Light-emitting labeled oligonucleotide structures and related systems and methods

Abstract

Provided herein are light-emitting labeled oligonucleotide constructs that can be used in systems and methods for polypeptide and / or nucleic acid sequencing.

Description

[Technical Field]

[0001] Light-emitting labeled oligonucleotide structures and related systems and methods are generally described. [Background technology]

[0002] Luminescent labels are often used in systems and methods for detecting and / or characterizing biological analytes. Some of these systems and methods involve monitoring biological reactions in real time using multiple types of luminescently labeled reaction components. To identify a specific type of luminescently labeled reaction component, it is important that each type of reaction component is labeled with a luminescent label that has easily distinguishable luminescent characteristics. However, when designing luminescent labels for use in these systems and methods, the sensitivity of complex biological processes must be carefully considered. Summary of the Invention [Means for solving the problem]

[0003] Luminescently labeled oligonucleotide structures and related systems and methods are generally described. The subject matter disclosed herein, in some cases, involves interrelated products, alternative solutions to a particular problem, and / or multiple different uses of one or more systems and / or articles.

[0004] In some embodiments, a luminescently labeled oligonucleotide structure is provided. In some embodiments, the structure comprises a first single-stranded oligonucleotide comprising one or more first luminescent labels. In some embodiments, the structure comprises a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In certain embodiments, the first complementary single-stranded oligonucleotide comprises one or more second luminescent labels. In certain embodiments, the shortest distance between any first luminescent label and any second luminescent label is at least 10 nm.

[0005] In some embodiments, a luminescently labeled oligonucleotide structure is provided. In some embodiments, the structure comprises a first single-stranded oligonucleotide comprising two or more first luminescent labels. In some embodiments, the structure comprises a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In certain embodiments, the first complementary single-stranded oligonucleotide comprises two or more first luminescent labels. In certain embodiments, the first luminescent label comprises a cyanine dye.

[0006] In some embodiments, a luminescently labeled oligonucleotide structure is provided. In some embodiments, the structure comprises a first single-stranded oligonucleotide bound to a first binding molecule (e.g., a multivalent protein such as an avidin protein). In some embodiments, the structure comprises a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In some embodiments, the structure comprises a second single-stranded oligonucleotide bound to the first binding molecule. In some embodiments, the structure comprises a second complementary single-stranded oligonucleotide hybridized to the second single-stranded oligonucleotide. In certain embodiments, the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide are conjugated to one or more first luminescent labels. In certain embodiments, the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide are conjugated to one or more second luminescent labels.

[0007] In some embodiments, a system is provided. In some embodiments, the system includes an integrated device including a plurality of sample wells. In certain embodiments, one or more sample wells are adapted to have a polypeptide immobilized on its surface. In some embodiments, the system includes one or more first amino acid recognition molecules bound to a first light-emitting label comprising a first light-emitting labeled oligonucleotide structure. In certain embodiments, the first light-emitting labeled oligonucleotide structure includes a first single-stranded oligonucleotide comprising one or more first fluorophores. In certain embodiments, the first light-emitting labeled oligonucleotide structure includes a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In some cases, the first complementary single-stranded oligonucleotide comprises one or more second fluorophores. In some cases, the shortest distance between any first light-emitting label and any second light-emitting label is at least 10 nm.

[0008] In some embodiments, a system is provided. In some embodiments, the system includes an integrated device including a plurality of sample wells. In certain embodiments, one or more sample wells are adapted to have a polypeptide immobilized on its surface. In some embodiments, the system includes one or more first amino acid recognition molecules bound to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure. In certain embodiments, the light-emitting labeled oligonucleotide structure includes a first single-stranded oligonucleotide comprising two or more first light-emitting labels. In certain embodiments, the light-emitting labeled oligonucleotide structure includes a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In some cases, the first complementary single-stranded oligonucleotide comprises two or more first fluorophores. In some cases, the first light-emitting label comprises a cyanine dye.

[0009] In some embodiments, a system is provided. In some embodiments, the system includes an integrated device including a plurality of sample wells. In certain embodiments, one or more sample wells are adapted to have a polypeptide immobilized on its surface. In some embodiments, the system includes one or more first amino acid recognition molecules bound to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure. In certain embodiments, the light-emitting labeled oligonucleotide structure includes a first single-stranded oligonucleotide bound to a first binding molecule (e.g., a multivalent protein such as an avidin protein). In certain embodiments, the light-emitting labeled oligonucleotide structure includes a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In certain embodiments, the light-emitting labeled oligonucleotide structure includes a second single-stranded oligonucleotide bound to the first binding molecule. In certain embodiments, the light-emitting labeled oligonucleotide structure includes a second complementary single-stranded oligonucleotide hybridized to the second single-stranded oligonucleotide. In some cases, the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide are conjugated to one or more first fluorophores. In some cases, the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide are conjugated to one or more second fluorophores.

[0010] In some embodiments, a method for determining the chemical properties of a polypeptide is provided. In some embodiments, the method includes contacting the polypeptide with one or more first amino acid recognition molecules bound to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure. In certain embodiments, the light-emitting labeled oligonucleotide structure comprises a first single-stranded oligonucleotide comprising one or more first fluorophores. In certain embodiments, the light-emitting labeled oligonucleotide structure comprises a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In some cases, the first complementary single-stranded oligonucleotide comprises one or more second fluorophores. In some cases, the shortest distance between any first fluorophore and any second fluorophore is at least 10 nm. In some embodiments, the method includes detecting a first series of signal pulses indicative of a first series of binding events between one or more amino acid recognition molecules and the polypeptide. In some embodiments, the method includes determining at least one chemical property of an amino acid of the polypeptide based on at least one property of the first series of signal pulses.

[0011] In some embodiments, a method for determining a chemical property of a polypeptide is provided. In some embodiments, the method includes contacting the polypeptide with one or more first amino acid recognition molecules bound to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure. In certain embodiments, the light-emitting labeled oligonucleotide structure comprises a first single-stranded oligonucleotide comprising two or more first fluorophores. In certain embodiments, the light-emitting labeled oligonucleotide structure comprises a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In some cases, the first complementary single-stranded oligonucleotide comprises two or more first fluorophores. In some cases, the first light-emitting label comprises a cyanine dye. In some embodiments, the method includes detecting a first series of signal pulses indicative of a first series of binding events between the one or more amino acid recognition molecules and the polypeptide. In some embodiments, the method includes determining at least one chemical property of an amino acid of the polypeptide based on at least one property of the first series of signal pulses.

[0012] In some embodiments, a method for determining the chemical properties of a polypeptide is provided. In some embodiments, the method comprises contacting the polypeptide with one or more first amino acid recognition molecules bound to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure. In certain embodiments, the light-emitting labeled oligonucleotide comprises a first single-stranded oligonucleotide bound to a first binding molecule (e.g., a multivalent protein such as an avidin protein). In certain embodiments, the light-emitting labeled oligonucleotide comprises a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide. In certain embodiments, the light-emitting labeled oligonucleotide comprises a second single-stranded oligonucleotide bound to the first binding molecule. In certain embodiments, the light-emitting labeled oligonucleotide comprises a second complementary single-stranded oligonucleotide hybridized to the second single-stranded oligonucleotide. In some cases, the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide are conjugated to one or more first fluorophores. In some cases, the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide are conjugated with one or more second fluorophores.In some embodiments, the method comprises detecting a first series of signal pulses that indicate a first series of binding events between one or more amino acid recognition molecules and the polypeptide.In some embodiments, the method comprises determining at least one chemical property of the amino acid of the polypeptide based on at least one property of the first series of signal pulses.

[0013] In some embodiments, a system is provided. In some embodiments, the system includes a first light-emitting marker having a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic. In some embodiments, the system includes a second light-emitting marker having a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic. In some embodiments, the system includes a third light-emitting marker having a third ordered pair of characteristics including a third value of the first characteristic and a third value of the second characteristic. In certain embodiments, the first ordered pair, the second ordered pair, and the third ordered pair differ from each other in at least one of the values ​​of the first characteristic and / or the second characteristic.

[0014] In some embodiments, a method is provided. In some embodiments, the method includes providing a first light-emitting label having a first ordered pair of properties comprising a first value of a first property and a first value of a second property. In some embodiments, the method includes providing a second light-emitting label having a second ordered pair of properties comprising a second value of the first property and a second value of the second property. In some embodiments, the method includes providing a third light-emitting label comprising a light-labeled oligonucleotide structure comprising a first single-stranded oligonucleotide comprising one or more first fluorophores and a first complementary single-stranded oligonucleotide comprising one or more second fluorophores. In certain embodiments, the third light-emitting label has a third ordered pair of properties comprising a third value of the first property and a third value of the second property. In some embodiments, the method includes modifying the number and / or type of one or more first fluorophores and / or one or more second fluorophores such that the first ordered pair, the second ordered pair, and the third ordered pair differ from each other in at least one of the values ​​of the respective first property and / or second property.

[0015] In some embodiments, a system is provided. In some embodiments, the system includes a first luminescent label having a first bin ratio value. In some embodiments, the system includes a second luminescent label having a second bin ratio value. In some embodiments, the system includes a third luminescent label having a third bin ratio value. In certain embodiments, the minimum difference between the first bin ratio value, the second bin ratio value, and the third bin ratio value of the first luminescent characteristic is at least 0.1.

[0016] The details of certain embodiments of the invention are set forth in the Detailed Description of Specific Embodiments set forth below. Other features, objects, and advantages of the invention will become apparent from the examples, figures, and claims. [Brief explanation of the drawings]

[0017] [Figure 1A] FIG. 1 shows a schematic diagram of an exemplary luminescently labeled oligonucleotide structure comprising a first single-stranded oligonucleotide comprising one copy of a first luminescent label and a first complementary single-stranded oligonucleotide comprising one copy of a second luminescent label, according to some embodiments. [Figure 1B] 1B shows a schematic diagram of the exemplary luminescently labeled oligonucleotide structure of FIG. 1A attached to a reactive component (eg, amino acid recognition molecule, nucleotide) via a linking molecule, according to some embodiments. [Figure 2A] FIG. 1 shows a schematic diagram of an exemplary luminescently labeled oligonucleotide structure comprising a first single-stranded oligonucleotide comprising two copies of a first luminescent label and a first complementary single-stranded oligonucleotide comprising one copy of a second luminescent label, according to some embodiments. [Figure 2B] 2B shows a schematic diagram of the exemplary luminescently labeled oligonucleotide structure of FIG. 2A attached to a reactive component (eg, amino acid recognition molecule, nucleotide) via a linking molecule, according to some embodiments. [Figure 3A] 1 shows a schematic diagram of an exemplary method for assembling a luminescently labeled oligonucleotide structure, according to some embodiments. [Figure 3B] FIG. 1 shows a schematic diagram of an exemplary method for constructing a luminescently labeled oligonucleotide structure by ligation, according to some embodiments. [Figure 4] Figure 1 shows an exemplary overview of real-time dynamic protein sequencing according to some embodiments. Protein samples are digested into peptide fragments, immobilized in a nanoscale reaction chamber, and incubated with a mixture of freely diffusing N-terminal amino acid (NAA) recognition factors and aminopeptidases that perform the sequencing process. Labeled recognition factors bind to peptides on and off when one of their cognate NAAs is exposed at the N-terminus, thereby generating a characteristic pulse pattern. The NAA is cleaved by the aminopeptidase, exposing the next amino acid for recognition. The temporal order and binding kinetics of NAA recognition enable peptide identification and are sensitive to features that modulate binding kinetics, such as post-translational modifications (PTMs). SEQ ID NOs: 21 and 22 are shown from left to right. [Figure 5] 1 shows an exemplary schematic diagram of a pixel of an integrated device according to some embodiments. [Figure 6A] 1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) using an amino acid recognition molecule labeled with a third luminescent label (designated R1C1) comprising a luminescent-labeled oligonucleotide structure comprising a first luminescent label comprising four copies of Cy®3B, a second luminescent label comprising three copies of ATRho6G, and a first single-stranded oligonucleotide comprising one copy of Cy®3B and having 100% sequence identity to sequence A, and a first complementary single-stranded oligonucleotide comprising one copy of ATRho6G and having 100% sequence identity to sequence B. [Figure 6B] 6B shows a plot of intensity versus bin ratio for the polypeptide sequencing reaction of FIG. 6A. [Figure 7A]1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) using an amino acid recognition molecule labeled with a third luminescent label (designated C2C) comprising a luminescent-labeled oligonucleotide structure comprising: a first luminescent label comprising 8 copies of Cy®3; a second luminescent label comprising 4 copies of Cy®3B; and a first single-stranded oligonucleotide comprising 2 copies of Cy®3 and having 100% sequence identity to sequence C; and a first complementary single-stranded oligonucleotide comprising 1 copy of Cy®3B and having 100% sequence identity to sequence D. [Figure 7B] 7B shows a plot of intensity versus bin ratio for the polypeptide sequencing reaction of FIG. 7A. [Figure 8A] 1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) using an amino acid recognition molecule labeled with a third light-emitting label (designated SG4Cy®) that includes a light-emitting labeled oligonucleotide structure that includes a first light-emitting label comprising four copies of Cy®B, a second light-emitting label comprising C2C, and a first single-stranded oligonucleotide comprising two copies of Cy® and having 100% sequence identity to sequence E, and a first complementary single-stranded oligonucleotide comprising two copies of Cy® and having 100% sequence identity to sequence F. [Figure 8B] 8B shows a plot of intensity versus bin ratio for the polypeptide sequencing reaction of FIG. 8A. [Figure 9A] 1 shows a retention plot representing the first step in the construction of a luminescently labeled oligonucleotide: conjugating a first luminescently labeled oligonucleotide strand (ODN1) bound to streptavidin (SV) to a second luminescently labeled oligonucleotide strand (ODN3). [Figure 9B]The second step in the construction of a luminescently labeled oligonucleotide: A retention plot showing the step of hybridizing a third luminescently labeled oligonucleotide strand (ODN4) bound to a second streptavidin to ODN3 in the product of step 1 shown in Figure 9A, and hybridizing a complementary oligonucleotide strand (ODN2) to the first luminescently labeled oligonucleotide strand (ODN1). [Figure 9C] A retention plot is shown representing the final step in the construction of a light-emitting labeled oligonucleotide: conjugating the product of step 2 shown in Figure 9B to an amino acid recognition molecule (PS610). [Figure 10A-1] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of ATRho6G. [Figure 10A-2] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of ATRho6G. [Figure 10A-3] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of ATRho6G. [Figure 10A-4] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of ATRho6G. [Figure 10B-1]Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAGK (SEQ ID NO: 19)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of C530NS. [Figure 10B-2] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAGK (SEQ ID NO: 19)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of C530NS. [Figure 10B-3] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAGK (SEQ ID NO: 19)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of C530NS. [Figure 10B-4] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (RLIFAGK (SEQ ID NO: 19)) using a first luminescent label containing 4 copies of Cy®3, a second luminescent label containing 3 copies of Cy®3B, and a third luminescent label containing 3 copies of C530NS. [Figure 10C-1] 1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using the light-labeled oligonucleotide structure from Example 4, which includes a first light-emitting label comprising 4 copies of Cy®3, a second light-emitting label comprising 4 copies of Cy®3B, a third light-emitting label comprising 2 copies of ATRho6G, and 8 copies of C530NS. [Figure 10C-2]1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using the light-labeled oligonucleotide structure from Example 4, which includes a first light-emitting label comprising 4 copies of Cy®3, a second light-emitting label comprising 4 copies of Cy®3B, a third light-emitting label comprising 2 copies of ATRho6G, and 8 copies of C530NS. [Figure 10C-3] 1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using the light-labeled oligonucleotide structure from Example 4, which includes a first light-emitting label comprising 4 copies of Cy®3, a second light-emitting label comprising 4 copies of Cy®3B, a third light-emitting label comprising 2 copies of ATRho6G, and 8 copies of C530NS. [Figure 10C-4] 1 shows a representative trace from a polypeptide sequencing reaction performed on a sample peptide (RLIFAYPDDDK (SEQ ID NO: 18)) using the light-labeled oligonucleotide structure from Example 4, which includes a first light-emitting label comprising 4 copies of Cy®3, a second light-emitting label comprising 4 copies of Cy®3B, a third light-emitting label comprising 2 copies of ATRho6G, and 8 copies of C530NS. [Figure 11A] Representative traces are shown from polypeptide sequencing reactions performed on a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) using amino acid recognition molecules labeled with a first luminescent label containing 4 copies of Cy®3 (termed "TetraCy3"), a second luminescent label containing 4 copies of Cy®3B (termed "TetraCy3B"), and a third luminescent label containing 8 copies of Cy®3 (termed "OctaCy3"). [Figure 11B] 8B shows a plot of intensity versus bin ratio for the polypeptide sequencing reaction of FIG. 8A. [Figure 12A]FIG. 1 shows a schematic diagram of an exemplary method used to construct luminescently labeled oligonucleotide structures by ligation and streptavidin conjugation. [Figure 12B-1] Shown are the results of size exclusion chromatography (top) and urea-PAGE gel analysis (bottom) demonstrating the purification of ligation products prepared according to Figure 12A. [Figure 12B-2] Shown are the results of size exclusion chromatography (top) and urea-PAGE gel analysis (bottom) demonstrating the purification of ligation products prepared according to Figure 12A. [Figure 12C] 12B shows the results of size exclusion chromatography demonstrating the purification of a streptavidin-conjugated product prepared according to FIG. 12A. [Figure 12D] 12B shows the results of size exclusion chromatography demonstrating the purification of streptavidin-conjugated amino acid recognition molecules prepared according to FIG. 12A. [Figure 13A-1] Shown is a representative trace (top) showing phenylalanine recognition for a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) and an intensity versus bin ratio plot (bottom) showing clear spatial separation of LC6IF, C2C (Example 2), and a recognition molecule with four copies of Cy®3B ("4-Cy3B"). [Figure 13A-2] Shown is a representative trace (top) showing phenylalanine recognition for a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) and an intensity versus bin ratio plot (bottom) showing clear spatial separation of LC6IF, C2C (Example 2), and a recognition molecule with four copies of Cy®3B ("4-Cy3B"). [Figure 13B-1] A representative trace (top) showing phenylalanine recognition for a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) and a plot of intensity versus bin ratio (bottom) showing clear spatial separation of LC6IF, C2C, and SG4Cy3 (Example 3) are shown. [Figure 13B-2]A representative trace (top) showing phenylalanine recognition for a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) and a plot of intensity versus bin ratio (bottom) showing clear spatial separation of LC6IF, C2C, and SG4Cy3 (Example 3) are shown. [Figure 13C-1] A representative trace (top) showing amino acid recognition during degradation of a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) is shown, along with a plot of intensity versus bin ratio (bottom) demonstrating clear spatial separation of LC6IF and SG4Cy3. [Figure 13C-2] A representative trace (top) showing amino acid recognition during degradation of a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) is shown, along with a plot of intensity versus bin ratio (bottom) demonstrating clear spatial separation of LC6IF and SG4Cy3. [Figure 13D-1] A representative trace (top) showing amino acid recognition during degradation of a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) is shown, along with a plot of intensity versus bin ratio (bottom) demonstrating clear spatial separation of LC6IF, R1C1 (Example 1), and 4-Cy3B. [Figure 13D-2] A representative trace (top) showing amino acid recognition during degradation of a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) is shown, along with a plot of intensity versus bin ratio (bottom) demonstrating clear spatial separation of LC6IF, R1C1 (Example 1), and 4-Cy3B. [Figure 13E] A plot of intensity versus bin ratio for a dye set including C2C, 4-Cy3B, and a label with 8 copies of Cy®3 in a construct prepared by ligation (“L8Cy3”) is shown. [Figure 13F] 1 shows a plot of intensity versus bin ratio for a dye set including C2C, 4-Cy3B, and a label with 8 copies of Cy®3 in a construct prepared by dual streptavidin ligation (“8Cy3”). [Figure 13G] Plots of intensity versus bin ratio for L8Cy3, LC6C, and LC6IF (top) and a table of corresponding values ​​(bottom) are shown. [Figure 13H-1]A representative trace (top) showing phenylalanine recognition for the sample peptide (FAAAYPDDD (SEQ ID NO: 17)) is shown, along with an intensity versus bin ratio plot (bottom) showing clear spatial separation of seven distinctly labeled recognition molecules. [Figure 13H-2] A representative trace (top) showing phenylalanine recognition for the sample peptide (FAAAYPDDD (SEQ ID NO: 17)) is shown, along with an intensity versus bin ratio plot (bottom) showing clear spatial separation of seven distinctly labeled recognition molecules. [Figure 13I-1] A representative trace (top) showing amino acid recognition during degradation of a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) is shown, along with an intensity versus bin ratio plot (bottom) showing the distinct spatial separation of seven distinctly labeled recognition molecules. [Figure 13I-2] A representative trace (top) showing amino acid recognition during degradation of a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) is shown, along with an intensity versus bin ratio plot (bottom) showing the distinct spatial separation of seven distinctly labeled recognition molecules. DETAILED DESCRIPTION OF THE INVENTION

[0018] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention. Light-emitting labeled oligonucleotide structures and related systems and methods are generally described. Some aspects of the present disclosure relate to light-emitting labeled oligonucleotide structures comprising double-stranded oligonucleotides, each strand of which is labeled with one or more types of light-emitting labels, and the minimum distance between each type of light-emitting label is relatively large (e.g., at least 10 nm). Some aspects of the present disclosure relate to light-emitting labeled oligonucleotide structures comprising a plurality of light-emitting labeled double-stranded oligonucleotides connected by one or more linking molecules (e.g., multivalent proteins such as avidin proteins). In certain embodiments, one or more light-emitting labeled double-stranded oligonucleotides among the plurality of light-emitting labeled double-stranded oligonucleotides contain one or more isocytosine or isoguanine nucleotides, and one or more light-emitting labeled double-stranded oligonucleotides among the plurality of light-emitting labeled double-stranded oligonucleotides do not contain isocytosine or isoguanine nucleotides. Some aspects of the present disclosure relate to a set of light-emitting labeled structures comprising one or more light-emitting labeled oligonucleotide structures, each structure of the set having one or more unique light-emitting properties (e.g., lifetime, intensity).

[0019] A luminescent label generally refers to a molecule that can absorb one or more photons and then emit one or more photons after one or more time durations. In some embodiments, the term "luminescent label" is used interchangeably with "label" or "luminescent molecule." Luminescent labels can be used in various systems and methods for detecting and / or characterizing biological analytes, including, but not limited to, systems and methods for sequencing polypeptides and / or nucleic acids. In certain embodiments, these systems and methods can involve monitoring biological reactions in real time using multiple types of luminescently labeled reaction components. As an illustrative example, a system or method for polypeptide sequencing can include multiple types of luminescently labeled amino acid recognition molecules, each type of amino acid recognition molecule labeled with a different type of luminescent label. As another illustrative example, a system or method for nucleic acid sequencing can include multiple types of luminescently labeled nucleotides, with each type of nucleotide (e.g., deoxyadenosine triphosphate (dATP), thymidine triphosphate (TTP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP)) labeled with a different type of luminescent label. In some embodiments, the luminescently labeled reaction components (e.g., amino acid recognition molecules, nucleotides) can be illuminated by a light source to produce luminescence, and the resulting luminescence can be detected by one or more photodetectors. The detected luminescence can be recorded and analyzed to identify or otherwise characterize the type of reaction component based on one or more luminescence characteristics of the detected luminescence. Each type of reaction component can be labeled with a luminescent label having easily distinguishable luminescence characteristics (e.g., lifetime, intensity) so that the type of luminescent-labeled reaction component emitting the detected luminescence can be identified or otherwise characterized.

