A molecular electronic circuit comprising: The circuit includes a conductive path through a portion of the polymerase enzyme, a molecular electronic circuit.

12. The molecular electronic circuit according to claim 1, A transimpedance amplifier electrically connected to at least one of the first electrode and the second electrode, the transimpedance amplifier providing an output including measurable electrical parameters A sensor comprising.

13. A method for sequencing a DNA molecule, comprising: Providing the sensor according to claim 12; Initiating at least one of a voltage or a current through the circuit; Exposing the circuit to a solution containing primed single-stranded DNA and / or dNTPs; Measuring an electrical signal through the circuit when the polymerase engages the template and extends it Including, Processing the electrical signal to identify features that provide information regarding the sequence underlying the DNA molecule processed by the polymerase.

Images