[0020] In some cases, the set of light-emitting labels may include one or more light-emitting labeled oligonucleotide structures described herein. In some cases, by attaching various numbers and / or types of fluorophores to the oligonucleotide strand, one or more light-emitting properties of the light-emitting labeled oligonucleotide may be tailored to be different from the light-emitting properties of other light-emitting labels in the set. In some cases, this may advantageously allow the development of light-emitting labeled oligonucleotide structures with light-emitting properties different from those of known fluorophores. In some cases, this may allow the development of sets of light-emitting labels with different values ​​for one or more light-emitting properties. In an exemplary, non-limiting embodiment, the set of light-emitting labels may include a first known fluorophore (e.g., Cy®3), a second known fluorophore (e.g., Cy®3B), and a light-emitting labeled oligonucleotide structure comprising a first oligonucleotide strand containing one or more copies of the first known fluorophore and a second oligonucleotide strand containing one or more copies of the second known fluorophore. In certain embodiments, one or more luminescence properties (e.g., lifetime, intensity) of the luminescent-labeled oligonucleotide structure may be different from the luminescence properties of the first known fluorophore and the luminescence properties of the second known fluorophore.In some cases, one or more luminescence properties of the luminescent-labeled oligonucleotide structure may be changed by adding or removing copies of the first known fluorophore and / or the second known fluorophore.In certain cases, for example, the luminescence intensity may be increased by adding additional copies of the first known fluorophore and / or the second known fluorophore.

[0021] Some aspects relate to a set of two or more luminescent labels, each of which has a value for one or more luminescent properties (e.g., lifetime, intensity) that differs from the values ​​of other luminescent labels in the set by a certain minimum amount. In certain embodiments, the minimum percentage difference between the values ​​of one or more luminescent properties of any two labels in a set of two or more luminescent labels may be relatively large. In some cases, the set of luminescent labels includes multiple luminescent labels, each of which has a different bin ratio. In certain cases, the minimum difference between the bin ratio values ​​of any two luminescent labels in the set is at least 0.1. In some cases, the set of luminescent labels includes multiple luminescent labels, each of which occupies a distinct spatial region in a two-dimensional plot of two luminescent properties (e.g., a plot of intensity versus bin ratio).

[0022] Luminescently labeled oligonucleotide structure In some embodiments, constructing multiple pairs of hybridized oligonucleotide strands with one or more linking molecules can advantageously provide a structure with a large number of fluorophores while maintaining sufficient distance between the fluorophores to prevent energy transfer between the fluorophores, which can reduce emission lifetimes.

[0023] A schematic diagram of an exemplary light-emitting labeled oligonucleotide structure is shown in FIG. 1A. In FIG. 1A, a first single-stranded oligonucleotide 100 includes one copy of a first light-emitting label 110. In certain embodiments, the first single-stranded oligonucleotide 100 further includes a first binding moiety 120. Furthermore, a first complementary single-stranded oligonucleotide 130 includes one copy of a second light-emitting label 140. As shown in FIG. 1A, the first single-stranded oligonucleotide 100 and the first complementary single-stranded oligonucleotide 130 can be hybridized to form a light-emitting labeled oligonucleotide 150. In the light-emitting labeled oligonucleotide 150, the first light-emitting label 110 and the second light-emitting label 140 can be separated by a minimum distance d. In some embodiments, the minimum distance d can be relatively large (e.g., at least 10 nm).

[0024] In some embodiments, the luminescently labeled oligonucleotide structure can be attached to a reaction component (e.g., an amino acid recognition molecule, a nucleotide) via a linking molecule. A schematic diagram of an exemplary reaction component labeled with a luminescently labeled oligonucleotide structure is shown in FIG. 1B. In FIG. 1B, the luminescently labeled oligonucleotide structure 150 includes a first binding moiety 120 bound to a first binding molecule 160. In some embodiments, the reaction component 170 includes a second binding moiety 180. In some embodiments, the second binding moiety 180 also binds to the first binding molecule 160, thereby conjugating the luminescently labeled oligonucleotide structure 150 to the reaction component 170. In certain embodiments, the first binding moiety 120 and the second binding moiety 180 can each include a biotin moiety (e.g., a bis-biotin moiety), and the first binding molecule 160 can include a multivalent protein such as an avidin protein (e.g., a streptavidin protein).

[0025] In some embodiments, the light-emitting labeled oligonucleotide structure comprises a plurality of first and / or second light-emitting labels. Figure 2A shows a schematic diagram of an exemplary light-emitting labeled oligonucleotide structure comprising two copies of a first light-emitting label (also referred to as two first light-emitting labels) and one copy of a second light-emitting label (also referred to as one second light-emitting label). In Figure 2A, a first single-stranded oligonucleotide 200 comprises two copies of a first light-emitting label 210, i.e., a first copy 210A and a second copy 210B. In certain embodiments, the first single-stranded oligonucleotide 200 further comprises a first binding moiety 220. Furthermore, the first complementary single-stranded oligonucleotide 230 comprises one copy of a second light-emitting label 240. As shown in Figure 2A, the first single-stranded oligonucleotide 200 and the first complementary single-stranded oligonucleotide 230 can be hybridized to form a light-emitting labeled oligonucleotide 250. In the luminescently labeled oligonucleotide 250, the minimum distance d between any first luminescent label 210 and any second luminescent label 240 (i.e., between the second copy of the first luminescent label 210B and the first copy of the second luminescent label 240 in FIG. 2A) may be relatively large (e.g., at least 10 nm).

[0026] 2B shows a schematic diagram of an exemplary luminescently labeled oligonucleotide structure bound to a reaction component (e.g., an amino acid recognition molecule, a nucleotide) via a linking molecule. In FIG. 2B, luminescently labeled oligonucleotide structure 250 includes a first binding moiety 220 bound to a first binding molecule 260. In some embodiments, reaction component 270 includes a second binding moiety 280. In some embodiments, second binding moiety 280 also binds to first binding molecule 260, thereby conjugating luminescently labeled oligonucleotide structure 250 to reaction component 270. In certain embodiments, first binding moiety 220 and second binding moiety 280 may each include a biotin moiety (e.g., a bis-biotin moiety), and first binding molecule 260 may include an avidin protein (e.g., a streptavidin protein).

[0027] In some embodiments, the first single-stranded oligonucleotide of the light-emitting labeled oligonucleotide structure comprises one or more copies of the first light-emitting label. In certain embodiments, the first single-stranded oligonucleotide comprises two or more copies of the first light-emitting label, three or more copies of the first light-emitting label, four or more copies of the first light-emitting label, five or more copies of the first light-emitting label, six or more copies of the first light-emitting label, seven or more copies of the first light-emitting label, eight or more copies of the first light-emitting label, nine or more copies of the first light-emitting label, or ten or more copies of the first light-emitting label.

[0028] In some embodiments, the first single-stranded oligonucleotide comprises one or more luminescent labels different from the first luminescent label. In certain embodiments, the first single-stranded oligonucleotide comprises one or more copies of a third luminescent label, and the third luminescent label is different from the first luminescent label. In some cases, the first single-stranded oligonucleotide comprises two or more copies of the third luminescent label, three or more copies of the third luminescent label, four or more copies of the third luminescent label, five or more copies of the third luminescent label, six or more copies of the third luminescent label, seven or more copies of the third luminescent label, eight or more copies of the third luminescent label, nine or more copies of the third luminescent label, or ten or more copies of the third luminescent label. In certain embodiments, the first single-stranded oligonucleotide further comprises one or more copies of an additional luminescent label different from the first and third luminescent labels.

[0029] In some embodiments, the first complementary single-stranded oligonucleotide of the light-emitting labeled oligonucleotide structure comprises one or more copies of a second light-emitting label. In some cases, the second light-emitting label is different from the first light-emitting label. In some cases, the second light-emitting label is the same as the first light-emitting label. In certain embodiments, the first complementary single-stranded oligonucleotide comprises two or more copies of the second light-emitting label, three or more copies of the second light-emitting label, four or more copies of the second light-emitting label, five or more copies of the second light-emitting label, six or more copies of the second light-emitting label, seven or more copies of the second light-emitting label, eight or more copies of the second light-emitting label, nine or more copies of the second light-emitting label, or ten or more copies of the second light-emitting label.

[0030] In some embodiments, the first complementary single-stranded oligonucleotide comprises one or more light-emitting labels different from the second light-emitting label. In certain embodiments, the first complementary single-stranded oligonucleotide comprises one or more copies of a fourth light-emitting label, wherein the fourth light-emitting label is different from the second light-emitting label. In some cases, the first complementary single-stranded oligonucleotide comprises two or more copies of the fourth light-emitting label, three or more copies of the fourth light-emitting label, four or more copies of the fourth light-emitting label, five or more copies of the fourth light-emitting label, six or more copies of the fourth light-emitting label, seven or more copies of the fourth light-emitting label, eight or more copies of the fourth light-emitting label, nine or more copies of the fourth light-emitting label, or ten or more copies of the fourth light-emitting label. In certain embodiments, the first single-stranded oligonucleotide further comprises one or more copies of an additional light-emitting label different from the second and fourth light-emitting labels.

[0031] In some embodiments, the luminescent labels described herein (e.g., first, second, third, and fourth luminescent labels) are fluorescent labels (e.g., comprise a fluorescent dye). In some embodiments, the luminescent labels comprise cyanine, rhodamine, boron-dipyrromethene (BODIPY), fluorescein, acridine, phenoxazine, coumarin, porphyrin, phthalocyanine, naphthalimide, pyrene, anthracene, naphthalene, naphthylamine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, benzoxazole, phenanthridine, quinoline, ethidium, benzamide, carbocyanine, salicylate, anthranilate, xanthene, or other similar compounds.

[0032] In some embodiments, the luminescent label comprises a dye selected from one or more of the following: 5 / 6-carboxyrhodamine 6G, 5-carboxyrhodamine 6G, 6-carboxyrhodamine 6G, 6-TAMRA, Abberior® STAR 440SXP, Abberior® STAR 470SXP, Abberior® STAR 488, Abberior® STAR 490SXP ... Abberior® STAR 512, Abberior® STAR 520SXP, Abberior® STAR 580, Abberior® STAR 600, Abberior® STAR 635, Abberior® STAR 635P, Abberior® STAR Red RED, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 480, Alexa Fluor® 488, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610-X, Alexa Fluor® 633, Alexa Fluor® Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700,Alexa Fluor® 750, Alexa Fluor® 790, AMCA, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 542, ATTO 550, ATTO 565, ATTO 590, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO ) 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTO Oxa12, ATTO Rho101, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14, ATTO Rho3B, ATTO Rho6G, ATTO Thio12, BD Horizon ( Trademark) V450, BODIPY® 493 / 501, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® CAL Fluor (registered trademark) FL, BODIPY (registered trademark) FL-X, BODIPY (registered trademark) R6G, BODIPY (registered trademark) TMR, BODIPY (registered trademark) TR, CAL Fluor (registered trademark) Gold 540, CAL Fluor (registered trademark) Green 510, CAL Fluor (registered trademark) Orange 560, CAL Fluor (registered trademark) Red 590, CAL Fluor (registered trademark) Red 610,CAL Fluor® Red 615, CAL Fluor® Red 635, Cascade® Blue, CF™ 350, CF™ 405M, CF™ 405S, CF™ 488A, CF™ 514, CF™ 532, CF™ 543, CF™ 546, CF™ 555, CF™ 568, CF™ 594, CF™ 620R, CF™ 633, CF™ 633-V1, CF™ 640R, CF™ 640R-V1 , CF(TM) 640R-V2, CF(TM) 660C, CF(TM) 660R, CF(TM) 680, CF(TM) 680R, CF(TM) 680R-V1, CF(TM) 750, CF(TM) 770, CF(TM) 790, Chromeo(TM) 642, Chromis 425N, Chromis 500N, Chromis 515N, Chromis 530N, Chromis 550A, Chromis 550C, Chromis 550Z, Chromis 560N, Chromis 570N, Chromis 577N, Chromis 600N, Chromis 630N, Chromis 645A, Chromis 645C, Chromis 645Z, Chromis 678A, Chromis 678C, Chromis 678Z, Chromis 770A, Chromis 770C, Chromis 80 0A, Chromis 800C, Chromis 830A, Chromis 830C, Cy® 3, Cy® 3.5, Cy® 3B, Cy® 5, Cy® 5.5, Cy® 7, DyLight® 350, DyLight® 405, DyLight® 415-Co1, DyLight® 425Q, DyLight® 485-LS,DyLight® 488, DyLight® 504Q, DyLight® 510-LS, DyLight® 515-LS, DyLight® 521-LS, DyLight® 530-R2, DyLight® 543Q, DyLight® 550, DyLight® 554-R0, DyLight® DyLight® 554-R1, DyLight® 590-R2, DyLight® 594, DyLight® 610-B1, DyLight® 615-B2, DyLight® 633, DyLight® 633-B1, DyLight® 633-B2, DyLight® 650, DyLight® 655-B1, DyLight® DyLight (registered trademark) 655-B2, DyLight (registered trademark) 655-B3, DyLight (registered trademark) 655-B4, DyLight (registered trademark) 662Q, DyLight (registered trademark) 675-B1, DyLight (registered trademark) 675-B2, DyLight (registered trademark) 675-B3, DyLight (registered trademark) 675-B4, DyLight (registered trademark) 679-C5, DyLight ) (registered trademark) 680, DyLight (registered trademark) 683Q, DyLight (registered trademark) 690-B1, DyLight (registered trademark) 690-B2, DyLight (registered trademark) 696Q, DyLight (registered trademark) 700-B1, DyLight (registered trademark) 700-B1, DyLight (registered trademark) 730-B1, DyLight (registered trademark) 730-B2, DyLight (registered trademark) 730-B3,DyLight® 730-B4, DyLight® 747, DyLight® 747-B1, DyLight® 747-B2, DyLight® 747-B3, DyLight® 747-B4, DyLight® 755, DyLight® 766Q, DyLight® 775-B2, DyLight® DyLight® 775-B3, DyLight® 775-B4, DyLight® 780-B1, DyLight® 780-B2, DyLight® 780-B3, DyLight® 800, DyLight® 830-B2, Dyomics-350, Dyomics-350XL, Dyomics-360XL, Dy Dyomics-370XL, Dyomics-375XL, Dyomics-380XL, Dyomics-390XL, Dyomics-405, Dyomics-415, Dyomics-430, Dyomics-431, Dyomics-478, Dyomics-480XL, Dyomics-481XL, Dyomics-485XL, Dyomics Dyomics-490, Dyomics-495, Dyomics-505, Dyomics-510XL, Dyomics-511XL, Dyomics-520XL, Dyomics-521XL, Dyomics-530, Dyomics-547, Dyomics-547P1, Dyomics-548, Dyomics-549,Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555, Dyomics-556, Dyomics-560, Dyomics-590, Dyomics-591, Dyomics, Dyomics-594, Dyomics-601XL, Dyomics-605, Dyomics-610, Dyomics-615, Dyomics-630, Dyomics-631, Dyomics-632, Dyomics-633, Dyomics-634, Dyomics-635, Dyomics-636, Dyomics Dyomics-647, Dyomics-647P1, Dyomics-648, Dyomics-648P1, Dyomics-649, Dyomics-649P1, Dyomics-650, Dyomics-651, Dyomics-652, Dyomics-654, Dyomics-675, Dyomics-676, Dyomics Dyomics-677, Dyomics-678, Dyomics-679P1, Dyomics-680, Dyomics-681, Dyomics-682, Dyomics-700, Dyomics-701, Dyomics-703, Dyomics-704, Dyomics-730, Dyomics-731, Dyomics -732, Dyomics-734, Dyomics-749, Dyomics-749P1, Dyomics-750, Dyomics-751, Dyomics-752, Dyomics-754, Dyomics-776, Dyomics-777, Dyomics-778, Dyomics-780, Dyomics-781,Dyomics-782, Dyomics-800, Dyomics-831, eFluor® 450, Eosin, FITC, Fluorescein, HiLyte™ Fluor 405, HiLyte™ Fluor 488, HiLyte™ Fluor 532, HiLyte™ Fluor 555, HiLyte™ Fluor 594, HiLyte™ Fluor 647, HiLyte™ Fluor 5 LightCycler® Red 680, HiLyte™ Fluor 750, IRDye® 680LT, IRDye® 750, IRDye® 800CW, JOE, LightCycler® 640R, LightCycler® Red 610, LightCycler® Red 640, LightCycler® Red 670, LightCycler® Red 705, Lissamine Rhodamine B, Naphthofluorescein, Oregon Green Green® 488, Oregon Green® 514, Pacific Blue™, Pacific Green™, Pacific Orange™, PET, PF350, PF405, PF415, PF488, PF505, PF532, PF546, PF555P, PF568, PF594, PF610, PF633P, PF647P, Quasar® 570, Quasar® 670, Quasar® 705, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rhodamine Green, Rhodamine Green-X, Rhodamine Red, ROX, Seta™ 375,Seta™ 470, Seta™ 555, Seta™ 632, Seta™ 633, Seta™ 650, Seta™ 660, Seta™ 670, Seta™ 680, Seta™ 700, Seta™ 750, Seta™ 780, Seta™ APC-780, Seta™ PerCP -680, Seta™ R-PE-670, Seta™ 646, SeTau 380, SeTau 425, SeTau 647, SeTau 405, Square 635, Square 650, Square 660, Square 672, Square 680, Sulforhodamine 101, TAMRA, TET, Texas Red®, TMR, TRITC, Yakima Yellow™, Zenon®, Zy3, Zy5, Zy5.5, and Zy7.

[0033] In certain embodiments, the luminescent label (e.g., the first luminescent label, the second luminescent label, the third luminescent label, the fourth luminescent label) comprises Cy®3, Cy®3B, ATTO Rho6G (also referred to as ATRho6G), Chromis 530N, and / or Chromis530N-S (also referred to as C530NS). In some embodiments, C530NS has the following structure:

[0034] [ka]

[0035] It has. In some cases, the first single-stranded oligonucleotide of the luminescently labeled oligonucleotide structure comprises a first luminescent label comprising Cy® 3B. In some cases, the first complementary single-stranded oligonucleotide comprises a second luminescent label comprising ATTO Rho6G.

[0036] In some cases, the first single-stranded oligonucleotide of the luminescently labeled oligonucleotide structure comprises a first luminescent label comprising ATTO Rho6G. In some cases, the first complementary single-stranded oligonucleotide comprises a second luminescent label comprising Cy®3B.

[0037] In some cases, the first single-stranded oligonucleotide of the light-emitting labeled oligonucleotide structure comprises two first light-emitting labels, each of which comprises Cy® 3. In some cases, the first complementary single-stranded oligonucleotide comprises one second light-emitting label that comprises Cy® 3B.

[0038] The light-emitting labeled oligonucleotide structure can have any suitable length. In some embodiments, the light-emitting labeled oligonucleotide structure has a length of at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, or at least 100 base pairs. In some embodiments, the light-emitting labeled oligonucleotide structure has a length of 20-25 base pairs, 20-30 base pairs, 20-40 base pairs, 20-50 base pairs, 20-60 base pairs, 20-70 base pairs, 20-80 base pairs, 20-90 base pairs, 20-100 base pairs, 25-30 base pairs, 25-40 base pairs, 25-50 base pairs, 25-60 base pairs, 25-70 base pairs, 25 The length may range from 80 base pairs, 25 to 90 base pairs, 25 to 100 base pairs, 30 to 50 base pairs, 30 to 60 base pairs, 30 to 70 base pairs, 30 to 80 base pairs, 30 to 90 base pairs, 30 to 100 base pairs, 50 to 70 base pairs, 50 to 80 base pairs, 50 to 90 base pairs, 50 to 100 base pairs, 70 to 100 base pairs, 80 to 100 base pairs, or 90 to 100 base pairs.

[0039] Table 1 provides a list of exemplary sequences for the oligonucleotide strands of the light-emitting labeled oligonucleotide structures. It should be understood that these sequences and other examples described herein are intended to be non-limiting.

[0040] [Table 1]

[0041] In some embodiments, one or more oligonucleotide strands of the light-emitting labeled oligonucleotide structure have a sequence that has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from Tables 1-3. In some embodiments, one or more oligonucleotide strands of the light-emitting labeled oligonucleotide structure have 25-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-99% or more sequence identity to a sequence listed in Tables 1-3. In some embodiments, the oligonucleotide strands comprise one or more nucleotide deletions, additions, or mutations compared to the sequences listed in Tables 1-3. In some embodiments, the oligonucleotide strands comprise deletions, additions, or mutations of 1, 2, 3, 4, 5, 6, 10, 20, 50, or more nucleotides (which may or may not be consecutive nucleotides) compared to the sequences listed in Tables 1-3.

[0042] In some embodiments, the different types of labels are separated by a minimum distance. Without being bound by any particular theory, a minimum distance may advantageously prevent energy transfer between the first type of luminescent label and the second type of luminescent label. In some cases, a minimum distance may advantageously prevent Förster resonance energy transfer (FRET).

[0043] In some embodiments, the minimum distance between any first and any second luminescent label is at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, at least 15 nm, at least 16 nm, at least 17 nm, at least 18 nm, at least 19 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, or at least 50 nm. In some embodiments, the minimum distance between any two luminescent labels is 0.34 *n, where n is the number of nucleotide bases between the luminescent labels. In some cases, the minimum distance between two luminescent labels can be measured as the distance between the geometric centers of the luminescent labels. In some embodiments, the geometric center of a molecule refers to the average position of all atoms in the molecule (e.g., all atoms in the luminescent labels), where the atoms are unweighted. Thus, in some embodiments, the geometric center of a molecule refers to a point in space that is the average of the coordinates of all atoms in the molecule. In some embodiments, the minimum distance d can be obtained (e.g., computationally or otherwise) using theoretical methods known in the art. In some embodiments, theoretical methods can include any approach that takes into account molecular structure, such as bond lengths, bond angles and rotations, electrostatic interactions, nucleic acid helicity, and other physical factors that can describe molecules in solution. In some embodiments, distance measurements can be obtained experimentally, for example, by crystallographic or spectroscopic means.

[0044] In some embodiments, the minimum distance between the attachment sites of luminescent labels to the oligonucleotide strand of the luminescent-labeled oligonucleotide structure may be relatively large.In some cases, the distance between the attachment sites of luminescent labels to the oligonucleotide strand can be represented by the number of intervening unlabeled nucleotides (e.g., intervening bases).It should be understood that the number of nucleotides can refer to either the number of nucleotide bases in a single-stranded nucleic acid or the number of nucleotide base pairs in a double-stranded nucleic acid.In some embodiments, the minimum distance between the attachment site of any first luminescent label and the attachment site of any second luminescent label is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or at least 100 unlabeled nucleotides. In some embodiments, the minimum distance between the attachment site of any first light-emitting label and the attachment site of any second light-emitting label is 5 to 10, 5 to 20, 4 to 30, 5 to 40, 5 to 50, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 100, 20 to 30, 20 to 40, 20 to 50, 20 to 100, 30 to 50, 30 to 100, and 50 to 100 unlabeled nucleotides.

[0045] In some embodiments, one or more of the oligonucleotide strands of the light-emitting labeled oligonucleotide structure comprises a binding moiety. In certain embodiments, the first single-stranded oligonucleotide comprises a binding moiety. In certain embodiments, the first complementary single-stranded oligonucleotide comprises a binding moiety.

[0046] In some embodiments, the binding moiety comprises at least one biotin moiety. In certain embodiments, the at least one biotin moiety comprises a bis-biotin moiety. In some embodiments, the binding group further comprises a tag sequence. In some embodiments, the tag sequence comprises at least one biotin ligase recognition sequence that enables biotinylation of the linker (e.g., incorporation of one or more biotin moieties, including biotin and bis-biotin moieties). In some embodiments, the tag sequence comprises two biotin ligase recognition sequences oriented in tandem. In some embodiments, a biotin ligase recognition sequence refers to an amino acid sequence recognized by a biotin ligase, which catalyzes the covalent bond between the sequence and a biotin molecule. Each biotin ligase recognition sequence of a tag sequence can be covalently linked to a biotin moiety, such that a tag sequence having multiple biotin ligase recognition sequences can be covalently linked to multiple biotin molecules. A region of a tag sequence having one or more biotin ligase recognition sequences can generally be referred to as a biotinylation tag or biotinylation sequence. In some embodiments, bis-biotin or a bis-biotin moiety can refer to two biotins bound to two tandemly oriented biotin ligase recognition sequences. In some embodiments, the linker binding group comprises at least one biotin ligase recognition sequence to which a biotin moiety is attached, or at least two biotin ligase recognition sequences to which a biotin moiety is attached.

[0047] In some embodiments, the binding portion of the light-emitting labeled oligonucleotide structure comprises a binding molecule or is conjugated to a binding molecule. In some embodiments, the first binding molecule comprises a multivalent protein (e.g., a protein having two or more ligand-binding sites that can independently bind to ligands). In some embodiments, the first binding molecule comprises an avidin protein. The term "avidin protein" refers to a biotin-binding protein, generally having a biotin-binding site on each of the four subunits of the avidin protein. Avidin proteins include, for example, avidin, streptavidin, traptavidin, tamavidin, bradavidin, xenavidin, and homologs and variants thereof. In certain embodiments, the avidin protein comprises streptavidin. In certain embodiments, the avidin protein is in the form of a monomer, dimer, or tetramer. In some embodiments, the avidin protein is streptavidin in tetrameric form (eg, homotetramer).

[0048] In some embodiments, the binding moiety comprises a click chemistry handle. As used herein, the term "click chemistry handle" refers to a reactant or reactive group capable of participating in a click chemistry reaction. For example, a strained alkyne, such as cyclooctyne, is a click chemistry handle because it can participate in strain-promoted cycloaddition. Generally, a click chemistry reaction requires at least two molecules containing click chemistry handles that can react with each other. Such a pair of click chemistry handles that are reactive with each other may be referred to herein as partner click chemistry handles. For example, azide is a partner click chemistry handle for cyclooctyne or any other alkyne. In some embodiments, click chemistry handles are used that can react to form a covalent bond in the presence of a metal catalyst, such as copper(II). In some embodiments, click chemistry handles are used that can react to form a covalent bond in the absence of a metal catalyst. Additional suitable click chemistry handles are known to those skilled in the art and include, but are not limited to, the click chemistry reaction partners, groups, and handles described in Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48:4900-4908 and International Application No. PCT / US2012 / 044584, and references therein, which references are incorporated herein by reference for their description of click chemistry handles and methodologies.

[0049] In some embodiments, the first binding molecule can be used to form a covalent or non-covalent bond between the luminescent-labeled oligonucleotide structure and one or more reaction components (e.g., an amino acid recognition molecule, an aminopeptidase, a nucleotide). In certain embodiments, the first binding molecule can be bound to an amino acid recognition molecule. In certain embodiments, the first binding molecule can be bound to an aminopeptidase. In certain embodiments, the first binding molecule can be bound to a nucleotide.

[0050] Multi-oligonucleotide structure Some embodiments relate to luminescently labeled oligonucleotide structures comprising multiple oligonucleotide strands constructed via one or more linking molecules (e.g., via biotin / streptavidin conjugation). For example, some embodiments relate to luminescently labeled oligonucleotide structures comprising: a first single-stranded biotinylated oligonucleotide bound to a first streptavidin; a first complementary single-stranded oligonucleotide hybridized to the first single-stranded biotinylated oligonucleotide; a second single-stranded biotinylated oligonucleotide hybridized to the second single-stranded biotinylated oligonucleotide, wherein the second complementary single-stranded oligonucleotide is biotinylated and bound to the second streptavidin; and at least one luminescent label bound to at least one single-stranded oligonucleotide.

[0051] In some embodiments, two or more oligonucleotide pairs separated by one or more binding molecules (e.g., avidin proteins) have sequences formed using different nucleotide systems. In certain embodiments, a first oligonucleotide pair (e.g., a first single-stranded oligonucleotide and a first complementary single-stranded oligonucleotide) comprises a sequence consisting of four types of nucleotides: A, C, G, and / or T. In some cases, an oligonucleotide comprising a sequence consisting of A, C, G, and / or T may be referred to as a "GCAT-based oligonucleotide." In certain embodiments, a second nucleotide pair (e.g., a second single-stranded oligonucleotide and a second complementary single-stranded oligonucleotide) comprises a sequence consisting of at least six types of nucleotides: A, C, G, T, isoguanine (iG), and isocytosine (iC). In some cases, an oligonucleotide comprising a sequence consisting of at least A, C, G, T, iG, and / or iC may be referred to as a "GCATiGiC-based oligonucleotide." In some cases, the use of two or more nucleotide systems may advantageously facilitate the construction of multiple oligonucleotide structures. In some cases, for example, the use of two or more nucleotide systems can advantageously improve orthogonality and reduce light-labeled single-stranded oligonucleotides that hybridize to the wrong strand.

[0052] In some embodiments, the light-emitting labeled oligonucleotide comprises an adenine and thymine base pair. In some embodiments, the light-emitting labeled oligonucleotide comprises a guanine and cytosine base pair. In some embodiments, the light-emitting labeled oligonucleotide comprises an isoguanine and isocytosine base pair (iG:iC base pair). In some embodiments, the light-emitting labeled oligonucleotide comprises a 2,6-diaminopurine (diaminopurine) and thymine nucleotide base pair.

[0053] In some embodiments, isoguanine has the following structure:

[0054] [ka]

[0055] In some embodiments, the isocytosine has the following structure:

[0056] [ka]

[0057] In some embodiments, the diaminopurine has the following structure:

[0058] [ka]

[0059] In some embodiments, the first single-stranded biotinylated oligonucleotide contains isoguanine and / or isocytosine, and the first complementary single-stranded oligonucleotide contains isocytosine and / or isoguanine, or the second single-stranded biotinylated oligonucleotide contains isoguanine and / or isocytosine, and the second complementary single-stranded oligonucleotide contains isocytosine and / or isoguanine. In some embodiments, the oligonucleotide structure further comprises a dye-labeled nucleoside or amino acid recognition molecule bound to a second streptavidin. In some embodiments, the first complementary single-stranded oligonucleotide is bound to a terminator.

[0060] Certain aspects of the present disclosure relate to methods of constructing a luminescently labeled oligonucleotide structure described herein, comprising contacting a first single-stranded biotinylated oligonucleotide with a first streptavidin, contacting a second single-stranded biotinylated oligonucleotide with the first streptavidin, contacting the first single-stranded biotinylated oligonucleotide with a first complementary single-stranded oligonucleotide, and contacting the second single-stranded biotinylated oligonucleotide with a second complementary single-stranded oligonucleotide. In some embodiments, at least one of the first single-stranded biotinylated oligonucleotide, the first complementary single-stranded oligonucleotide, the second single-stranded biotinylated oligonucleotide, and the second complementary single-stranded oligonucleotide comprises at least one luminescent label. In some embodiments, the first single-stranded biotinylated oligonucleotide contains isoguanine and / or isocytosine, and the first complementary single-stranded oligonucleotide contains isocytosine and / or isoguanine, or the second single-stranded biotinylated oligonucleotide contains isoguanine and / or isocytosine, and the second complementary single-stranded oligonucleotide contains isocytosine and / or isoguanine. In some embodiments, the first complementary single-stranded oligonucleotide is biotinylated. In some embodiments, the first complementary single-stranded oligonucleotide is luminescently labeled. In some embodiments, the second complementary single-stranded oligonucleotide is luminescently labeled. In some embodiments, the method is repeated once or twice.

[0061] In some embodiments, the methods provided herein include constructing a light-labeled oligonucleotide structure comprising multiple light-labeled oligonucleotides. In some embodiments, light-labeled oligonucleotides have a limit on the number of dyes that can be attached to the oligonucleotide. In some embodiments, this limit is due to dye-dye interactions. The present disclosure relates to the discovery that this limit can be overcome by conjugating multiple light-labeled oligonucleotides together, rather than adding additional dyes to the same oligonucleotide. The present disclosure also relates to the discovery that oligonucleotide bending or curvature, i.e., as more oligonucleotides are added to a light-labeled oligonucleotide structure, limits the length of the oligonucleotide structure. The present disclosure relates to the discovery that incorporation of additional nucleotide bases (i.e., isoguanine and isocytosine in addition to adenine, guanine, cytosine, and thymine) facilitates the conjugation of some light-labeled oligonucleotides without limiting the bending or curvature of the oligonucleotide.

[0062] FIG. 3A shows a schematic diagram of an exemplary method for constructing a luminescently-labeled oligonucleotide structure, according to some embodiments. In some embodiments, construction of a luminescently-labeled oligonucleotide structure begins with a first biotinylated, luminescently-labeled oligonucleotide strand 310. In certain embodiments, strand 310 has a sequence consisting of four different types of nucleotide bases. In some embodiments, strand 310 is conjugated to streptavidin 320. In some embodiments, a second biotinylated, single-stranded, luminescently-labeled oligonucleotide 330 is conjugated to streptavidin 320. In some embodiments, strand 330 has a sequence containing six different types of nucleotide bases. In some embodiments, a first complementary, luminescently-labeled oligonucleotide 340 is hybridized to strand 310. Like strand 310, strand 340 has a sequence containing only four different types of nucleotide bases. In some embodiments, a second complementary, luminescently-labeled oligonucleotide 350 is hybridized to strand 330. Like strand 330, strand 350 has a sequence containing six different types of nucleotide bases. In some embodiments, strand 350 is biotinylated. In some embodiments, strand 350 is conjugated to a second streptavidin 360. The fully assembled oligonucleotide structure is shown in Figure 3A.

[0063] In some embodiments, the light-emitting labeled oligonucleotide structures described herein contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight light-emitting labels. In some embodiments, the number of light-emitting labels can be increased by adding additional biotinylated light-emitting labeled oligonucleotides to the light-emitting labeled oligonucleotide structure. In some embodiments, amino acid recognition molecules can be added to the ends of the light-emitting labeled oligonucleotide structures for use in polypeptide sequencing. In some embodiments, nucleotides can be added to the ends of the light-emitting labeled oligonucleotide structures for use in nucleic acid sequencing.

[0064] In some embodiments, the light-emitting labeled oligonucleotide structure can have any suitable length. In some embodiments, the light-emitting labeled oligonucleotide structure has a length of at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 50 base pairs, at least 60 base pairs, at least 70 base pairs, at least 80 base pairs, at least 90 base pairs, or at least 100 base pairs. In some embodiments, the light-emitting labeled oligonucleotide structure has a length of 20-25 base pairs, 20-30 base pairs, 20-40 base pairs, 20-50 base pairs, 20-60 base pairs, 20-70 base pairs, 20-80 base pairs, 20-90 base pairs, 20-100 base pairs, 25-30 base pairs, 25-40 base pairs, 25-50 base pairs, 25-60 base pairs, 25-70 base pairs, 25 The length may range from 80 base pairs, 25 to 90 base pairs, 25 to 100 base pairs, 30 to 50 base pairs, 30 to 60 base pairs, 30 to 70 base pairs, 30 to 80 base pairs, 30 to 90 base pairs, 30 to 100 base pairs, 50 to 70 base pairs, 50 to 80 base pairs, 50 to 90 base pairs, 50 to 100 base pairs, 70 to 100 base pairs, 80 to 100 base pairs, or 90 to 100 base pairs.

[0065] In some embodiments, at least one luminescent label is fluorescent (e.g., comprises a fluorophore). The at least one luminescent label can be any luminescent label described herein. In certain embodiments, the at least one luminescent label comprises Cy®3, Cy®3B, ATRho6G, Chromis 530N, and / or C530NS.

[0066] In some embodiments, any single-stranded oligonucleotide that includes a light-emitting label includes one, two, three, or four light-emitting labels. In some embodiments, the oligonucleotide structure includes at least four light-emitting labels or at least eight light-emitting labels.

[0067] In some embodiments, the light-emitting labeled oligonucleotide structure further comprises a third single-stranded biotinylated oligonucleotide bound to the second streptavidin. In some embodiments, the oligonucleotide structure further comprises a third complementary single-stranded oligonucleotide hybridized to the third single-stranded biotinylated oligonucleotide, wherein the third complementary single-stranded oligonucleotide is biotinylated and bound to the third streptavidin. In some embodiments, the dye-labeled nucleoside or amino acid recognition molecule is bound to the third streptavidin. In some embodiments, the oligonucleotide structure further comprises a fourth single-stranded biotinylated oligonucleotide bound to the third streptavidin. In some embodiments, the oligonucleotide structure further comprises a fourth complementary single-stranded oligonucleotide hybridized to the fourth single-stranded biotinylated oligonucleotide, wherein the fourth complementary single-stranded oligonucleotide is biotinylated and bound to the fourth streptavidin. In some embodiments, the dye-labeled nucleoside or amino acid recognition molecule is conjugated to a fourth streptavidin.

[0068] In some embodiments, the second complementary single-stranded oligonucleotide is bound to the second binding molecule. In some embodiments, the third single-stranded oligonucleotide is bound to the second binding molecule. In some embodiments, the third complementary single-stranded oligonucleotide is hybridized to the third single-stranded oligonucleotide. In certain embodiments, the second binding molecule comprises an avidin protein. In certain cases, the avidin protein comprises streptavidin.

[0069] Aspects of the present disclosure relate to a system including a chip containing a plurality of wells, one or more of which are adapted to receive a peptide and have the peptide bound to its surface; and a dye-labeled nucleoside or amino acid recognition molecule is bound to a light-emitting labeled oligonucleotide as described herein. In some embodiments, the dye-labeled nucleoside or amino acid recognition molecule is configured to bind to a terminal nucleotide of a nucleic acid or a terminal amino acid of a peptide. In some embodiments, the plurality of wells includes 96 wells, 384 wells, 1,536 wells, or more wells. In some embodiments, the peptide is derived from a sample containing multiple peptides. In some embodiments, the peptide is immobilized on the bottom of one of the plurality of wells via a secondary complex. In some embodiments, the secondary complex is a streptavidin-biotin complex.

[0070] Aspects of the present disclosure relate to a method for nucleotide and / or polypeptide sequencing, comprising contacting a single nucleic acid or polypeptide molecule with one or more dye-labeled nucleoside or amino acid recognition molecules attached to a structure described herein, and detecting a series of signal pulses indicating association of the one or more dye-labeled nucleoside or amino acid recognition molecules with sequentially exposed nucleotides or amino acids at the terminals of the single nucleic acid or polypeptide during synthesis or degradation of the single nucleic acid or polypeptide, thereby sequencing the single nucleic acid or polypeptide molecule. In some embodiments, the association of one or more structures with each type of terminally exposed nucleotide or amino acid generates a characteristic pattern of the series of signal pulses that is different from other types of terminally exposed nucleotides or amino acids. In some embodiments, the characteristic pattern comprises a portion of the series of signal pulses. In some embodiments, the signal pulses of the characteristic pattern correspond to individual association events between the dye-labeled nucleoside or amino acid recognition molecules and the terminally exposed nucleotides or amino acids. In some embodiments, the signal pulses of the characteristic pattern comprise a pulse duration that characterizes the dissociation rate of the bond between the dye-labeled nucleoside or amino acid recognition molecule and the terminally exposed nucleotide or amino acid. In some embodiments, each signal pulse of the characteristic pattern is separated from the others by an inter-pulse duration that characterizes the association rate of the dye-labeled nucleoside or amino acid recognition molecule bond. In some embodiments, the characteristic pattern corresponds to a series of reversible dye-labeled nucleoside or amino acid recognition molecule binding interactions with terminally exposed nucleotides or amino acids of a single polypeptide molecule. In some embodiments, the series of reversible dye-labeled nucleoside or amino acid recognition molecule binding interactions comprises the reversible formation of one binary complex species at the terminus of the single polypeptide molecule. In some embodiments, the series of reversible dye-labeled nucleoside or amino acid recognition molecule binding interactions comprises the reversible formation of different binary complex species at the terminus of the single polypeptide molecule.In some embodiments, the characteristic pattern indicates nucleotides or amino acids exposed at the termini of a single polypeptide molecule and nucleotides or amino acids at consecutive positions. In some embodiments, the nucleotides or amino acids exposed at the termini and the nucleotides or amino acids at consecutive positions are of different types. In some embodiments, sequencing involves identifying each type of nucleotide or amino acid sequentially exposed at the termini of a single polypeptide during synthesis or degradation of the single nucleic acid polypeptide. In some embodiments, sequencing involves identifying a portion of all types of nucleotides or amino acids sequentially exposed at the termini of a single polypeptide during synthesis or degradation of the single polypeptide. In some embodiments, sequencing involves determining the relative positions of nucleotides or amino acids sequentially exposed at the termini of a single nucleic acid or polypeptide during synthesis or degradation of the single nucleic acid or polypeptide. In some embodiments, sequencing involves identifying at least two consecutive nucleotides or amino acids in a single nucleic acid or polypeptide molecule. In some embodiments, sequencing involves identifying at least two non-consecutive nucleotides or amino acids in a single nucleic acid or polypeptide molecule.

[0071] Ligated oligonucleotide structures Some embodiments relate to luminescently labeled oligonucleotide structures comprising multiple oligonucleotide strands assembled by ligation, and methods for preparing the same. For example, some embodiments relate to methods for preparing luminescently labeled reaction components by ligating the termini of one double-stranded oligonucleotide to the termini of another double-stranded oligonucleotide, wherein each double-stranded oligonucleotide comprises one or more luminescent labels as described herein.

[0072] 3B shows a schematic diagram of an exemplary method for constructing a luminescently labeled oligonucleotide structure by ligation, according to some embodiments. In some embodiments, a first double-stranded oligonucleotide 370 is provided, and one or both strands of the first double-stranded oligonucleotide 370 comprise one or more luminescent labels. In some embodiments, one strand of the first double-stranded oligonucleotide 370 comprises a first binding moiety (e.g., a first biotin moiety, such as a first bis-biotin moiety). In some embodiments, a second double-stranded oligonucleotide 380 is provided, and one or both strands of the second double-stranded oligonucleotide 380 comprise one or more luminescent labels.

[0073] In some embodiments, the first double-stranded oligonucleotide 370 and / or the second double-stranded oligonucleotide 380 comprise a structure according to the luminescently labeled oligonucleotide or multi-oligonucleotide structure described herein. For example, in some embodiments, the one or more luminescent labels of the first and / or second double-stranded oligonucleotide are separated from each other by a distance of at least 10 nm. In some embodiments, the first double-stranded oligonucleotide comprises one or more isoguanine and / or isocytosine nucleotides, and the second double-stranded oligonucleotide does not comprise one or more isoguanine and / or isocytosine nucleotides. In some embodiments, the second double-stranded oligonucleotide comprises one or more isoguanine and / or isocytosine nucleotides, and the first double-stranded oligonucleotide does not comprise one or more isoguanine and / or isocytosine nucleotides. In some embodiments, the first or second double-stranded oligonucleotide comprises at least one diaminopurine nucleotide.

[0074] In some embodiments, one or more oligonucleotide strands of first double-stranded oligonucleotide 370 and / or second double-stranded oligonucleotide 380 have a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from Tables 1-3. In some embodiments, one or more oligonucleotide strands of first double-stranded oligonucleotide 370 and / or second double-stranded oligonucleotide 380 have 25-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-99% or more sequence identity to a sequence listed in Tables 1-3. In some embodiments, an oligonucleotide strand contains one or more nucleotide deletions, additions, or mutations compared to a sequence set forth in Tables 1-3. In some embodiments, the oligonucleotide strand contains deletions, additions, or mutations of 1, 2, 3, 4, 5, 6, 10, 20, 50, or more nucleotides (which may or may not be consecutive nucleotides) compared to the sequences set forth in Tables 1-3.

[0075] In some embodiments, the first double-stranded oligonucleotide 370 and the second double-stranded oligonucleotide 380 comprise complementary overhangs suitable for overhang ligation. For example, as shown schematically in FIG. 3B, each double-stranded oligonucleotide can comprise a double-stranded portion (e.g., a duplex portion) and a single-stranded portion (e.g., an unpaired portion), with the single-stranded portion forming the overhang. In some embodiments, the overhang is a 5' overhang formed by the 5' portion of one strand in each double-stranded oligonucleotide. In some embodiments, the overhang is a 3' overhang formed by the 3' portion of one strand in each double-stranded oligonucleotide. In some embodiments, the overhang comprises a phosphate (e.g., a monophosphate). In some embodiments, the overhang is a 5' overhang comprising a 5'-monophosphate. In some embodiments, the first double-stranded oligonucleotide 370 comprises a first overhang and the second double-stranded oligonucleotide 380 comprises a second overhang that is complementary to the first overhang.

[0076] In some embodiments, a first double-stranded oligonucleotide 370 including a first overhang is contacted with a second double-stranded oligonucleotide 380 including a second overhang under hybridization conditions. In some embodiments, the hybridization conditions are sufficient to allow the first overhang of the first double-stranded oligonucleotide to hybridize to the second overhang of the second double-stranded oligonucleotide. In some embodiments, the second overhang is perfectly complementary to the first overhang. However, perfect complementarity is not a requirement; in some embodiments, the second overhang is partially complementary to the first overhang, so long as the complementarity is sufficient for the first and second overhangs to hybridize under hybridization conditions.

[0077] In some embodiments, assembly of the light-emitting labeled oligonucleotide structure proceeds by ligating a first double-stranded oligonucleotide 370 to a second double-stranded oligonucleotide 380. In some embodiments, ligation comprises enzymatic ligation. For example, in some embodiments, ligation comprises contacting the first and second double-stranded oligonucleotides with a ligase under ligation conditions. In some embodiments, the ligase is a DNA ligase (e.g., T4 DNA ligase). In some embodiments, ligation comprises ligating both strands of the first double-stranded oligonucleotide 370 to both strands of the second double-stranded oligonucleotide 380. In some embodiments, a first overhang comprises a 5'-phosphate ligated to the 3'-hydroxyl of one strand of the second double-stranded oligonucleotide 380, and a second overhang comprises a 5'-phosphate ligated to the 3'-hydroxyl of one strand of the first double-stranded oligonucleotide 370.

[0078] In some embodiments, assembly of the light-labeled oligonucleotide structure proceeds by contacting the ligated first and second double-stranded oligonucleotides with a multivalent protein 374 that binds to first binding moiety 372 to form a complex comprising the ligated double-stranded oligonucleotide and the multivalent protein. In some embodiments, multivalent protein 374 comprises an avidin protein (e.g., streptavidin) and first binding moiety 372 comprises a biotin moiety as described herein.

[0079] In some embodiments, assembly of the luminescently labeled oligonucleotide structure proceeds by contacting the complex with a reaction component 390 (e.g., an amino acid recognition molecule, a nucleotide) that includes a second binding moiety 376, and the multivalent protein 374 binds to the second binding moiety to form the luminescently labeled reaction component.

[0080] Set of luminous signs Some aspects relate to a set of luminescent labels comprising a plurality of luminescent labels. In some embodiments, each luminescent label in the set of luminescent labels has a different value for one or more luminescent properties. In some cases, a set of luminescent labels can be advantageously used to label a set of reaction components to ensure that each type of reaction component (e.g., amino acid recognition molecule) can be identified during protein sequencing and / or nucleic acid sequencing. In some embodiments, the set of luminescent labels can comprise one or more luminescently labeled oligonucleotide structures described herein. In some embodiments, the set of luminescent labels can comprise one or more fluorophores known in the art (e.g., Cy®3, Cy®3B, ATTO Rho6G).

[0081] Non-limiting examples of luminescence properties include luminescence lifetime, luminescence intensity, bin ratio, and luminescence wavelength. In certain embodiments, each luminescent label has a value of the luminescence property that is different from the value of the luminescence property of each other luminescent label in the set of luminescent labels. In certain embodiments, the minimum percentage difference between the luminescence property values ​​of any two luminescent labels in the set of luminescent labels is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, or at least 500%. In certain embodiments, the minimum percentage difference between the luminescence characteristic values ​​for any two luminescent labels of the set of luminescent labels is 1-5%, 1-10%, 1-20%, 1-30%, 1-50%, 1-100%, 1-150%, 1-200%, 1-500%, 5-10%, 5-20%, 5-30%, 5-50%, 5-100%, 5-150%, 5-200%, 5-500% , 10-20%, 10-30%, 10-50%, 10-100%, 10-150%, 10-200%, 10-500%, 20-50%, 20-100%, 20-150%, 20-200%, 20-500%, 50-100%, 50-150%, 50-200%, 50-500%, 100-200%, 100-500%, or 200-500%.

[0082] The set of luminescent labels can have any suitable number of luminescent labels. In certain embodiments, the set of luminescent labels includes two or more luminescent labels, three or more luminescent labels, four or more luminescent labels, five or more luminescent labels, six or more luminescent labels, seven or more luminescent labels, eight or more luminescent labels, nine or more luminescent labels, or ten or more luminescent labels. In some embodiments, the set of luminescent labels includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more luminescent labels.

[0083] In some embodiments, the luminescence characteristic includes a bin ratio. In certain cases, the bin ratio can be a measure of luminescence lifetime. In some cases, the bin ratio of a luminescence label can be obtained using an integrated device described herein. In some embodiments, the bin ratio of a luminescence label can refer to the ratio of photoelectrons collected during a first time period (bin 0) to photoelectrons collected during a second time period (bin 1). In certain embodiments, the first time period can begin a relatively long time after the excitation pulse (e.g., 3 ns after the excitation pulse). In certain embodiments, the second time period can begin a relatively short time after the excitation pulse (e.g., 1 ns after the excitation pulse). In some cases, a relatively low bin ratio can indicate that the dye has a relatively short luminescence lifetime. In some cases, a relatively high bin ratio can indicate that the dye has a relatively long luminescence lifetime.

[0084] In some embodiments, each luminescent label of a set of luminescent labels can have a distinct bin ratio value. In certain embodiments, the minimum difference between the bin ratio values ​​of a set of luminescent labels is at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1.0. In certain embodiments, the minimum difference between the bin ratio values ​​of a set of luminescent labels is 0.05-0.2, 0.05-0.3, 0.05-0.4, 0.05-0.5, 0.05-0.6, 0.05-0.7, 0.05-0.8, 0.05-0.9, 0.05-1.0, 0.1-0.2, 0.1-0.3, 0.1-0.4, 0.1- In certain embodiments, the minimum percentage difference between bin ratio values ​​of a set of luminescent labels is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 100%, at least 150%, at least 200%, or at least 500%. In certain embodiments, the minimum percentage difference between the bin ratio values ​​of a set of luminescent labels is 1-5%, 1-10%, 1-20%, 1-30%, 1-50%, 1-100%, 1-150%, 1-200%, 1-500%, 5-10%, 5-20%, 5-30%, 5-50%, 5-100%, 5-150%, 5-200%, 5-500%, 10-20%, 1 The ranges are 0-30%, 10-50%, 10-100%, 10-150%, 10-200%, 10-500%, 20-50%, 20-100%, 20-150%, 20-200%, 20-500%, 50-100%, 50-150%, 50-200%, 50-500%, 100-200%, 100-500%, or 200-500%.

[0085] In some embodiments, each light-emitting label in the set of light-emitting labels has a unique combination of two or more different light-emitting characteristics. In some embodiments, the system includes a first light-emitting label having a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic. In some embodiments, the system includes a second light-emitting label having a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic. In some embodiments, the system includes a third light-emitting label having a third ordered pair of characteristics including a third value of the first characteristic and a third value of the second characteristic. In certain embodiments, the first ordered pair, the second ordered pair, and the third ordered pair differ from each other in at least one of the values ​​of the first characteristic and / or the second characteristic. In certain embodiments, the first ordered pair, the second ordered pair, and the third ordered pair are separated by a minimum distance.

[0086] In some embodiments, the method includes providing a first light-emitting label having a first ordered pair of properties comprising a first value of the first property and a first value of the second property. In some embodiments, the method includes providing a second light-emitting label having a second ordered pair of properties comprising a second value of the first property and a second value of the second property. In some embodiments, the method includes providing a third light-emitting label comprising a light-labeled oligonucleotide structure comprising a first single-stranded oligonucleotide comprising one or more first fluorophores and a first complementary single-stranded oligonucleotide comprising one or more second fluorophores, the third light-emitting label having a third ordered pair of properties comprising a third value of the first property and a third value of the second property. In some embodiments, the method includes modifying the number and / or type of the one or more first fluorophores and / or one or more second fluorophores such that the first ordered pair, the second ordered pair, and the third ordered pair differ from each other in at least one of the values ​​of the first property and / or the second property.

[0087] In some cases, the set of luminescent labels includes multiple luminescent labels, each occupying a distinct spatial region (e.g., a different position) on the two-dimensional plot of the two luminescent properties. In particular cases, the two-dimensional plot is an intensity versus bin ratio plot. Non-limiting examples of intensity versus bin ratio plots are shown in Figures 6B, 7B, and 8B. In some embodiments, a pair of ordered properties associated with a luminescent label represents the centroid of a cluster of points associated with that luminescent label on the two-dimensional plot of the two luminescent properties.

[0088] In some embodiments, the set of luminescent labels includes one or more, two or more, three or more, four or more, or five or more of: a first luminescent label including R1C1, a second luminescent label including C2C, a third luminescent label including SG4Cy3, a fourth luminescent label including one or more copies of ATRho6G, and a fifth luminescent label including one or more copies of Cy3B.

[0089] Polypeptide sequencing As described herein, in some embodiments, the present disclosure provides compositions and methods for polypeptide sequencing. Figure 4 shows a schematic diagram of an exemplary dynamic peptide sequencing reaction in which individual on-off binding events generate signal pulses of a signal output. As shown on the left, a polypeptide sample can be fragmented into peptides, which are immobilized in sample wells of an array. The immobilized peptides are then exposed to one or more amino acid recognition molecules (also referred to as recognition factors) and one or more cleavage reagents (e.g., aminopeptidases). As shown on the right, the amino acid recognition molecules reversibly bind to the termini of the peptides, and a detectable signal is generated while the recognition molecules bind to the peptides. Because on-off binding of the recognition molecules generally occurs at a faster rate than amino acid cleavage, binding events preceding amino acid cleavage generate a series of signal pulses that can be used to determine at least one chemical property of the peptide (and / or the polypeptide from which it originates). In certain embodiments, determining at least one chemical property of the peptide includes detecting the presence or absence of a target residue. In certain embodiments, determining at least one chemical property of the peptide comprises determining the position of a target residue in the peptide (and / or the polypeptide from which it originates). In certain embodiments, determining at least one chemical property of the peptide comprises determining whether one or more amino acids comprise a post-translational modification. In certain embodiments, determining at least one chemical property of the peptide comprises identifying one or more amino acids of the peptide.

[0090] Methods, reagents, and compositions for performing dynamic sequencing are described in more detail in International Application No. PCT / US2019 / 061831, filed November 15, 2019, International Application No. PCT / US2021 / 033493, filed May 20, 2021, a U.S. application entitled "Polypeptidyl Linkers," filed on the same day as the present application, and a U.S. application entitled "Polypeptide Cleaving Reagents and Uses Thereof," filed on the same day as the present application (each of which is incorporated by reference in its entirety herein).

[0091] Thus, in some embodiments, polypeptide sequencing is performed by detecting a series of signal pulses that indicate the association of one or more amino acid recognition factors with consecutive amino acids exposed at the ends of the polypeptide in an ongoing degradation reaction. The series of signal pulses can be analyzed to determine a characteristic pattern in the series of signal pulses, and the time course of the characteristic pattern can be used to determine the amino acid sequence of the polypeptide.

[0092] As described herein, signal pulse information can be used to identify amino acids based on a characteristic pattern in a series of signal pulses. In some embodiments, the characteristic pattern includes a plurality of signal pulses, each signal pulse including a pulse duration. In some embodiments, the plurality of signal pulses can be characterized by summary statistics (e.g., mean, median, time decay constant) of the distribution of pulse durations in the characteristic pattern. In some embodiments, the average pulse duration of the characteristic pattern is between about 1 millisecond and about 10 seconds (e.g., between about 1 millisecond and about 1 second, between about 1 millisecond and about 100 milliseconds, between about 1 millisecond and about 10 milliseconds, between about 1 millisecond and about 10 milliseconds, between about 10 milliseconds and about 10 seconds, between about 100 milliseconds and about 10 seconds, between about 1 second and about 10 seconds, between about 10 milliseconds and about 100 milliseconds, or between about 100 milliseconds and about 500 milliseconds). In some embodiments, the average pulse duration is between about 50 milliseconds and about 2 seconds, between about 50 milliseconds and about 500 milliseconds, or between about 500 milliseconds and about 2 seconds.

[0093] In some embodiments, different characteristic patterns corresponding to different types of amino acids in a single polypeptide can be distinguished from one another based on statistically significant differences in summary statistics. For example, in some embodiments, one characteristic pattern can be distinguished from another based on a difference in mean pulse duration of at least 10 milliseconds (e.g., about 10 ms to about 10 s, about 10 ms to about 1 s, about 10 ms to about 100 ms, about 100 ms to about 10 s, about 1 s to about 10 s, or about 100 ms to about 1 s). In some embodiments, the difference in mean pulse duration is at least 50 ms, at least 100 ms, at least 250 ms, at least 500 ms, or more. In some embodiments, the difference in mean pulse duration is about 50 ms to about 1 s, about 50 ms to about 500 ms, about 50 ms to about 250 ms, about 100 ms to about 500 ms, about 250 ms to about 500 ms, or about 500 ms to about 1 s. In some embodiments, the mean pulse duration of one characteristic pattern differs from the mean pulse duration of another characteristic pattern by about 10-25%, 25-50%, 50-75%, 75-100%, or more than 100%, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, or more. It should be understood that in some embodiments, smaller differences in mean pulse duration between different characteristic patterns may require more pulse durations within each characteristic pattern to be distinguished from one another with statistical reliability.

[0094] In some embodiments, a characteristic pattern generally refers to a plurality of association events between amino acids of a polypeptide and a means for binding amino acids (e.g., amino acid recognition molecules). In some embodiments, a characteristic pattern comprises at least 10 association events (e.g., at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1,000, or more association events). In some embodiments, a characteristic pattern comprises about 10 to about 1,000 association events (e.g., about 10 to about 500 association events, about 10 to about 250 association events, about 10 to about 100 association events, or about 50 to about 500 association events). In some embodiments, a plurality of association events is detected as a plurality of signal pulses.

[0095] In some embodiments, a characteristic pattern refers to a plurality of signal pulses that can be characterized by summary statistics described herein. In some embodiments, a characteristic pattern includes at least 10 signal pulses (e.g., at least 25, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1,000, or more signal pulses). In some embodiments, a characteristic pattern includes about 10 to about 1,000 signal pulses (e.g., about 10 to about 500 signal pulses, about 10 to about 250 signal pulses, about 10 to about 100 signal pulses, or about 50 to about 500 signal pulses).

[0096] In some embodiments, a characteristic pattern refers to multiple association events between an amino acid recognition molecule and amino acids of a polypeptide that occur over a time interval prior to the removal of an amino acid (e.g., a cleavage event). In some embodiments, a characteristic pattern refers to multiple association events that occur over a time interval between two cleavage events (e.g., before the removal of an amino acid and after the removal of a terminally previously exposed amino acid). In some embodiments, the time interval for a characteristic pattern is about 1 minute to about 30 minutes (e.g., about 1 minute to about 20 minutes, about 1 minute to about 10 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, or about 5 minutes to about 10 minutes).

[0097] In some embodiments, polypeptide sequencing reaction conditions can be configured to achieve a time interval that allows for sufficient related events to provide a desired level of confidence in a characteristic pattern. This can be achieved by configuring reaction conditions based on various characteristics, including, for example, reagent concentration, the molar ratio of one reagent to another (e.g., the ratio of amino acid recognition molecules to cleavage reagents, the ratio of one recognition molecule to another recognition molecule, the ratio of one cleavage reagent to another cleavage reagent), the number of different reagent types (e.g., the number of different types of recognition molecules and / or cleavage reagents, the number of recognition molecule types relative to the number of cleavage reagent types), cleavage activity (e.g., peptidase activity), binding characteristics (e.g., kinetic and / or thermodynamic binding parameters for recognition molecule binding), reagent modifications (e.g., polyols and other protein modifications that can alter interaction kinetics), reaction mixture components (e.g., one or more components such as pH, buffers, salts, divalent cations, surfactants, and other reaction mixture components described herein), reaction temperature, and various other parameters that will be apparent to those skilled in the art, as well as combinations thereof. Reaction conditions can be configured based on one or more embodiments described herein, including, for example, signal pulse information (e.g., pulse duration, inter-pulse duration, magnitude change), labeling strategy (e.g., number and / or type of fluorophores, linkers with or without shielding elements), surface modification (e.g., modification of sample well surface, including polypeptide immobilization), sample preparation (e.g., polypeptide fragment size, polypeptide modification for immobilization), and other embodiments described herein.

[0098] In some embodiments, polypeptide sequencing reactions according to the present disclosure are carried out under conditions that allow amino acid recognition and cleavage to occur simultaneously in a single reaction mixture. For example, in some embodiments, polypeptide sequencing reactions are carried out in a reaction mixture having a pH that allows association and cleavage events to occur. In some embodiments, polypeptide sequencing reactions are carried out in a reaction mixture at a pH of about 6.5 to about 9.0. In some embodiments, polypeptide sequencing reactions are carried out in a reaction mixture at a pH of about 7.0 to about 8.5 (e.g., about 7.0 to about 8.0, about 7.5 to about 8.5, about 7.5 to about 8.0, or about 8.0 to about 8.5).

[0099] In some embodiments, the polypeptide sequencing reaction is carried out in a reaction mixture comprising one or more buffers. In some embodiments, the reaction mixture comprises a buffer at a concentration of at least 10 mM (e.g., at least 20 mM and up to 250 mM, at least 50 mM, 10-250 mM, 10-100 mM, 20-100 mM, 50-100 mM, or 100-200 mM). In some embodiments, the reaction mixture comprises a buffer at a concentration of about 10 mM to about 50 mM (e.g., about 10 mM to about 25 mM, about 25 mM to about 50 mM, or about 20 mM to about 40 mM). Examples of buffering agents include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris (tris(hydroxymethyl)aminomethane), and MOPS (3-(N-morpholino)propanesulfonic acid).

[0100] In some embodiments, the polypeptide sequencing reaction is carried out in a reaction mixture comprising a salt concentration of at least 10 mM. In some embodiments, the reaction mixture comprises a salt concentration of at least 10 mM (e.g., at least 20 mM, at least 50 mM, at least 100 mM, or more). In some embodiments, the reaction mixture comprises a salt concentration of about 10 mM to about 250 mM (e.g., about 20 mM to about 200 mM, about 50 mM to about 150 mM, about 10 mM to about 50 mM, or about 10 mM to about 100 mM). Examples of salts include, but are not limited to, sodium salts, potassium salts, and acetate salts, such as sodium chloride (NaCl), sodium acetate (NaOAc), and potassium acetate (KOAc).

[0101] Further examples of components for use in the reaction mixture include divalent cations (e.g., Mg 2+ , Co 2+ ) and surfactants (e.g., polysorbate 20). In some embodiments, the reaction mixture contains a divalent cation at a concentration of about 0.1 mM to about 50 mM (e.g., about 10 mM to about 50 mM, about 0.1 mM to about 10 mM, or about 1 mM to about 20 mM). In some embodiments, the reaction mixture contains a surfactant at a concentration of at least 0.01% (e.g., about 0.01% to about 0.10%). In some embodiments, the reaction mixture contains one or more components useful in single-molecule analysis, such as an oxygen scavenger system (e.g., a PCA / PCD system or a pyranose oxidase / catalase / glucose system) and / or one or more triplet-state quenchers (e.g., Trolox®, COT, and NBA).

[0102] In some embodiments, the polypeptide sequencing reaction is performed at a temperature at which association and cleavage events can occur. In some embodiments, the polypeptide sequencing reaction is performed at a temperature of at least 10°C. In some embodiments, the polypeptide sequencing reaction is performed at a temperature of about 10°C to about 50°C (e.g., 15-45°C, 20-40°C, 25°C or near 25°C, 30°C or near 30°C, 35°C or near 35°C, 37°C or near 37°C). In some embodiments, the polypeptide sequencing reaction is performed at or near room temperature.

[0103] In some embodiments, polypeptide sequencing according to the present disclosure can be performed by contacting the polypeptide with a sequencing reaction mixture comprising one or more amino acid recognition molecules and / or one or more cleavage reagents (e.g., peptidases). In some embodiments, the sequencing reaction mixture comprises the amino acid recognition molecules at a concentration of about 10 nM to about 10 μM. In some embodiments, the sequencing reaction mixture comprises the cleavage reagent at a concentration of about 500 nM to about 500 μM.

[0104] In some embodiments, the sequencing reaction mixture comprises the amino acid recognition molecule at a concentration of about 100 nM to about 10 μM, about 250 nM to about 10 μM, about 100 nM to about 1 μM, about 250 nM to about 1 μM, about 250 nM to about 750 nM, or about 500 nM to about 1 μM. In some embodiments, the sequencing reaction mixture comprises the amino acid recognition molecule at a concentration of about 100 nM, about 250 nM, about 500 nM, about 750 nM, or about 1 μM.

[0105] In some embodiments, the sequencing reaction mixture comprises a cleavage reagent at a concentration of about 500 nM to about 250 μM, about 500 nM to about 100 μM, about 1 μM to about 100 μM, about 500 nM to about 50 μM, about 1 μM to about 100 μM, about 10 μM to about 200 μM, or about 10 μM to about 100 μM. In some embodiments, the sequencing reaction mixture comprises a cleavage reagent at a concentration of about 1 μM, about 5 μM, about 10 μM, about 30 μM, about 50 μM, about 70 μM, or about 100 μM.

[0106] In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 10 nM to about 10 μM and a cleavage reagent at a concentration of about 500 nM to about 500 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 100 nM to about 1 μM and a cleavage reagent at a concentration of about 1 μM to about 100 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 250 nM to about 1 μM and a cleavage reagent at a concentration of about 10 μM to about 100 μM. In some embodiments, the sequencing reaction mixture comprises an amino acid recognition molecule at a concentration of about 500 nM and a cleavage reagent at a concentration of about 25 μM to about 75 μM. In some embodiments, the concentrations of the amino acid recognition molecule and / or the cleavage reagent in the reaction mixture are as described elsewhere herein.

[0107] In some embodiments, the sequencing reaction mixture comprises the amino acid recognition molecule and the cleavage reagent at a molar ratio of about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 75:1, about 50:1, about 25:1, about 10:1, about 5:1, about 2:1, or about 1:1. In some embodiments, the sequencing reaction mixture comprises the amino acid recognition molecule and the cleavage reagent at a molar ratio of about 10:1 to about 200:1. In some embodiments, the sequencing reaction mixture comprises the amino acid recognition molecule and the cleavage reagent at a molar ratio of about 50:1 to about 150:1. In some embodiments, the molar ratio of amino acid recognition molecule to cleavage reagent in the reaction mixture is about 1:1,000 to about 1:1 or about 1:1 to about 100:1 (e.g., 1:1,000, about 1:500, about 1:200, about 1:100, about 1:10, about 1:5, about 1:2, about 1:1, about 5:1, about 10:1, about 50:1, about 100:1). In some embodiments, the molar ratio of amino acid recognition molecule to cleavage reagent in the reaction mixture is about 1:100 to about 1:1 or about 1:1 to about 10:1. In some embodiments, the molar ratio of amino acid recognition molecule to cleavage reagent in the reaction mixture is as described elsewhere herein.

[0108] In some embodiments, the sequencing reaction mixture comprises one or more amino acid recognition molecules and one or more cleavage reagents. In some embodiments, the sequencing reaction mixture comprises at least three amino acid recognition molecules and at least one cleavage reagent. In some embodiments, the sequencing reaction mixture comprises two or more cleavage reagents. In some embodiments, the sequencing reaction mixture comprises at least one and up to ten cleavage reagents (e.g., 1-3 cleavage reagents, 2-10 cleavage reagents, 1-5 cleavage reagents, 3-10 cleavage reagents). In some embodiments, the sequencing reaction mixture comprises at least three and up to 30 amino acid recognition molecules (e.g., 3-25, 3-20, 3-10, 3-5, 5-30, 5-20, 5-10, or 10-20 amino acid recognition molecules).

[0109] In some embodiments, a sequencing reaction mixture comprises two or more amino acid recognition molecules and / or two or more cleavage reagents. In some embodiments, a sequencing reaction mixture described as comprising two or more amino acid recognition molecules (or cleavage reagents) refers to a mixture having two or more types of amino acid recognition molecules (or cleavage reagents). For example, in some embodiments, a sequencing reaction mixture comprises two or more amino acid binding proteins. In some embodiments, two or more amino acid binding proteins refer to two or more types of amino acid binding proteins. In some embodiments, one type of amino acid binding protein has an amino acid sequence that differs from that of another type of amino acid binding protein in the reaction mixture. In some embodiments, one type of amino acid binding protein has a label that differs from the label of another type of amino acid binding protein in the reaction mixture. In some embodiments, one type of amino acid binding protein associates with (e.g., binds to) amino acids that differ from the amino acids associated with another type of amino acid binding protein in the reaction mixture. In some embodiments, one type of amino acid binding protein associates with (e.g., binds to) a subset of amino acids that differs from the subset of amino acids associated with another type of amino acid binding protein in the reaction mixture.

[0110] Amino acid recognition molecules In some embodiments, the methods provided herein include contacting a polypeptide with an amino acid recognition molecule (which may or may not contain a label) that selectively binds to at least one type of terminal amino acid. As used herein, in some embodiments, terminal amino acid may refer to the amino-terminal amino acid of a polypeptide or the carboxy-terminal amino acid of a polypeptide. In some embodiments, the labeled recognition molecule preferentially binds to one type of terminal amino acid over another type of terminal amino acid. In some embodiments, the labeled recognition molecule preferentially binds to one type of terminal amino acid over internal amino acids of the same type. In still other embodiments, the labeled recognition molecule preferentially binds to one type of amino acid present at any position in the polypeptide, for example, to amino acids of the same type as terminal amino acids and internal amino acids.

[0111] As used herein, in some embodiments, a type of amino acid refers to one of the 20 naturally occurring amino acids or a subset of those types. In some embodiments, a type of amino acid refers to a modified variant of one of the 20 naturally occurring amino acids, or a subset of unmodified and / or modified variants thereof. Examples of modified amino acid variants include, but are not limited to, post-translationally modified variants (e.g., acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked glycosylation, hydroxylation, methylation, myristoylation, NEDDylation, nitration, oxidation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, sumoylation, and ubiquitination), chemically modified variants, unnatural amino acids, and proteinogenic amino acids such as selenocysteine ​​and pyrrolysine. In some embodiments, the subset of amino acid types includes two or more but fewer than 20 amino acids with one or more similar biochemical properties. For example, in some embodiments, the type of amino acid refers to one type selected from amino acids with charged side chains (e.g., positively and / or negatively charged side chains), amino acids with polar side chains (e.g., polar uncharged side chains), amino acids with nonpolar side chains (e.g., nonpolar aliphatic and / or aromatic side chains), and amino acids with hydrophobic side chains.

[0112] In some embodiments, the method provided herein comprises contacting a polypeptide with one or more labeled recognition molecules that selectively bind to one or more types of terminal amino acids.As an illustrative and non-limiting example, when four labeled recognition molecules are used in the method of the present disclosure, any one recognition molecule selectively binds to one type of terminal amino acid that is different from the other type of amino acid that any of the other three selectively binds to (for example, the first recognition molecule binds to the first type of terminal amino acid, the second recognition molecule binds to the second type of terminal amino acid, the third recognition molecule binds to the third type of terminal amino acid, and the fourth recognition molecule binds to the fourth type of terminal amino acid).For the purposes of this discussion, one or more labeled recognition molecules in the context of the method described herein can alternatively be referred to as a set of labeled recognition molecules.

[0113] In some embodiments, the set of labeled recognition molecules includes at least one and up to six labeled recognition molecules. For example, in some embodiments, the set of labeled recognition molecules includes 1, 2, 3, 4, 5, or 6 labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 10 or fewer labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 8 or fewer labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 6 or fewer labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 4 or fewer labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 3 or fewer labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 2 or fewer labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes 4 labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes at least 2 and up to 20 (e.g., at least 2 and up to 10, at least 2 and up to 8, at least 4 and up to 20, at least 4 and up to 10) labeled recognition molecules. In some embodiments, the set of labeled recognition molecules includes more than 20 (e.g., 20-25, 20-30) recognition molecules. However, it should be understood that any number of recognition molecules can be used in accordance with the methods of the present disclosure to accommodate the desired application.

[0114] According to the present disclosure, in some embodiments, one or more types of amino acids are identified by detecting the emission of a labeled recognition molecule. In some embodiments, the labeled recognition molecule includes a recognition molecule that selectively binds to one type of amino acid and a luminescent label having an emission associated with the recognition molecule. In this manner, the emission (e.g., emission lifetime, emission intensity, and other emission characteristics described elsewhere herein) can be correlated with the selective binding of the recognition molecule to identify the amino acid of the polypeptide. In some embodiments, multiple types of labeled recognition molecules can be used in the methods according to the present disclosure, each type including a luminescent label with an emission that is uniquely distinguishable from the plurality. In some embodiments, the luminescent label of each type of labeled recognition molecule is uniquely distinguishable from the plurality by emission intensity alone. Suitable luminescent labels can include luminescent molecules such as fluorophore dyes, as described elsewhere herein.

[0115] In some embodiments, amino acid recognition molecules can be engineered by those skilled in the art using conventional, well-known techniques. In some embodiments, desirable properties include the ability to selectively and with high affinity bind to one type of amino acid only when that amino acid is located at a terminal (e.g., N- or C-terminus) of a polypeptide. In yet other embodiments, desirable properties include the ability to selectively and with high affinity bind to one type of amino acid when that amino acid is located at a terminal (e.g., N- or C-terminus) of a polypeptide and when that amino acid is located at an internal position of the polypeptide. In some embodiments, desirable properties include selective and low affinity (e.g., K of about 50 nM or more, e.g., about 50 nM to about 50 μM, about 100 nM to about 10 μM, about 500 nM to about 50 μM) binding to two or more types of amino acids. DFor example, in some embodiments, the present disclosure provides methods for sequencing by detecting reversible binding interactions during the polypeptide degradation process. Advantageously, such methods can be performed using recognition molecules that reversibly bind with low affinity to two or more types of amino acids (e.g., a subset of amino acid types).

[0116] As used herein, in some embodiments, the terms "selective" and "specific" (and variations thereof, e.g., selectively, specifically, selectivity, specificity) refer to a preferential binding interaction. For example, in some embodiments, an amino acid recognition molecule that selectively binds to one type of amino acid binds to one type of amino acid preferentially over another type of amino acid. A selective binding interaction distinguishes one type of amino acid (e.g., one type of terminal amino acid) from another type of amino acid (e.g., another type of terminal amino acid) by typically about 10-fold to 100-fold or more (e.g., about 1,000-fold or more than 10,000-fold). Thus, it should be understood that a selective binding interaction can refer to any binding interaction that is uniquely distinguishable for one type of amino acid over another type of amino acid. For example, in some aspects, the present disclosure provides a method for polypeptide sequencing by obtaining data indicative of the association of one or more amino acid recognition molecules with a polypeptide molecule. In some embodiments, the data includes a series of signal pulses corresponding to a series of reversible amino acid recognition molecule binding interactions with amino acids of the polypeptide molecule, and the data can be used to identify the amino acids. Thus, in some embodiments, a "selective" or "specific" binding interaction refers to a detected binding interaction that distinguishes between one type of amino acid and another type of amino acid.

[0117] In some embodiments, the amino acid recognition molecule has a dissociation constant (K D ) is about 10 -6 Less than M (e.g., about 10 -7 Less than M, about 10 -8Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Under M to 10 -16 In some embodiments, the amino acid recognition molecule binds to one type of amino acid (e.g., one type of terminal amino acid) with a K of less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM. D In some embodiments, the amino acid recognition molecule binds to one type of amino acid with a K of about 50 nM to about 50 μM (e.g., about 50 nM to about 500 nM, about 50 nM to about 5 μM, about 500 nM to about 50 μM, about 5 μM to about 50 μM, or about 10 μM to about 50 μM). D In some embodiments, the amino acid recognition molecule binds to one type of amino acid with a K of about 50 nM. D Combine with.

[0118] In some embodiments, the amino acid recognition molecule is a molecule that recognizes two or more types of amino acids, with a total length of about 10 -6 Less than M (e.g., about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Under M to 10 -16 (as low as M) D In some embodiments, the amino acid recognition molecule binds to two or more types of amino acids with a K of less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM. D In some embodiments, the amino acid recognition molecule binds to two or more types of amino acids with a K of between about 50 nM and about 50 μM (e.g., between about 50 nM and about 500 nM, between about 50 nM and about 5 μM, between about 500 nM and about 50 μM, between about 5 μM and about 50 μM, or between about 10 μM and about 50 μM). DIn some embodiments, the amino acid recognition molecule binds to two or more types of amino acids with a K of about 50 nM. D Combine with.

[0119] In some embodiments, the amino acid recognition molecule has at least 0.1 s -1 Dissociation rate (k off In some embodiments, the dissociation rate is about 0.1 s -1 ~about 1,000s -1 (for example, about 0.5 s -1 ~about 500s -1 , about 0.1 seconds -1 ~approx. 100s -1 , about 1 s -1 ~approx. 100s -1 , or about 0.5 seconds -1 ~about 50s -1 In some embodiments, the dissociation rate is about 0.5 s -1 ~approx. 20 seconds -1 In some embodiments, the dissociation rate is about 2 s -1 ~approx. 20 seconds -1 In some embodiments, the dissociation rate is about 0.5 s -1 ~about 2s -1 is.

[0120] In some embodiments, K D or k off The value of k may be a known literature value, or the value may be empirically determined. off The value of k can be empirically determined based on signal pulse information obtained in single molecule assays, as described elsewhere herein. For example, k off The value of K can be approximated by the reciprocal of the average pulse duration. In some embodiments, the amino acid recognition molecules have different K for each of the two or more types. D or k off In some embodiments, the first K of the first type of amino acid binds to two or more types of amino acids having D or koff is the second K of the second type of amino acid D or k off In some embodiments, K differs by at least 10% (e.g., at least 25%, at least 50%, at least 100%, or more) from D or k off The first and second values ​​differ by about 10-25%, 25-50%, 50-75%, 75-100%, or more than 100%, for example, about 2-fold, 3-fold, 4-fold, 5-fold, or more.

[0121] As described herein, an amino acid recognition molecule can be any biomolecule that can selectively or specifically bind to one molecule over another (e.g., one type of amino acid over another type of amino acid). In some embodiments, the recognition molecule is not a peptidase or does not have peptidase activity. For example, in some embodiments, the disclosed polypeptide sequencing method involves contacting a polypeptide molecule with one or more recognition molecules and a cleavage reagent. In such embodiments, the one or more recognition molecules do not have peptidase activity, and removal of one or more amino acids from the polypeptide molecule (e.g., removal of amino acids from the terminus of the polypeptide molecule) is performed by the cleavage reagent.

[0122] Recognition molecules include, for example, proteins and nucleic acids, and can be synthetic or recombinant. In some embodiments, the recognition molecule can be an antibody or an antigen-binding portion of an antibody, an SH2 domain-containing protein or a fragment thereof, or an enzyme biomolecule, such as a peptidase, an aminotransferase, a ribozyme, an aptazyme, or a tRNA synthetase, including an aminoacyl-tRNA synthetase, and related molecules described in U.S. Patent Application No. 15 / 255,433, filed September 2, 2016, entitled "MOLECULES AND METHODS FOR ITERATIVE POLYPEPTIDE ANALYSIS AND PROCESSING."

[0123] In some embodiments, the recognition molecule of the present disclosure is a degradation pathway protein. Examples of degradation pathway proteins suitable for use as a recognition molecule include, but are not limited to, N-end rule pathway proteins such as Arg / N-end rule pathway proteins, Ac / N-end rule pathway proteins, and Pro / N-end rule pathway proteins. In some embodiments, the recognition molecule is an N-end rule pathway protein selected from a Gid protein (e.g., Gid4 or Gid10 protein), a UBR box protein (e.g., UBR1, UBR2) or a UBR box domain-containing protein fragment thereof, a p62 protein or a ZZ domain-containing fragment thereof, and a ClpS protein (e.g., ClpS1, ClpS2). Thus, in some embodiments, the labeled recognition molecule comprises a degradation pathway protein. In some embodiments, the labeled recognition molecule comprises a ClpS protein.

[0124] In some embodiments, the recognition molecule of the present disclosure is a ClpS protein, e.g., Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS, or Plasmodium falciparum ClpS. In some embodiments, the recognition molecule is an L / F transferase, e.g., Escherichia coli leucyl / phenylalanyl-tRNA protein transferase. In some embodiments, the recognition molecule is a D / E leucyltransferase, such as Vibrio vulnificus aspartate / glutamate leucyltransferase Bpt. In some embodiments, the recognition molecule is a UBR protein or UBR box domain, such as a UBR protein, or the UBR box domain of human UBR1 and UBR2 or Saccharomyces cerevisiae UBR1. In some embodiments, the recognition molecule is a p62 protein, such as the H. sapiens p62 protein or the Rattus norvegicus p62 protein, or a truncated variant thereof containing at least the ZZ domain. In some embodiments, the recognition molecule is a Gid4 protein, such as the H. sapiens GID4 or the Saccharomyces cerevisiae GID4. In some embodiments, the recognition molecule is a Gid10 protein, for example, Saccharomyces cerevisiae GID10.In some embodiments, the recognition molecule is an N-meristoyltransferase, such as Leishmania major N-meristoyltransferase or H. sapiens N-meristoyltransferase NMT1. In some embodiments, the recognition molecule is a BIR2 protein, such as Drosophila melanogaster BIR2. In some embodiments, the recognition molecule is a tyrosine kinase or an SH2 domain of a tyrosine kinase, such as an H. sapiens Fyn SH2 domain, an H. sapiens Src tyrosine kinase SH2 domain, or a variant thereof, such as an H. sapiens Fyn SH2 domain triple mutant superbinder. In some embodiments, the recognition molecule is an antibody or antibody fragment, such as a single chain antibody variable fragment (scFv) directed against phosphotyrosine or other post-translationally modified amino acid variants described herein.

[0125] In some embodiments, the recognition molecules of the present disclosure are amino acid-binding proteins that can be used in sequencing methods with other types of amino acid-binding molecules, such as peptidases and / or nucleic acid aptamers. Peptidases, also known as proteases or proteinases, are enzymes that catalyze the hydrolysis of peptide bonds. Peptidases digest polypeptides into shorter fragments and can generally be classified into endopeptidases, which cleave polypeptide chains internally, and exopeptidases, which cleave polypeptide chains at their termini. In some embodiments, the labeled recognition molecule comprises a peptidase modified to inactivate exopeptidase or endopeptidase activity. In this way, the labeled recognition molecule selectively binds amino acids without further cleaving them from the polypeptide. In yet other embodiments, peptidases that have not been modified to inactivate exopeptidase or endopeptidase activity can be used with the amino acid-binding proteins of the present disclosure. For example, in some embodiments, the labeled recognition molecule comprises a labeled exopeptidase.

[0126] In some embodiments, the amino acid recognition molecule comprises one or more labels. In some embodiments, the one or more labels comprise a luminescent label or a conductivity label, as described elsewhere herein. In some embodiments, the one or more labels comprise one or more polyol moieties (e.g., one or more moieties selected from dextran, polyvinylpyrrolidone, polyethylene glycol, polypropylene glycol, polyoxyethylene glycol, and polyvinyl alcohol). For example, in some embodiments, the amino acid recognition molecule is PEGylated. In some embodiments, polyol modification (e.g., PEGylation) can limit the degree of nonspecific attachment to the surface of a substrate (e.g., a sequencing chip). In some embodiments, polyol modification can limit the degree of aggregation or interaction between the amino acid recognition molecule and other recognition molecules, cleavage reagents, or other species present in the sequencing reaction mixture. PEGylation can be performed by incubating the recognition molecule (e.g., an amino acid binding protein such as a ClpS protein) with mPEG4-NHS ester, which labels primary amines, such as surface-exposed lysine side chains. Other types of PEG and other polyol modification methods are known in the art.

[0127] In some embodiments, one or more labels comprise a tag sequence. For example, in some embodiments, an amino acid recognition molecule comprises a tag sequence that provides one or more functions other than amino acid binding. In some embodiments, the tag sequence comprises at least one biotin ligase recognition sequence that enables biotinylation of the recognition molecule (e.g., incorporation of one or more biotin molecules comprising biotin and bis-biotin moieties). In some embodiments, the tag sequence comprises two tandemly oriented biotin ligase recognition sequences. In some embodiments, a biotin ligase recognition sequence refers to an amino acid sequence recognized by a biotin ligase, which catalyzes the covalent bond between the sequence and a biotin molecule. Each biotin ligase recognition sequence of a tag sequence can be covalently linked to a biotin moiety, such that a tag sequence having multiple biotin ligase recognition sequences can be covalently linked to multiple biotin molecules. A region of a tag sequence having one or more biotin ligase recognition sequences can generally be referred to as a biotinylation tag or biotinylation sequence. In some embodiments, bis-biotin or bis-biotin moiety can refer to two biotins attached to two biotin ligase recognition sequences oriented in tandem.

[0128] Further examples of functional sequences in the tag sequence include purification tags, cleavage sites, and other moieties useful for purifying and / or modifying the recognition molecule. Examples of amino acid recognition molecules (e.g., amino acid binding proteins) for use in accordance with the present disclosure are described in more detail in International Application No. PCT / US2019 / 061831, filed November 15, 2019, and International Application No. PCT / US2021 / 033493, filed May 20, 2021, the relevant contents of which are incorporated herein by reference in their entireties.

[0129] Cleavage Reagent In some embodiments, the cleavage reagent of the present disclosure is an exopeptidase. Exopeptidases generally require that the polypeptide substrate contain at least one of a free amino group at its amino terminus or a free carboxyl group at its carboxy terminus. In some embodiments, an exopeptidase according to the present disclosure hydrolyzes a bond at or near the end of a polypeptide. In some embodiments, the exopeptidase hydrolyzes bonds three or fewer residues from the polypeptide terminus. For example, in some embodiments, a single hydrolysis reaction catalyzed by the exopeptidase cleaves a single amino acid, dipeptide, or tripeptide from the polypeptide terminus.

[0130] In some embodiments, the exopeptidases of the present disclosure are aminopeptidases or carboxypeptidases, cleaving single amino acids from the amino or carboxy termini, respectively. In some embodiments, the exopeptidases of the present disclosure are dipeptidyl-peptidases or peptidyl-dipeptidases, cleaving dipeptides from the amino or carboxy termini, respectively. In yet other embodiments, the exopeptidases of the present disclosure are tripeptidyl-peptidases, cleaving tripeptides from the amino termini. The classification of peptidases and the activity of each class or subclass are well known and described in the literature (see, e.g., Gurupriya, VS & Roy, SC Proteases and Protease Inhibitors in Male Reproduction. Proteases in Physiology and Pathology 195-216 (2017); and Brix, K. & Stocker, W. Proteases: Structure and Function. Chapter 1). In some embodiments, a peptidase according to the present disclosure removes more than three amino acids from a polypeptide terminus. Thus, in some embodiments, the peptidase is an endopeptidase, e.g., an endopeptidase that preferentially cleaves at a particular position (e.g., before or after a particular amino acid). In some embodiments, the size of the polypeptide cleavage product of endopeptidase activity depends on the distribution of cleavage sites (e.g., amino acids) within the analyzed polypeptide.

[0131] The exopeptidase according to the present disclosure can be selected or engineered based on the directionality of the sequencing reaction. For example, in embodiments where a polypeptide is sequenced from the amino terminus to the carboxy terminus, the exopeptidase comprises aminopeptidase activity. Conversely, in embodiments where a polypeptide is sequenced from the carboxy terminus to the amino terminus, the exopeptidase comprises carboxypeptidase activity. Examples of carboxypeptidases that recognize specific carboxy-terminal amino acids and can be used as labeled exopeptidases or inactivated to be used as non-cleavable labeled recognition molecules as described herein are described in the literature (see, for example, Garcia-Guerrero, MC, et al. (2018) PNAS 115(17)).

[0132] Peptidases suitable for use as cleavage reagents and / or recognition molecules include aminopeptidases that selectively bind one or more types of amino acids. In some embodiments, the aminopeptidase recognition molecule is modified to inactivate aminopeptidase activity. In some embodiments, the aminopeptidase cleavage reagent is non-specific, cleaving most or all types of amino acids from the end of a polypeptide. In some embodiments, the aminopeptidase cleavage reagent is more efficient at cleaving one or more types of amino acids from the end of a polypeptide than other types of amino acids at the end of the polypeptide. For example, aminopeptidases according to the present disclosure specifically cleave alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and / or valine. In some embodiments, the aminopeptidase is a proline aminopeptidase. In some embodiments, the aminopeptidase is a proline iminopeptidase. In some embodiments, the aminopeptidase is a glutamate / aspartate-specific aminopeptidase. In some embodiments, the aminopeptidase is a methionine-specific aminopeptidase.

[0133] In some embodiments, the aminopeptidase is a non-specific aminopeptidase. In some embodiments, the non-specific aminopeptidase is a zinc metalloprotease.

[0134] Examples of cleavage reagents (e.g., aminopeptidases) for use in accordance with the present disclosure are described in more detail in International Application No. PCT / US2019 / 061831, filed November 15, 2019, and International Application No. PCT / US2021 / 033493, filed May 20, 2021, the relevant contents of which are incorporated herein by reference in their entireties.

[0135] Nucleic acid sequencing Some aspects of the present application are useful for sequencing biological polymers such as nucleic acids. In some embodiments, the methods, compositions, and devices described herein can be used to identify a series of nucleotide monomers incorporated into a nucleic acid (e.g., by detecting the time course of incorporation of a series of labeled nucleotides). In some embodiments, the methods, compositions, and devices described herein can be used to identify a series of nucleotides incorporated into a template-dependent nucleic acid sequencing reaction product synthesized by a polymerase enzyme.

[0136] In certain embodiments, template-dependent nucleic acid sequencing products are performed by a naturally occurring nucleic acid polymerase. In some embodiments, the polymerase is a mutant or modified variant of a naturally occurring polymerase. In some embodiments, the template-dependent nucleic acid sequencing products include one or more nucleotide segments complementary to a template nucleic acid strand. In one aspect, the present application provides a method for determining the sequence of a template (or target) nucleic acid strand by determining the sequence of its complementary nucleic acid strand.

[0137] In another aspect, the present application provides a method for sequencing a target nucleic acid by sequencing multiple nucleic acid fragments, wherein the target nucleic acid comprises fragments. In certain embodiments, the method includes combining multiple fragment sequences to provide a sequence or subsequence of a parent target nucleic acid. In some embodiments, the combining step is performed by computer hardware and software. The methods described herein may enable sequencing of a set of related target nucleic acids, such as a chromosome or an entire genome.

[0138] During sequencing, a polymerizing enzyme can couple (e.g., attach) to a priming location of a target nucleic acid molecule. The priming location can be a primer complementary to a portion of the target nucleic acid molecule. Alternatively, the priming location is a gap or nick provided within a double-stranded segment of the target nucleic acid molecule. The gap or nick can be 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 nucleotides in length. The nick can provide a break in one strand of a double-stranded sequence, which can provide a priming location for a polymerizing enzyme, such as a strand-displacing polymerase enzyme.

[0139] In some cases, the sequencing primer can be annealed to a target nucleic acid molecule, which may or may not be immobilized on a solid support. The solid support can include, for example, a sample well (e.g., a nanoaperture, a reaction chamber) on a chip used for nucleic acid sequencing. In some embodiments, the sequencing primer can be immobilized on a solid support, and upon hybridization of the target nucleic acid molecule, the target nucleic acid molecule is also immobilized on the solid support. In some embodiments, the polymerase is immobilized on a solid support, and the soluble primer and target nucleic acid are contacted with the polymerase. However, in some embodiments, a complex comprising the polymerase, target nucleic acid, and primer is formed in solution, and the complex is immobilized on a solid support (e.g., by immobilization of the polymerase, primer, and / or target nucleic acid). In some embodiments, none of the components in the sample well (e.g., a nanoaperture, a reaction chamber) is immobilized on a solid support. For example, in some embodiments, a complex comprising the polymerase, target nucleic acid, and primer is formed in solution, and the complex is not immobilized on a solid support.

[0140] Under appropriate conditions, a polymerase enzyme contacted with the annealed primer / target nucleic acid can add or incorporate one or more nucleotides into the primer, and the nucleotides can be added to the primer in a template-dependent manner in a 5' to 3' direction. Such incorporation of nucleotides into a primer (e.g., by the action of a polymerase) can generally be referred to as a primer extension reaction. Each nucleotide can be associated with a detectable tag that can be detected and identified during the nucleic acid extension reaction (e.g., based on its luminescence lifetime and / or other properties) and used to determine the sequence of each nucleotide incorporated into the extended primer and, therefore, the newly synthesized nucleic acid molecule. Sequence complementarity of the newly synthesized nucleic acid molecule can also determine the sequence of the target nucleic acid molecule. In some cases, the annealing of the sequencing primer to the target nucleic acid molecule and the incorporation of nucleotides into the sequencing primer can occur under similar reaction conditions (e.g., the same or similar reaction temperature) or different reaction conditions (e.g., different reaction temperatures). In some embodiments, sequencing by synthesis methods may involve determining the presence of a population of target nucleic acid molecules (e.g., copies of the target nucleic acid) and / or amplifying the target nucleic acid to achieve the population of target nucleic acids. However, in some embodiments, sequencing by synthesis is used to determine the sequence of a single molecule in each reaction being evaluated (preparing the target template for sequencing does not require nucleic acid amplification). In some embodiments, multiple single molecule sequencing reactions are performed in parallel (e.g., on a single chip) in accordance with aspects of the present application. For example, in some embodiments, multiple single molecule sequencing reactions are each performed in separate reaction chambers (e.g., nanoapertures, sample wells) on a single chip.

[0141] Embodiments can sequence single nucleic acid molecules with high accuracy and long read lengths, for example, at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% accuracy and / or read lengths of about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or 100,000 bp or more. In some embodiments, the target nucleic acid molecule used in single molecule sequencing is a single-stranded target nucleic acid (e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid (RNA), RNA derivatives) template, which is added to or immobilized in a sample well (e.g., a nanoaperture), which contains at least one additional component of the sequencing reaction (e.g., a polymerase such as a DNA polymerase, a sequencing primer) immobilized or attached to a solid support, such as the bottom or side of the sample well. The target nucleic acid molecule or polymerase can be attached to a sample wall, e.g., the bottom or side wall of the sample well, directly or via a linker. The sample well (e.g., nanoaperture) may also contain any other reagents necessary for nucleic acid synthesis by a primer extension reaction, such as suitable buffers, cofactors, enzymes (e.g., polymerases), and deoxyribonucleoside polyphosphates, including dNTPs such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP), containing a luminescent tag such as a fluorophore.In some embodiments, each class of dNTP (e.g., adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs (e.g., dUTP), and thymine-containing dNTPs (e.g., dTTP)) is conjugated to a separate light-emitting tag such that detection of light emitted from the tag indicates the type of dNTP incorporated into newly synthesized nucleic acid. The light emitted from the light-emitting tag can be detected and attributed to the appropriate light-emitting tag (and thus the associated dNTP) by any suitable device and / or method, including the devices and methods for detection described elsewhere herein. The light-emitting tag can be conjugated to the dNTP at any position such that the presence of the light-emitting tag does not inhibit incorporation of the dNTP into newly synthesized nucleic acid strands or the activity of the polymerase. In some embodiments, the light-emitting tag is conjugated to the terminal phosphate (e.g., the gamma phosphate) of the dNTP.

[0142] In some embodiments, single-stranded target nucleic acid template can be contacted with sequencing primer, dNTP, polymerase, and other reagents required for nucleic acid synthesis.In some embodiments, all appropriate dNTPs can be simultaneously contacted with single-stranded target nucleic acid template (for example, all dNTPs are present at the same time), so that dNTP incorporation can occur continuously.In other embodiments, dNTPs can be contacted with single-stranded target nucleic acid template sequentially, and the single-stranded target nucleic acid template is contacted with each appropriate dNTP separately, with a washing step between contacting the single-stranded target nucleic acid template with different dNTPs.This cycle of contacting the single-stranded target nucleic acid template with each dNTP separately and then washing can be repeated for each consecutive base position of the identified single-stranded target nucleic acid template.

[0143] In some embodiments, a sequencing primer anneals to a single-stranded target nucleic acid template, and a polymerase sequentially incorporates dNTPs (or other deoxyribonucleoside polyphosphates) into the primer based on the single-stranded target nucleic acid template. A unique luminescent tag associated with each incorporated dNTP can be excited with appropriate excitation light during or after incorporation of the dNTP into the primer, and the emitted light can then be detected using any suitable device and / or method, including the devices and methods for detection described elsewhere herein. The detection of a specific emission (e.g., having a specific emission lifetime, intensity, spectrum, and / or combination thereof) can be attributed to the specific incorporated dNTP. The sequence obtained from the collection of detected luminescent tags can then be used to determine the sequence of the single-stranded target nucleic acid template by sequence complementarity.

[0144] Although this disclosure refers to dNTPs, the devices, systems, and methods provided herein can be used with various types of nucleotides, such as ribonucleotides and deoxyribonucleotides (e.g., deoxyribonucleoside polyphosphates having at least 4, 5, 6, 7, 8, 9, or 10 phosphate groups). Such ribonucleotides and deoxyribonucleotides may include various types of tags (or markers) and linkers.

[0145] Devices and Delivery Systems In some embodiments, the methods according to the present disclosure can be implemented using a system that enables single-molecule analysis. The system can include an integrated device and an instrument configured to interface with the integrated device. The integrated device can include an array of pixels, with each pixel including a sample well and at least one photodetector. The sample wells of the integrated device can be formed on or across the surface of the integrated device and configured to receive samples disposed on the surface of the integrated device. Collectively, the sample wells can be considered an array of sample wells. The multiple sample wells can have a suitable size and shape such that at least some of the sample wells receive a single sample (e.g., a single molecule such as a polypeptide). In some embodiments, the number of samples in the sample wells can be distributed among the sample wells of the integrated device such that some sample wells contain one sample, while others contain zero, two, or more samples.

[0146] Excitation light is provided to the integrated device from one or more light sources external to the integrated device. Optical components of the integrated device may receive the excitation light from the light source and direct the light toward the array of sample wells of the integrated device, illuminating an illumination area within the sample well. In some embodiments, the sample wells may have a configuration that allows a sample to be held in close proximity to the surface of the sample well, which may facilitate delivery of excitation light to the sample and detection of emission light from the sample. A sample positioned within the illumination area may emit emission light in response to being illuminated by the excitation light. For example, the sample may be labeled with a fluorescent label that emits light in response to achieving an excited state through illumination with the excitation light. The emission light emitted by the sample may then be detected by one or more photodetectors in pixels corresponding to the sample well with the sample being analyzed. Multiple samples may be analyzed in parallel when performed across an array of sample wells, which may range in number from approximately 10,000 pixels to 1,000,000 pixels according to some embodiments.

[0147] The integrated device may include an optical system for receiving excitation light and directing the excitation light among the reaction chamber array. The optical system may include one or more grating couplers configured to couple the excitation light to other optical components of the integrated device and direct the excitation light to other optical components. For example, the optical system may include optical components that direct the excitation light from the grating coupler toward the reaction chamber array. Such optical components may include an optical splitter, an optical combiner, and a waveguide. In some embodiments, one or more optical splitters can couple the excitation light from the grating coupler and deliver the excitation light to at least one waveguide. According to some embodiments, the optical splitter can have a configuration that enables substantially uniform delivery of excitation light across all waveguides, such that each of the waveguides receives substantially the same amount of excitation light. Such embodiments may improve the performance of the integrated device by improving the uniformity of the excitation light received by the sample wells of the integrated device. For example, examples of suitable components for coupling excitation light into the reaction chamber and / or directing emitted light to the photodetector and for inclusion within an integrated device are described in U.S. patent application Ser. No. 14 / 821,688, filed Aug. 7, 2015, entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," and U.S. patent application Ser. No. 14 / 543,865, filed Nov. 17, 2014, entitled "INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES," both of which are incorporated by reference in their entireties. Examples of suitable grating couplers and waveguides that may be implemented in an integrated device are described in U.S. patent application Ser. No. 15 / 844,403, filed Dec. 15, 2017, entitled "OPTICAL COUPLER AND WAVEGUIDE SYSTEM," which is incorporated by reference in its entirety.

[0148] Additional photonic structures may be disposed between the sample well and the photodetector and configured to reduce or prevent excitation light from reaching the photodetector, which would otherwise contribute to signal noise when detecting the emission light. In some embodiments, the metal layer, which may act as a circuit for the integrated device, may also act as a spatial filter. Examples of suitable photonic structures may include spectral filters, polarizing filters, and spatial filters, and are described in U.S. Patent Application No. 16 / 042,968, filed July 23, 2018, entitled "OPTICAL REJECTION PHOTONIC STRUCTURES," and U.S. Provisional Patent Application No. 63 / 124,655, filed December 11, 2020, entitled "INTEGRATED CIRCUIT WITH IMPROVED CHARGE TRANSFER EFFICIENCY AND ASSOCIATED TECHNIQUES," both of which are incorporated by reference in their entireties.

[0149] Components located remotely from the integrated device can be used to position and align the excitation source relative to the integrated device. Such components can include optical components, including lenses, mirrors, prisms, windows, apertures, attenuators, and / or optical fibers. Additional mechanical components can be included in the instrument to enable control of one or more alignment components. Such mechanical components can include actuators, stepper motors, and / or knobs. Examples of suitable excitation sources and alignment mechanisms are described in U.S. patent application Ser. No. 15 / 161,088, filed May 20, 2016, entitled "PULSED LASER AND SYSTEM," which is incorporated by reference in its entirety. Another example of a beam steering module is described in U.S. patent application Ser. No. 15 / 842,720, filed December 14, 2017, entitled "COMPACT BEAM SHAPING AND STEERING ASSEMBLY," which is incorporated by reference herein. Further examples of suitable excitation sources are described in U.S. Patent Application No. 14 / 821,688, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES," which is incorporated by reference in its entirety.

[0150] The photodetector(s) associated with each pixel of the integrated device may be configured and arranged to detect light emitted from the corresponding reaction chamber of the pixel. Examples of suitable photodetectors are described in U.S. Patent Application No. 14 / 821,656, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS," which is incorporated by reference in its entirety. In some embodiments, the reaction chambers and their respective photodetectors may be aligned along a common axis. In this manner, the photodetectors may overlap the reaction chambers within the pixel.

[0151] Characteristics of the detected emitted light can provide an indication for identifying a label associated with the emitted light. Such characteristics can include any suitable type of characteristic, including the arrival time of a photon detected by a photodetector, the amount of photons accumulated over time by a photodetector, and / or the distribution of photons across two or more photodetectors. In some embodiments, such characteristics can be any one or a combination of two or more of luminescence lifetime, luminescence intensity, brightness, absorption spectrum, emission spectrum, luminescence quantum yield, wavelength (e.g., peak wavelength), and signal characteristics (e.g., pulse duration, inter-pulse duration, change in signal intensity).

[0152] In some embodiments, the photodetector may have a configuration that allows detection of one or more timing characteristics (e.g., luminescence lifetime) associated with the sample's emission. The photodetector may detect a distribution of photon arrival times after a pulse of excitation light propagates through the integrated device, and the distribution of arrival times may provide an indication of the timing characteristics of the sample's emission light (e.g., a proxy for luminescence lifetime). In some embodiments, one or more photodetectors provide an indication of the probability (e.g., luminescence intensity) of emission light emitted by a label. In some embodiments, multiple photodetectors may be sized and positioned to capture the spatial distribution of emission light. The output signal from the one or more photodetectors may then be used to distinguish one label from multiple labels, and multiple labels may be used to identify a sample within a sample. In some embodiments, the sample may be excited by multiple excitation energies, and the emission light and / or timing characteristics of the emission light emitted by the sample in response to the multiple excitation energies can distinguish one label from multiple labels.

[0153] During operation, parallel analysis of samples in the reaction chambers is performed by exciting some or all of the samples in the chambers using excitation light and detecting signals from the sample emissions using photodetectors. The emission light from the samples can be detected by corresponding photodetectors and converted into at least one electrical signal. The electrical signal can be transmitted along conductive lines within the integrated device's circuitry, which can be connected to an instrument interfaced with the integrated device. The electrical signal can then be processed and / or analyzed. The processing or analysis of the electrical signal can be performed on a suitable computing device located either on or off the instrument.

[0154] The instrument may include a user interface for controlling the operation of the instrument and / or the integrated device. The user interface may be configured to allow a user to input information into the instrument, such as commands and / or settings used to control the instrument's functions. In some embodiments, the user interface may include buttons, switches, dials, and a microphone for voice commands. The user interface may allow a user to receive feedback regarding the instrument and / or the performance of the integrated device, such as information obtained by proper alignment and / or readout signals from a photodetector on the integrated device. In some embodiments, the user interface may provide feedback using a speaker to provide audible feedback. In some embodiments, the user interface may include indicator lights and / or a display screen to provide visual feedback to the user.

[0155] In some embodiments, the instrument may include a computer interface configured to connect to a computing device. The computer interface may be a USB interface, a FireWire interface, or any other suitable computer interface. The computing device may be any general-purpose computer, such as a laptop or desktop computer. In some embodiments, the computing device may be a server (e.g., a cloud-based server) accessible over a wireless network via a suitable computer interface. The computer interface may facilitate communication of information between the instrument and the computing device. Input information for controlling and / or configuring the instrument may be provided to the computing device and transmitted to the instrument via the computer interface. Output information generated by the instrument may be received by the computing device via the computer interface. The output information may include feedback regarding instrument performance, integrated device performance, and / or data generated from the photodetector readout signal.

[0156] In some embodiments, the instrument may include a processing device configured to analyze data received from one or more photodetectors of the integrated device and / or transmit control signals to the excitation source. In some embodiments, the processing device may comprise a general-purpose processor, a specially adapted processor (e.g., one or more central processing units (CPUs), such as microprocessors or microcontroller cores, field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), custom integrated circuits, digital signal processors (DSPs), or combinations thereof). In some embodiments, processing of data from the one or more photodetectors may be performed by both the instrument's processing device and an external computing device. In other embodiments, the external computing device may be omitted, and processing of data from the one or more photodetectors may be performed solely by the integrated device's processing device.

[0157] According to some embodiments, an instrument configured to analyze a sample based on its luminescence emission characteristics can detect differences in luminescence lifetime and / or intensity between different luminescent molecules and / or differences in lifetime and / or intensity of the same luminescent molecule in different environments. The inventors have recognized and appreciated that differences in luminescence emission lifetimes can be used to distinguish between the presence or absence of different luminescent molecules and / or to distinguish between different environments or conditions to which the luminescent molecules are exposed. In some cases, aspects of the system can be simplified by distinguishing luminescent molecules based on lifetime (e.g., rather than emission wavelength). As an example, wavelength-discriminating optics (e.g., wavelength filters, dedicated detectors for each wavelength, dedicated pulsed light sources at different wavelengths, and / or diffractive optics) may be reduced in number or eliminated when distinguishing luminescent molecules based on lifetime. In some cases, a single pulsed light source operating at a single characteristic wavelength can be used to excite different luminescent molecules that emit within the same wavelength region of the optical spectrum but have measurably different lifetimes. Analytical systems that use a single pulsed light source, rather than multiple light sources operating at different wavelengths, to excite and distinguish different luminescent molecules that emit in the same wavelength region can be less complex to operate and maintain, more compact, and can be manufactured at lower cost.

[0158] While analytical systems based on luminescence lifetime analysis may have certain advantages, the amount of information obtained by the analytical system and / or detection accuracy may be increased by enabling additional detection techniques. For example, some embodiments of the system may be further configured to identify one or more characteristics of a sample based on emission wavelength and / or emission intensity. In some implementations, luminescence intensity may additionally or alternatively be used to distinguish between different luminescent labels. For example, some luminescent labels may emit at significantly different intensities or have significant differences in excitation probability (e.g., at least about 35% difference) even if their decay rates are similar. By referencing binned signals to the measured excitation light, it may be possible to distinguish between different luminescent labels based on intensity levels.

[0159] According to some embodiments, different luminescence lifetimes can be distinguished by a photodetector configured to time-bin luminescence emission events following excitation of the luminescent labels. Time binning can occur during a single charge accumulation cycle for the photodetector. A charge accumulation cycle is the interval between readout events during which photogenerated carriers accumulate within the bins of the time-binning photodetector. Examples of time-binning photodetectors are described in U.S. Patent Application No. 14 / 821,656, filed August 7, 2015, entitled "INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS," which is incorporated herein by reference. In some embodiments, the time-binning photodetector can generate charge carriers within a photon absorption / carrier generation region and directly transfer the charge carriers to charge carrier storage bins within a charge carrier storage region. In such embodiments, the time-binning photodetector may not include a carrier transfer / capture region. Such time-binning photodetectors are sometimes referred to as "direct binning pixels." An example of a time-binning photodetector including a direct binning pixel is described in U.S. Patent Application No. 15 / 852,571, filed December 22, 2017, entitled "INTEGRATED PHOTODETECTOR WITH DIRECT BINNING PIXEL," which is incorporated herein by reference.

[0160] In some embodiments, different numbers of fluorophores of the same type can be linked to different reagents in a sample, so that each reagent can be identified based on emission intensity.For example, two fluorophores can be linked to a first labeled recognition molecule, and four or more fluorophores can be linked to a second labeled recognition molecule.Due to the different numbers of fluorophores, there may be different excitation and emission probabilities associated with different recognition molecules.For example, there may be more emission events for the second labeled recognition molecule during the signal accumulation interval, so that the apparent intensity of the bin is significantly higher than that of the first labeled recognition molecule.

[0161] The inventors of the present application have recognized and appreciated that distinguishing biological or chemical samples based on fluorophore decay rates and / or fluorophore intensities can allow for simplification of optical excitation and detection systems. For example, optical excitation can be performed using a single wavelength source (e.g., a light source that produces one characteristic wavelength rather than multiple light sources, or a light source that operates at multiple different characteristic wavelengths). Furthermore, wavelength-discriminating optics and filters may not be required in the detection system. Also, a single photodetector may be used for each reaction chamber to detect emissions from different fluorophores. The phrase "characteristic wavelength" or "wavelength" is used to refer to the central or dominant wavelength within a limited emission bandwidth (e.g., the central or peak wavelength within a 20 nm bandwidth output by a pulsed light source). In some cases, "characteristic wavelength" or "wavelength" can be used to refer to the peak wavelength within the full bandwidth of the emission output by a source.

[0162] According to one aspect of the present disclosure, an exemplary integrated device may be configured to perform single molecule analysis in combination with the above-described instruments. It should be understood that the exemplary integrated device described herein is intended to be exemplary, and that other integrated device configurations may be configured to perform any or all of the techniques described herein.

[0163] 5 is a cross-sectional view of pixel 1-112 of integrated device 1-102. Pixel 1-112 includes a photodetection region, which may be a pinned photodiode (PPD), and a charge storage region, which may be a storage diode (SDO). In some embodiments, the photodetection region and the charge storage region may be formed within the semiconductor material of the pixel by doping regions of the semiconductor material. For example, the photodetection region and the charge storage region may be formed with the same conductivity type (e.g., n-type doping or p-type doping).

[0164] During operation of the pixel 1-112, excitation light can illuminate the reaction chamber 1-108, causing incident photons, including fluorescent emissions from the sample, to flow along the optical axis to the photodetection region PPD. As shown in FIG. 5, the pixel 1-112 can include a waveguide 1-220 configured to optically couple (e.g., by evanescent coupling) excitation light from a grating coupler of an integrated device (not shown) into the reaction chamber 1-108. In response, the sample in the reaction chamber 1-108 can emit fluorescent light toward the photodetection region PPD. In some embodiments, the pixel 1-112 can also include one or more photonic structures 1-230, which can include one or more light-removing structures, such as a spectral filter, a polarizing filter, and / or a spatial filter. For example, the photonic structure 1-230 can be configured to reduce the amount of excitation light reaching the photodetection region PPD and / or increase the amount of fluorescent emissions reaching the photodetection region PPD. Also, as shown in pixel 1-112, pixel 1-112 may include one or more metal layers 1-240, which may be configured as filters and / or may carry control signals from control circuitry configured to control the transfer gates, as further described herein.

[0165] In some embodiments, pixel 1-112 may include one or more transfer gates configured to control operation of pixel 1-112 by applying an electrical bias to one or more semiconductor regions of pixel 1-112 in response to one or more control signals. For example, when transfer gate ST0 induces a first electrical bias in the semiconductor region between photodetection region PPD and storage region SD0, a transfer path (e.g., a charge transfer channel) may be formed in the semiconductor region. Charge carriers (e.g., photoelectrons) generated in photodetection region PPD by incident photons flow along the transfer path to storage region SD0. In some embodiments, the first electrical bias may be applied during a collection period during which charge carriers from the sample are selectively directed to storage region SD0. Alternatively, when transfer gate ST0 imposes a second electrical bias on the semiconductor region between photodetection region PPD and storage region SD0, charge carriers from photodetection region PPD may be blocked from reaching storage region SD0 along the transfer path. In some embodiments, the drain gate REJ can provide a channel to the drain D to draw noise charge carriers generated in the photodetection region PPD by the excitation light away from the photodetection region PPD and the storage region SD0, such as during a rejection period before fluorescent emission photons from the sample reach the photodetection region PPD. In some embodiments, during a readout period, the transfer gate ST0 can provide a second electrical bias, and the transfer gate TX0 can provide an electrical bias to flow the charge carriers stored in the storage region SD0 to the readout region, which may be a floating diffusion (FD) region, for processing.

[0166] It should be understood that, according to various embodiments, the transfer gates described herein may comprise semiconductor materials and / or metals, and may include the gate of a field effect transistor (FET), the base of a bipolar junction transistor (BJT), etc.

[0167] In some embodiments, operation of the pixel 1-112 may include one or more collection sequences, each of which includes one or more rejection (e.g., drain) periods and one or more collection periods. In one example, a collection sequence performed in accordance with one or more pulses of an excitation light source may begin with a rejection period, such as to discard charge carriers generated within the pixel 1-112 (e.g., within the photodetection region PD) in response to excitation photons from the light source. For example, excitation photons may arrive at the pixel 1-112 prior to the arrival of fluorescent emission photons from the reaction chamber. A transfer gate for the charge storage region may be biased to have low conductivity in a charge transfer channel connecting the charge storage region to the photodetection region, preventing the transfer and accumulation of charge carriers in the charge storage region. A drain gate for the drain region may be biased to have high conductivity in a drain channel between the photodetection region and the drain region, facilitating the draining of charge carriers from the photodetection region to the drain region. The transfer gate for any charge storage region coupled to the photodetection region may be biased to have low conductivity between the photodetection region and the charge storage region, thereby preventing charge carriers from being transferred or stored in the charge storage region during the rejection period.

[0168] The rejection period may be followed by a collection period, during which charge carriers generated in response to incident photons are transferred to one or more charge accumulation regions. During the collection period, incident photons may include fluorescent emission photons, resulting in the accumulation of fluorescent emission charge carriers in the charge accumulation region(s). For example, a transfer gate for one of the charge accumulation regions may be biased to have high conductivity between the photodetection region and the charge accumulation region, facilitating the accumulation of charge carriers in the charge accumulation region. Any drain gate coupled to the photodetection region may be biased to have low conductivity between the photodetection region and the drain region to prevent charge carriers from being discarded during the collection period.

[0169] Some embodiments may include multiple rejection and / or collection periods in a collection sequence, such as a first rejection and collection period followed by a second rejection and collection period, with each pair of rejection and collection periods occurring in response to a pulse of excitation light. In one example, charge carriers generated in the photodetection region during each collection period of a collection sequence (e.g., in response to multiple pulses of excitation light) may be collected in a single charge storage region. In some embodiments, the charge carriers collected in the charge storage region may be read out for processing before the next collection sequence. Alternatively or additionally, in some embodiments, the charge carriers collected in a first charge storage region during a first collection sequence may be transferred to a second charge storage region sequentially coupled to the first charge storage region and read out simultaneously with the next collection sequence. In some embodiments, a processing circuit configured to read out charge carriers from one or more pixels may be configured to determine one or more of luminescence intensity information, luminescence lifetime information, luminescence spectrum information, and / or any other mode of luminescence information associated with performing the techniques described herein.

[0170] In some embodiments, the first collection sequence may include transferring charge carriers generated in the light detection response to the excitation pulse to the charge storage region at a first time following each excitation pulse, and the second collection sequence may include transferring charge carriers generated in the light detection response to the excitation pulse to the charge storage region at a second time following each excitation pulse. For example, the number of charge carriers collected after the first and second times can indicate luminance lifetime information of the received light.

[0171] As further described herein, the pixels of the integrated device can be controlled to perform one or more collection sequences using one or more control signals from the control circuit of the integrating circuit, for example, by providing control signals to the drains and / or transfer gates of the pixels of the integrating circuit. In some embodiments, charge carriers can be read out from the FD region of each pixel for processing during a readout pixel associated with each pixel and / or row or column of pixels. In some embodiments, the FD region of a pixel can be read out using a correlated double sampling (CDS) technique. [Example]

[0172] Example 1 Polypeptide sequencing runs were performed using amino acid recognition molecules labeled with a first luminescent label containing four copies of Cy®3B, a second luminescent label containing three copies of ATRho6G, and a third luminescent label (designated R1C1) containing a luminescent-labeled oligonucleotide structure containing a first single-stranded oligonucleotide containing one copy of Cy®3B and having 100% sequence identity to sequence A, and a first complementary single-stranded oligonucleotide containing one copy of ATRho6G and having 100% sequence identity to sequence B. In R1C1, the ATRho6G and Cy®3B fluorophores were separated by a distance of 10 nm. This distance was predicted from the B-DNA model and was 0.34 * n, where n is the number of oligonucleotide bases between fluorophores.

[0173] A polypeptide sequencing run was performed on a sample peptide having the sequence FAAAYPDDD (SEQ ID NO: 17). Figure 6A shows a representative trace indicating that phenylalanine (F) was identified. Figure 6B shows a plot of intensity versus bin ratio. Figure 6B shows that Cy®3B, ATRho6G, and R1C1 each occupied distinct spatial regions of the plot. Furthermore, Figure 6B shows that R1C1 had a bin ratio of 0.51, which was between the bin ratio of 0.43 for Cy®3B and the bin ratio of 0.58 for ATRho6G.

[0174] The fact that the R1C1 bin ratios were consistent with the average bin ratios of Cy®3B and ATRho6G indicated that the 10 nm spacing between the ATRho6G and Cy®3B fluorophores effectively prevented FRET formation between the two fluorophores. Furthermore, the R1C1 bin ratios indicated that the contribution of each fluorophore to the apparent fluorescence lifetime was proportional to its intensity.

[0175] Example 2 Polypeptide sequencing runs were performed using amino acid recognition molecules labeled with a first luminescent label containing 8 copies of Cy®3, a second luminescent label containing 4 copies of Cy®3B, and a third luminescent label (designated C2C) containing a luminescently labeled oligonucleotide structure containing a first single-stranded oligonucleotide containing 2 copies of Cy®3 and having 100% sequence identity to sequence C, and a first complementary single-stranded oligonucleotide containing 1 copy of Cy®3B and having 100% sequence identity to sequence D. In C2C, the ATRho6G and Cy®3B fluorophores were separated by a distance of 10 nm. This distance was predicted from the B-DNA model to be 0.34 nm. * n, where n is the number of oligonucleotide bases between fluorophores.

[0176] A polypeptide sequencing run was performed on a sample peptide having the sequence FAAAYPDDD (SEQ ID NO: 17). Figure 7A shows a representative trace indicating that phenylalanine (F) was identified. Figure 7B shows a plot of intensity versus bin ratio. Figure 7B shows that Cy®3, Cy®3B, and C2C each occupied distinct spatial regions of the plot. Furthermore, Figure 7B shows that C2C had a bin ratio of 0.39, which was between the bin ratios of 0.28 for Cy®3 and 0.44 for Cy®3B.

[0177] The fact that the C2C bin ratios matched the average bin ratios of Cy®3 and Cy®3B indicated that the 10 nm spacing between the Cy®3 and Cy®3B fluorophores effectively prevented FRET formation between the two fluorophores. Furthermore, the C2C bin ratios indicated that the contribution of each fluorophore to the apparent fluorescence lifetime was proportional to its intensity.

[0178] Example 3 Polypeptide sequencing runs were performed using amino acid recognition molecules labeled with a third light-emitting label (designated SG4Cy®3) comprising a light-emitting labeled oligonucleotide structure comprising a first light-emitting label comprising four copies of Cy®3B, a second light-emitting label comprising C2C, and a first single-stranded oligonucleotide comprising two copies of Cy®3 and having 100% sequence identity to sequence E, and a first complementary single-stranded oligonucleotide comprising two copies of Cy®3 and having 100% sequence identity to sequence F. In SG4Cy®3, each oligonucleotide strand had two Cy®3 fluorophores bulged out around the GC-rich region.

[0179] A polypeptide sequencing run was performed on a sample peptide having the sequence FAAAYPDDD (SEQ ID NO: 17). Figure 8A shows a representative trace indicating that phenylalanine (F) and tyrosine (Y) residues were identified. Figure 8B shows a plot of intensity versus bin ratio. Figure 8B shows that Cy®3B, C2C, and SG4Cy®3 each occupied distinct spatial regions of the plot.

[0180] Example 4 A luminescently labeled oligonucleotide construct comprising multiple luminescently labeled oligonucleotides containing multiple luminescent labels was constructed by a stepwise hybridization and conjugation approach, as shown schematically in FIG. 3A.

[0181] To avoid bending and curvature of the oligonucleotide duplex and to facilitate the conjugation and hybridization of multiple fluorescently labeled oligonucleotides, two different types of oligonucleotides were used. The first type of oligonucleotide contained four types of nucleotides (A, C, G, and T). These oligonucleotides were "GCAT-based oligonucleotides." The second type of oligonucleotide contained up to seven types of nucleotides (A, C, G, T, iG, iC, and diaminopurine). These oligonucleotides were "GCATiGiC-based oligonucleotides."

[0182] The luminescently labeled oligonucleotide structure was constructed by biotinylating the first GCAT-based oligonucleotide (ODN1) and conjugating ODN1 to one end of a streptavidin (SV) homotetramer. Next, the first GCATiGiC-based oligonucleotide (ODN3) was biotinylated and conjugated to the second end of the streptavidin homotetramer to form an ODN1-SV-ODN3 intermediate structure. Both ODN1 and ODN3 were luminescently labeled. Figure 9A shows the ODN1-SV-ODN3 intermediate structure and a retention plot representing the excess ODN3 that was not conjugated to streptavidin. Next, a GCAT-based oligonucleotide (ODN2) complementary to ODN1 was hybridized to ODN1, and a GCATiGiC-based oligonucleotide (ODN4) complementary to ODN3 was hybridized to ODN3. Both ODN2 and ODN4 were luminescently labeled, and ODN4 was further conjugated to a second streptavidin. This step yielded an ODN1 / ODN2-SV-ODN3 / ODN4-SV intermediate structure. Figure 9B shows the ODN1 / ODN2-SV-ODN3 / ODN4-SV intermediate structure and a retention plot representing excess species that were not conjugated to streptavidin or hybridized to the conjugated oligonucleotide. Finally, to prepare a luminescently labeled oligonucleotide structure for sequencing, a terminator was added to one end of the structure, and a biotinylated amino acid recognition factor protein was added to the second streptavidin. The final step yielded the ODN1 / ODN2-SV-ODN3 / ODN4-SV-PS610 structure. Figure 9C shows a retention plot representing the ODN1 / ODN2-SV-ODN3 / ODN4-SV-PS610 structure. The sequences of ODN1, ODN2, ODN3, and ODN4 are provided in Table 2, where / X / is C530NS.

[0183] [Table 2]

[0184] Example 5 The luminescently labeled oligonucleotide structures from Example 4 were evaluated in polypeptide sequencing reactions to determine the effectiveness of the structures compared to standard luminescently labeled oligonucleotide structures. Figure 10A shows a representative trace from a polypeptide sequencing reaction using a first luminescent label comprising four copies of Cy®3, a second luminescent label comprising three copies of Cy®3B, and a third luminescent label comprising three copies of ATRho6G. Figure 10B shows a representative trace from a polypeptide sequencing reaction using a first luminescent label comprising four copies of Cy®3, a second luminescent label comprising three copies of Cy®3B, and a third luminescent label comprising three copies of C530NS. Figure 10C shows a representative trace from a polypeptide sequencing reaction using a first luminescent label comprising four copies of Cy®3, a second luminescent label comprising four copies of Cy®3B, a third luminescent label comprising two copies of ATRho6G, and a fourth luminescent label comprising eight copies of C530NS, the luminescently labeled oligonucleotide structure from Example 4. Figure 10C shows that clear resolution was achieved between the first, second, third, and fourth luminescent labels.

[0185] Example 6 Polypeptide sequencing runs were performed using amino acid recognition molecules labeled with a first luminescent label containing four copies of Cy®3 (termed "TetraCy3"), a second luminescent label containing four copies of Cy®3B (termed "TetraCy3B"), and a third luminescent label containing eight copies of Cy®3 (termed "OctaCy3").

[0186] A polypeptide sequencing run was performed on a sample peptide having the sequence FAAAYPDDD (SEQ ID NO: 17). Figure 11A shows a representative trace indicating that phenylalanine (F) was identified. Figure 11B shows a plot of intensity versus bin ratio. Figure 11B shows that the first, second, and third luminescent labels each occupied distinct spatial regions of the plot.

[0187] Example 7 Luminescently labeled oligonucleotide structures containing multiple luminescently labeled oligonucleotides were constructed using a stepwise ligation and conjugation approach, as shown schematically in Figure 12A. Two double-stranded oligonucleotides were prepared as shown: a first double-stranded oligonucleotide formed by hybridizing strands 1A and 1B, and a second double-stranded oligonucleotide formed by hybridizing strands 2A and 2B. Strands 1A, 1B, and 2B each contained two copies of an internal Cy®3, and strand 2A contained one internal amine conjugated to iFluor®570. Strand 1A contained a bis-biotin moiety at the 5' end. Strands 1B and 2B contained 5'-monophosphates in the overhanging regions of the complementary sequences. Table 3 provides sequence information for the strands used in this example.

[0188] [Table 3]

[0189] * Sequence notation: / 54 / : biotin (1-dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite); / 22 / : symmetric doubler (1,3-bis-[5-(4,4'-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); / 10 / : Cy®3 phosphoramidite; / MP / : 5'-monophosphate; and / Z / : internal amine phosphoramidite. The two double-stranded oligonucleotides were hybridized via the complementary overhang regions of strands 1B and 2B, followed by ligation using T4 DNA ligase to generate a single double-stranded oligonucleotide containing all six dyes. The ligated construct was purified by size-exclusion chromatography (Figure 12B) and conjugated to streptavidin via the bis-biotin moiety of strand 1A (Figure 12C). The streptavidin-conjugated construct was then conjugated to an amino acid recognition molecule (PS610) bearing a bis-biotin moiety (Figure 12D).

[0190] Example 8 An amino acid recognition run was performed on a sample peptide (FAAAYPDDD (SEQ ID NO: 17)) using an amino acid recognition molecule ("LC6IF") labeled according to Example 7. Figure 13A shows a representative trace (top) demonstrating the identification of phenylalanine (F), along with an intensity vs. bin ratio plot (bottom) demonstrating the clear spatial separation of LC6IF, C2C (Example 2), and a recognition molecule with four copies of Cy®3B ("4-Cy3B"). Figure 13B shows another representative trace (top) demonstrating the identification of phenylalanine (F), along with an intensity vs. bin ratio plot (bottom) demonstrating the clear spatial separation of LC6IF, C2C, and SG4Cy3 (Example 3).

[0191] Dynamic polypeptide sequencing reactions were performed on a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) using a set of amino acid recognition molecules with different labels, including LC6IF. Figure 13C shows a representative trace (top) demonstrating amino acid recognition during sample peptide degradation, along with an intensity vs. bin ratio plot (bottom) demonstrating the distinct spatial separation of LC6IF and SG4Cy3. Figure 13D shows another representative trace (top) demonstrating amino acid recognition during sample peptide degradation, along with an intensity vs. bin ratio plot (bottom) demonstrating the distinct spatial separation of LC6IF, R1C1 (Example 1), and 4-Cy3B.

[0192] Figures 13E and 13F show plots of intensity versus bin ratio for dye sets that included C2C, 4-Cy3B, and a label with 8 copies of Cy® in a construct prepared by ligation according to Example 7 ("L8Cy3") (Figure 13E) or a label with 8 copies of Cy® in a construct prepared by dual streptavidin ligation according to Example 4 ("8Cy3") (Figure 13F). Figure 13G shows plots of intensity versus bin ratio for L8Cy3, LC6C, and LC6IF (top) and a table of corresponding values ​​(bottom).

[0193] An amino acid recognition run was performed on the sample peptide (FAAAYPDDD (SEQ ID NO: 17)) using a set of seven amino acid recognition molecules with different labels, including LC6IF. Figure 13H shows a representative trace (top) demonstrating the identification of phenylalanine (F), and an intensity vs. bin ratio plot (bottom) showing the clear spatial separation of the seven distinctly labeled recognition molecules.

[0194] Dynamic polypeptide sequencing reactions were performed on a sample peptide (DQLRLAGGK (SEQ ID NO: 20)) using a set of differentially labeled amino acid recognition molecules, including LC6IF. Figure 13I shows a representative trace (top) demonstrating amino acid recognition during sample peptide degradation, along with an intensity versus bin ratio plot (bottom) demonstrating the distinct spatial separation of the seven distinctly labeled recognition molecules.

[0195] The results of this example demonstrate that labeled oligonucleotides constructed by ligation (e.g., Figure 3B) are as effective as labeled oligonucleotides constructed via linkage molecules (e.g., Figure 3A) in separating different clusters in a two-dimensional plot of intensity versus bin ratio; thus, both constructs provide highly effective and distinguishable luminescent labels.

[0196] Equivalents and Scope In the claims, articles such as "a," "an," and "the" can mean one or more unless indicated to the contrary or clear from context. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, utilized in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from context. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise relevant to a given product or process. The invention includes embodiments in which two or more, or all of the members of a group are present in, utilized in, or otherwise relevant to a given product or process.

[0197] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in other claims that depend on the same base claim. Where elements are presented as lists, for example, in Markush group format, each subgroup of the same elements is also disclosed, and any element can be removed from the group. Generally, when the invention or aspects of the invention are referred to as including certain elements and / or features, it is understood that particular embodiments of the invention or aspects of the invention include or consist essentially of such elements and / or features. For simplicity, these embodiments are not specifically described herein.

[0198] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so connected, i.e., elements that are conjunctively present in some cases and non-conjunctively present in other cases. Elements listed with "and / or" should be construed in the same manner, i.e., as "one or more" of the elements so connected. Other elements, whether related or unrelated to the elements specifically identified by the "and / or" clause, may optionally be present. Thus, as a non-limiting example, a statement such as "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer to, in one embodiment, A only (optionally including elements other than B); in another embodiment, B only (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements), etc.

[0199] As used in this specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., the inclusion of at least one, but more than one, of a number or list of elements, optionally including additional unlisted items. "Only one of," "exactly one of," or, when used in the claims, "consisting of," refers to the inclusion of exactly one element of a number or list of elements, unless the term clearly indicates the contrary. In general, the term "or" as used herein shall be construed to indicate exclusive alternatives (i.e., "one or the other but not both") only when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0200] As used herein and in the claims, the phrase "at least one," when referring to a list of one or more elements, should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, may optionally be present, whether related to the specifically identified elements or not. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") means that in one embodiment, A is at least one, optionally including more than one, and B is absent (optionally including elements other than B); in another embodiment, B is at least one, optionally including more than one, and A is absent (optionally including elements other than A); in yet another embodiment, A is at least one, optionally including more than one, and B is at least one, optionally including more than one (optionally including other elements), etc.

[0201] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein including multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the steps or acts of the method are recited.

[0202] In the claims and the foregoing specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are understood to be open-ended, i.e., meaning including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as defined in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures. It should be recognized that embodiments described herein using open-ended transitional phrases (e.g., "comprising") also contemplate, in alternative embodiments, the features "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrases. For example, if the application describes "a composition comprising A and B," the application also contemplates the alternative embodiments "a composition consisting only of A and B" and "a composition consisting essentially of A and B."

[0203] Where ranges are specified, endpoints are included. Furthermore, unless otherwise specified or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are understood to contemplate any specific value or subrange within the stated range in different embodiments of the invention, to the tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise.

[0204] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and the present specification, the specification shall control. Furthermore, any particular embodiment of the present invention that falls within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are deemed known to those of skill in the art, they may be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0205] Those skilled in the art will recognize, or be able to ascertain or recognize using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the invention, as defined in the following claims.

[0206] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The description of an embodiment of a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. The description of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0207] Reference to the Electronic Sequence Listing The contents of the electronic sequence listing (R070870141WO00-SEQ-JIB.xml; size: 30,922 bytes; and creation date: October 20, 2023) are incorporated herein by reference in their entirety.

Claims

1. 1. A light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide comprising one or more first luminescent labels; a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide, the first complementary single-stranded oligonucleotide comprising one or more second luminescent labels; A luminescently labeled oligonucleotide structure, wherein the shortest distance between any first luminescent label and any second luminescent label is at least 10 nm.

2. The structure of claim 1 , wherein the first luminescent label is different from the second luminescent label.

3. The structure of claim 1 , wherein the first luminescent label is the same as the second luminescent label.

4. The structure of any one of claims 1 to 3, wherein the first luminescent label and the second luminescent label are fluorescent labels.

5. The structure of any one of claims 1 to 4, wherein the first and second luminescent labels comprise cyanine, rhodamine, ATTO-rhodamine, and / or BODIPY dyes.

6. The structure of any one of claims 1 to 5, wherein the first luminescent label comprises Cy®3, Cy®3B, ATRho6G, and / or C530NS.

7. The structure of any one of claims 1 to 6, wherein the first luminescent label comprises Cy®3.

8. The structure of any one of claims 1 to 6, wherein the first luminescent label comprises Cy®3B.

9. The structure of any one of claims 1 to 8, wherein the second luminescent label comprises Cy®3, Cy®3B, ATRho6G, and / or C530NS.

10. The structure of any one of claims 1 to 9, wherein the second luminescent label comprises Cy®3B.

11. The structure of any one of claims 1 to 9, wherein the second luminescent label comprises ATRho6G.

12. 11. The structure of any one of claims 1 to 7 and 9 to 10, wherein the first luminescent label comprises Cy®3 and the second luminescent label comprises Cy®3B.

13. 12. The structure of any one of claims 1 to 6, 8 to 9, and 11, wherein the first luminescent label comprises Cy®3B and the second luminescent label comprises ATRho6G.

14. The structure of any one of claims 1 to 13, wherein the first single-stranded oligonucleotide comprises two or more first luminescent labels, three or more first luminescent labels, or four or more first luminescent labels.

15. The structure of any one of claims 1 to 14, wherein the first complementary single-stranded oligonucleotide comprises two or more second luminescent labels, three or more second luminescent labels, or four or more second luminescent labels.

16. 16. The structure of any one of claims 1 to 7 and 9 to 15, wherein the first single-stranded oligonucleotide comprises two first luminescent labels, each of which comprises Cy®3.

17. 17. The structure of any one of claims 1 to 10, 12, and 14 to 16, wherein the first complementary single-stranded oligonucleotide comprises one second luminescent label, and the second luminescent label comprises Cy® 3B.

18. 18. The structure of any one of claims 1 to 17, wherein the first single-stranded oligonucleotide further comprises one or more third luminescent labels, and the third luminescent labels are different from the first luminescent labels.

19. 19. The structure of any one of claims 1 to 18, wherein the first complementary single-stranded oligonucleotide further comprises one or more fourth luminescent labels, and the fourth luminescent labels are different from the second luminescent labels.

20. 20. The structure of any one of claims 1 to 19, wherein the structure has a length of at least 50, at least 70, or at least 100 base pairs.

21. The structure of any one of claims 1 to 20, wherein the first single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence A.

22. 22. The structure of any one of claims 1 to 21, wherein the first complementary single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence B.

23. The structure of any one of claims 1 to 22, wherein the first single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence C.

24. The structure of any one of claims 1 to 23, wherein the first complementary single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence D.

25. 25. The structure of any one of claims 1 to 24, wherein the shortest distance between any first luminescent label and any second luminescent label is at least 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, or 20 nm.

26. The structure of any one of claims 1 to 25, wherein the first single-stranded oligonucleotide is bound to a first binding molecule.

27. 27. The structure of claim 26, wherein the first binding molecule comprises an avidin protein.

28. 28. The structure of claim 27, wherein the avidin protein comprises streptavidin.

29. The structure of any one of claims 1 to 28, wherein the first single-stranded oligonucleotide comprises a biotin moiety.

30. 30. The structure of claim 29, wherein the biotin moiety is a bis-biotin moiety.

31. 31. The structure of any one of claims 26 to 30, further comprising an amino acid recognition molecule attached to the first binding molecule.

32. 1. A light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide comprising two or more first luminescent labels; a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide, the first complementary single-stranded oligonucleotide comprising two or more first luminescent labels; A luminescently labeled oligonucleotide construct, wherein the first luminescent label comprises a cyanine dye.

33. 33. The structure of claim 32, wherein the cyanine dye comprises Cy®3.

34. 34. The structure of claim 32 or 33, wherein the first single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence E.

35. The structure of any one of claims 32 to 34, wherein the first complementary single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence F.

36. 1. A light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide bound to a first binding molecule; a first complementary single-stranded oligonucleotide hybridized to said first single-stranded oligonucleotide; a second single-stranded oligonucleotide bound to the first binding molecule; and a second complementary single-stranded oligonucleotide hybridized to said second single-stranded oligonucleotide; the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide are conjugated to one or more first luminescent labels; A luminescently labeled oligonucleotide construct, wherein the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide is conjugated to one or more second luminescent labels.

37. 37. The structure of claim 36, wherein the first binding molecule comprises an avidin protein.

38. 38. The structure of claim 37, wherein the avidin protein comprises streptavidin.

39. 39. The structure of any one of claims 36 to 38, wherein the first single-stranded oligonucleotide and / or the second single-stranded oligonucleotide comprises a biotin moiety.

40. 40. The structure of claim 39, wherein the biotin moiety comprises a bis-biotin moiety.

41. 41. The structure of any one of claims 36 to 40, wherein the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide is conjugated to two or more first luminescent labels.

42. 42. The structure of any one of claims 36 to 41, wherein the first single-stranded oligonucleotide and the first complementary single-stranded oligonucleotide are each conjugated to two or more first luminescent labels.

43. 43. The structure of any one of claims 36 to 42, wherein the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide is conjugated to two or more second luminescent labels.

44. 44. The structure of any one of claims 36 to 43, wherein the second single-stranded oligonucleotide and the second complementary single-stranded oligonucleotide are each conjugated to two or more second luminescent labels.

45. 45. The structure of any one of claims 36 to 44, wherein the first luminescent label comprises Cy®3, Cy®3B, ATRho6G, and / or C530NS.

46. 46. ​​The structure of any one of claims 36 to 45, wherein the second luminescent label comprises Cy®3, Cy®3B, ATRho6G, and / or C530NS.

47. 47. The structure of any one of claims 36 to 46, wherein the structure comprises at least four luminescent labels.

48. 48. The structure of any one of claims 36 to 47, wherein the structure comprises at least eight luminescent labels.

49. 49. The structure of any one of claims 36 to 48, wherein the second complementary single-stranded oligonucleotide is bound to a second binding molecule.

50. 50. The structure of claim 49, further comprising a third single-stranded oligonucleotide bound to the second binding molecule.

51. 51. The construct of claim 50, further comprising a third complementary single-stranded oligonucleotide hybridized to said third single-stranded oligonucleotide.

52. 52. The structure of any one of claims 49 to 51, wherein the second binding molecule comprises an avidin protein.

53. 53. The structure of claim 52, wherein the avidin protein comprises streptavidin.

54. 54. The structure of any one of claims 50 to 53, further comprising an amino acid recognition molecule attached to the second binding molecule.

55. 55. The structure of any one of claims 50 to 54, wherein the third single-stranded oligonucleotide comprises a biotin moiety.

56. 56. The structure of claim 55, wherein the biotin moiety is a bis-biotin moiety.

57. 57. The structure of any one of claims 50 to 56, wherein the third single-stranded oligonucleotide and / or the third complementary single-stranded oligonucleotide is conjugated to one or more third luminescent labels.

58. 58. The structure of claim 57, wherein the third single-stranded oligonucleotide and / or the third complementary single-stranded oligonucleotide is conjugated to two or more third luminescent labels.

59. 59. The structure of claim 58, wherein the third single-stranded oligonucleotide and the third complementary single-stranded oligonucleotide are each conjugated to two or more third luminescent labels.

60. 60. The structure of any one of claims 36 to 59, wherein the structure is at least 70 base pairs in length.

61. 61. The structure of any one of claims 36 to 60, wherein the structure is at least 100 base pairs in length.

62. 62. The structure of any one of claims 36 to 61, wherein the first single-stranded oligonucleotide comprises one or more isoguanine and / or isocytosine nucleotides and the second single-stranded oligonucleotide does not comprise one or more isoguanine and / or isocytosine nucleotides.

63. 63. The structure of any one of claims 36 to 62, wherein the second single-stranded oligonucleotide comprises one or more isoguanine and / or isocytosine nucleotides and the first single-stranded oligonucleotide does not comprise one or more isoguanine and / or isocytosine nucleotides.

64. 64. The structure of any one of claims 36 to 63, wherein the first single-stranded oligonucleotide or the second single-stranded oligonucleotide comprises at least one diaminopurine nucleotide.

65. 1. A system comprising: an integrated device comprising a plurality of sample wells, one or more of the sample wells adapted to have a polypeptide immobilized on its surface; one or more first amino acid recognition molecules attached to a first light-emitting label comprising a first light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide comprising one or more first fluorophores; and a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide, the first complementary single-stranded oligonucleotide comprising one or more second fluorophores; one or more first amino acid recognition molecules, wherein the shortest distance between any first fluorophore and any second fluorophore is at least 10 nm.

66. 66. The system of claim 65, further comprising one or more second amino acid recognition molecules linked to a second luminescent label.

67. 67. The system of claim 66, wherein the first light-emitting indicator has a first value for a first characteristic, the second light-emitting indicator has a second value for the first characteristic, and the percentage difference between the first value and the second value is at least 20%.

68. 68. The system of claim 67, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

69. 69. The system of any one of claims 65 to 68, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, and the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the first ordered pair and the second ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

70. 70. The system of claim 69, wherein the first characteristic is different from the second characteristic.

71. 71. The system of claim 69 or 70, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

72. 72. The system of any one of claims 69 to 71, wherein the first ordered pair and the second ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

73. 73. The system of any one of claims 65 to 72, further comprising one or more third amino acid recognition molecules linked to a third luminescent label.

74. 74. The system of claim 73, wherein the first light-emitting indicator has a first value for a first characteristic, the second light-emitting indicator has a second value for the first characteristic, and the third light-emitting indicator has a third value for the first characteristic, and wherein a minimum percentage difference between the first value, the second value, and the third value is at least 20%.

75. 75. The system of claim 74, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

76. 76. The system of claim 73, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the third light-emitting indicator has a third ordered pair of characteristics including a third value of the first characteristic and a third value of the second characteristic, and the first ordered pair, the second ordered pair, and the third ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

77. 77. The system of claim 76, wherein the first characteristic is different from the second characteristic.

78. 78. The system of claim 76 or 77, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

79. 79. The system of any one of claims 76 to 78, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

80. 1. A system comprising: an integrated device comprising a plurality of sample wells, one or more of the sample wells adapted to have a polypeptide immobilized on its surface; one or more first amino acid recognition molecules attached to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide comprising two or more first fluorophores; and a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide, the first complementary single-stranded oligonucleotide comprising two or more first fluorophores; one or more first amino acid recognition molecules, wherein the first fluorophore comprises a cyanine dye.

81. 81. The system of claim 80, wherein the cyanine dye comprises Cy®3.

82. 82. The system of claim 80 or 81, wherein the first single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence E.

83. 83. The system of any one of claims 80 to 82, wherein the first complementary single-stranded oligonucleotide comprises a sequence that is at least 80% identical to sequence F.

84. 84. The system of any one of claims 80 to 83, further comprising one or more second amino acid recognition molecules linked to a second luminescent label.

85. 85. The system of claim 84, wherein the first light-emitting indicator has a first value for a first characteristic, the second light-emitting indicator has a second value for the first characteristic, and the percentage difference between the first value and the second value is at least 20%.

86. 86. The system of claim 85, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

87. 87. The system of any one of claims 84 to 86, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, and the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the first ordered pair and the second ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

88. 88. The system of claim 87, wherein the first characteristic is different from the second characteristic.

89. 89. The system of claim 87 or 88, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

90. 90. The system of any one of claims 87 to 89, wherein the first ordered pair and the second ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

91. 91. The system of any one of claims 80 to 90, further comprising one or more third amino acid recognition molecules linked to a third luminescent label.

92. 92. The system of claim 91, wherein the first light-emitting indicator has a first value for a first characteristic, the second light-emitting indicator has a second value for the first characteristic, and the third light-emitting indicator has a third value for the first characteristic, and a minimum percentage difference between the first value, the second value, and the third value is at least 20%.

93. 93. The system of claim 92, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

94. 94. The system of claim 91, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the third light-emitting indicator has a third ordered pair of characteristics including a third value of the first characteristic and a third value of the second characteristic, and the first ordered pair, the second ordered pair, and the third ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

95. 95. The system of claim 94, wherein the first characteristic is different from the second characteristic.

96. 96. The system of claim 94 or 95, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

97. 97. The system of any one of claims 94 to 96, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

98. 1. A system comprising: an integrated device comprising a plurality of sample wells, one or more of the sample wells adapted to have a polypeptide immobilized on its surface; one or more first amino acid recognition molecules attached to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide bound to a first binding molecule; a first complementary single-stranded oligonucleotide hybridized to said first single-stranded oligonucleotide; a second single-stranded oligonucleotide bound to the first binding molecule; and a second complementary single-stranded oligonucleotide hybridized to said second single-stranded oligonucleotide; the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide are conjugated to one or more first fluorophores; one or more first amino acid recognition molecules, wherein the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide are conjugated to one or more second fluorophores.

99. 99. The system of claim 98, further comprising one or more second amino acid recognition molecules linked to a second luminescent label.

100. 100. The system of claim 99, wherein the first luminous indicator has a first value for a first characteristic, the second luminous indicator has a second value for the first characteristic, and the percentage difference between the first value and the second value is at least 20%.

101. 101. The system of claim 100, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

102. 102. The system of claim 99, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, and the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the first ordered pair and the second ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

103. 103. The system of claim 102, wherein the first characteristic is different from the second characteristic.

104. 104. The system of claim 102 or 103, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

105. 105. The system of any one of claims 102 to 104, wherein the first ordered pair and the second ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

106. 106. The system of any one of claims 98 to 105, further comprising one or more third amino acid recognition molecules linked to a third luminescent label.

107. 107. The system of claim 106, wherein the first light-emitting indicator has a first value for a first characteristic, the second light-emitting indicator has a second value for the first characteristic, and the third light-emitting indicator has a third value for the first characteristic, and a minimum percentage difference between the first value, the second value, and the third value is at least 20%.

108. 108. The system of claim 107, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

109. 109. The system of any one of claims 106 to 108, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the third light-emitting indicator has a third ordered pair of characteristics including a third value of the first characteristic and a third value of the second characteristic, and the first ordered pair, the second ordered pair, and the third ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

110. 110. The system of claim 109, wherein the first characteristic is different from the second characteristic.

111. 111. The system of claim 109 or 110, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

112. 112. The system of any one of claims 109 to 111, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

113. 1. A method for determining the chemical properties of a polypeptide, comprising: contacting the polypeptide with one or more first amino acid recognition molecules attached to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide comprising one or more first fluorophores; and a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide, the first complementary single-stranded oligonucleotide comprising one or more second fluorophores; contacting, wherein the shortest distance between any first fluorophore and any second fluorophore is at least 10 nm; detecting a first series of signal pulses indicative of a first series of binding events between the one or more first amino acid recognition molecules and the polypeptide; determining at least one chemical property of an amino acid of the polypeptide based on at least one property of the first series of signal pulses; A method comprising:

114. 114. The method of claim 113, further comprising contacting the polypeptide with one or more second amino acid recognition molecules linked to a second luminescent label.

115. 115. The method of claim 114, wherein the first luminescent label has a first value for a first characteristic, the second luminescent label has a second value for the first characteristic, and the percentage difference between the first value and the second value is at least 20%.

116. 116. The method of claim 115, wherein the first property comprises luminescence intensity and / or luminescence lifetime.

117. 117. The method of any one of claims 114 to 116, wherein the first light-emitting indicator has a first ordered pair of characteristics comprising a first value of a first characteristic and a first value of a second characteristic, and the second light-emitting indicator has a second ordered pair of characteristics comprising a second value of the first characteristic and a second value of the second characteristic, and the first ordered pair and the second ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

118. 118. The method of claim 117, wherein the first characteristic is different from the second characteristic.

119. 119. The method of claim 117 or 118, wherein the first property comprises luminescence intensity and the second property comprises luminescence lifetime.

120. 120. A method according to any one of claims 117 to 119, wherein the first ordered pair and the second ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

121. 121. The method of any one of claims 113 to 120, further comprising contacting the polypeptide with one or more third amino acid recognition molecules linked to a third luminescent label.

122. 122. The method of claim 121, wherein the first luminescent label has a first value for a first characteristic, the second luminescent label has a second value for the first characteristic, and the third luminescent label has a third value for the first characteristic, and a minimum percentage difference between the first value, the second value, and the third value is at least 20%.

123. 123. The method of claim 122, wherein the first property comprises luminescence intensity and / or luminescence lifetime.

124. 124. The method of any one of claims 121 to 123, wherein the first light-emitting indicator has a first ordered pair of characteristics comprising a first value of a first characteristic and a first value of a second characteristic, the second light-emitting indicator has a second ordered pair of characteristics comprising a second value of the first characteristic and a second value of the second characteristic, and the third light-emitting indicator has a third ordered pair of characteristics comprising a third value of the first characteristic and a third value of the second characteristic, and the first ordered pair, the second ordered pair, and the third ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

125. 125. The method of claim 124, wherein the first characteristic is different from the second characteristic.

126. 126. The method of claim 124 or 125, wherein the first property comprises luminescence intensity and the second property comprises luminescence lifetime.

127. 127. A method according to any one of claims 124 to 126, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

128. 1. A method for determining the chemical properties of a polypeptide, comprising: contacting the polypeptide with one or more first amino acid recognition molecules attached to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide comprising two or more first fluorophores; and a first complementary single-stranded oligonucleotide hybridized to the first single-stranded oligonucleotide, the first complementary single-stranded oligonucleotide comprising two or more first fluorophores; contacting, wherein the first luminescent label comprises a cyanine dye; detecting a first series of signal pulses indicative of a first series of binding events between the one or more amino acid recognition molecules and the polypeptide; determining at least one chemical property of an amino acid of the polypeptide based on at least one property of the first series of signal pulses; A method comprising:

129. 129. The method of claim 128, further comprising contacting the polypeptide with one or more second amino acid recognition molecules linked to a second luminescent label.

130. 130. The method of claim 129, wherein the first luminescent label has a first value for a first characteristic, the second luminescent label has a second value for the first characteristic, and the percentage difference between the first value and the second value is at least 20%.

131. 131. The method of claim 130, wherein the first property comprises luminescence intensity and / or luminescence lifetime.

132. 132. The method of any one of claims 129 to 131, wherein the first light-emitting indicator has a first ordered pair of characteristics comprising a first value of a first characteristic and a first value of a second characteristic, and the second light-emitting indicator has a second ordered pair of characteristics comprising a second value of the first characteristic and a second value of the second characteristic, and the first ordered pair and the second ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

133. 133. The method of claim 132, wherein the first characteristic is different from the second characteristic.

134. 134. The method of claim 132 or 133, wherein the first property comprises luminescence intensity and the second property comprises luminescence lifetime.

135. A method according to any one of claims 132 to 134, wherein the first ordered pair and the second ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

136. 136. The method of any one of claims 132 to 135, further comprising contacting the polypeptide with one or more third amino acid recognition molecules linked to a third luminescent label.

137. 137. The method of claim 136, wherein the first luminescent label has a first value for a first characteristic, the second luminescent label has a second value for the first characteristic, and the third luminescent label has a third value for the first characteristic, and a minimum percentage difference between the first value, the second value, and the third value is at least 20%.

138. 138. The method of claim 137, wherein the first property comprises luminescence intensity and / or luminescence lifetime.

139. 139. The method of any one of claims 136 to 138, wherein the first light-emitting indicator has a first ordered pair of characteristics comprising a first value of a first characteristic and a first value of a second characteristic, the second light-emitting indicator has a second ordered pair of characteristics comprising a second value of the first characteristic and a second value of the second characteristic, and the third light-emitting indicator has a third ordered pair of characteristics comprising a third value of the first characteristic and a third value of the second characteristic, and wherein the first ordered pair, the second ordered pair, and the third ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic, respectively.

140. 140. The method of claim 139, wherein the first characteristic is different from the second characteristic.

141. 141. The method of claim 139 or 140, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

142. 142. A method according to any one of claims 139 to 141, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

143. 1. A method for determining the chemical properties of a polypeptide, comprising: contacting the polypeptide with one or more first amino acid recognition molecules attached to a first light-emitting label comprising a light-emitting labeled oligonucleotide structure, said structure comprising: a first single-stranded oligonucleotide bound to a first binding molecule; a first complementary single-stranded oligonucleotide hybridized to said first single-stranded oligonucleotide; a second single-stranded oligonucleotide bound to the first binding molecule; and a second complementary single-stranded oligonucleotide hybridized to said second single-stranded oligonucleotide; the first single-stranded oligonucleotide and / or the first complementary single-stranded oligonucleotide are conjugated to one or more first fluorophores; contacting, wherein the second single-stranded oligonucleotide and / or the second complementary single-stranded oligonucleotide are conjugated to one or more second fluorophores; detecting a first series of signal pulses indicative of a first series of binding events between the one or more amino acid recognition molecules and the polypeptide; determining at least one chemical property of an amino acid of the polypeptide based on at least one property of the first series of signal pulses; A method comprising:

144. 144. The method of claim 143, further comprising contacting the polypeptide with one or more second amino acid recognition molecules linked to a second luminescent label.

145. 145. The method of claim 144, wherein the first luminescent label has a first value for a first characteristic, the second luminescent label has a second value for the first characteristic, and the percentage difference between the first value and the second value is at least 20%.

146. 146. The method of claim 145, wherein the first property comprises luminescence intensity and / or luminescence lifetime.

147. 147. The method of any one of claims 144 to 146, wherein the first light-emitting indicator has a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic, and the second light-emitting indicator has a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic, and the first ordered pair and the second ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

148. 148. The method of claim 147, wherein the first characteristic is different from the second characteristic.

149. 149. The method of claim 147 or 148, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

150. A method according to any one of claims 147 to 149, wherein the first ordered pair and the second ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

151. 151. The method of any one of claims 143 to 150, further comprising contacting the polypeptide with one or more third amino acid recognition molecules linked to a third luminescent label.

152. 152. The method of claim 151, wherein the first luminescent label has a first value for a first characteristic, the second luminescent label has a second value for the first characteristic, and the third luminescent label has a third value for the first characteristic, and a minimum percentage difference between the first value, the second value, and the third value is at least 20%.

153. 153. The method of claim 152, wherein the first characteristic comprises luminescence intensity and / or luminescence lifetime.

154. 154. The method of any one of claims 151 to 153, wherein the first light-emitting indicator has a first ordered pair of characteristics comprising a first value of a first characteristic and a first value of a second characteristic, the second light-emitting indicator has a second ordered pair of characteristics comprising a second value of the first characteristic and a second value of the second characteristic, and the third light-emitting indicator has a third ordered pair of characteristics comprising a third value of the first characteristic and a third value of the second characteristic, and the first ordered pair, the second ordered pair, and the third ordered pair differ in at least one of the values ​​of the first characteristic and / or the second characteristic.

155. 155. The method of claim 154, wherein the first characteristic is different from the second characteristic.

156. 156. The method of claim 154 or 155, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

157. 157. A method according to any one of claims 154 to 156, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

158. 1. A system comprising: a first luminescent indicator having a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic; a second light-emitting indicator having a second ordered pair of characteristics including a second value of the first characteristic and a second value of the second characteristic; a third light-emitting indicator having a third ordered pair of characteristics including a third value of the first characteristic and a third value of the second characteristic; wherein the first ordered pair, the second ordered pair, and the third ordered pair differ from each other in at least one of the respective values ​​of the first characteristic and / or the second characteristic.

159. 159. The system of claim 158, wherein the first characteristic is different from the second characteristic.

160. 160. The system of claim 158 or 159, wherein the first characteristic is related to luminescence intensity.

161. 161. The system of any one of claims 158 to 160, wherein the second property is related to luminescence lifetime.

162. A system described in any one of claims 158 to 161, wherein the first luminescent label, the second luminescent label, and the third luminescent label occupy different positions on a plot of the first characteristic and the second characteristic.

163. The system of any one of claims 158 to 162, wherein at least one of the first light-emitting label, the second light-emitting label, and the third light-emitting label is a light-emitting labeled oligonucleotide structure comprising a first single-stranded oligonucleotide comprising one or more first light-emitting labels and a first complementary single-stranded oligonucleotide comprising one or more second light-emitting labels.

164. The system of any one of claims 158 to 163, wherein the first luminescent label comprises R1C1.

165. The system of any one of claims 158 to 164, wherein the second luminescent label comprises C2C.

166. The system of any one of claims 158 to 165, wherein the third luminescent label comprises SG4Cy3.

167. The system of any one of claims 158 to 166, further comprising a fourth light-emitting sign having a fourth value of the first light-emitting characteristic and a fourth value of the second light-emitting characteristic, wherein a minimum percentage difference between the first value, the second value, the third value, and the fourth value of the first light-emitting characteristic is at least 20%, and a minimum percentage difference between the first value, the second value, the third value, and the fourth value of the second light-emitting characteristic is at least 20%.

168. The system of claim 167, wherein the fourth luminescent label comprises at least one copy of ATRho6G.

169. The system of any one of claims 158 to 168, further comprising a fifth light-emitting sign having a fifth value of the first light-emitting characteristic and a fifth value of the second light-emitting characteristic, wherein a minimum percentage difference between the first value, the second value, the third value, the fourth value, and the fifth value of the first characteristic is at least 20%, and a minimum percentage difference between the first value, the second value, the third value, the fourth value, and the fifth value of the second characteristic is at least 20%.

170. 170. The system of claim 169, wherein the fifth luminescent label comprises at least one copy of Cy(R)3B.

171. 1. A system comprising: a first luminescent label having a first bin ratio value; a second luminescent label having a second bin ratio value; a third luminescent label having a third bin ratio value; A system wherein a minimum difference between the first bin ratio value, the second bin ratio value, and the third bin ratio value of a first emission characteristic is at least 0.

1.

172. The system of claim 171, wherein the first luminescent label comprises R1C1.

173. The system of claim 171 or 172, wherein the second luminescent label comprises C2C.

174. 174. The system of any one of claims 171 to 173, wherein the third luminescent label comprises SG4Cy3, at least one copy of ATRho6G, and / or at least one copy of Cy®3B.

175. A system according to any one of claims 171 to 174, wherein the minimum difference is at least 0.

2.

176. 1. A method comprising: providing a first light-emitting sign having a first ordered pair of characteristics including a first value of a first characteristic and a first value of a second characteristic; providing a second light-emitting sign having a second ordered pair of properties including a second value of the first property and a second value of the second property; providing a third light-emitting label comprising a light-labeled oligonucleotide structure comprising a first single-stranded oligonucleotide comprising one or more first fluorophores and a first complementary single-stranded oligonucleotide comprising one or more second fluorophores, wherein the third light-emitting label has a third ordered pair of properties comprising a third value of the first property and a third value of the second property; modifying the number and / or type of the one or more first fluorophores and / or the one or more second fluorophores such that the first ordered pair, the second ordered pair, and the third ordered pair differ from each other in at least one of the respective values ​​of the first property and / or the second property.

177. 177. The method of claim 176, wherein the first characteristic is different from the second characteristic.

178. 178. The method of claim 176 or 177, wherein the first characteristic comprises luminescence intensity and the second characteristic comprises luminescence lifetime.

179. 179. A method according to any one of claims 176 to 178, wherein the first ordered pair, the second ordered pair, and the third ordered pair occupy different positions on a plot of the first characteristic and the second characteristic.

180. 1. A method for preparing a luminescently labeled reaction component, said method comprising: ligating a first double-stranded oligonucleotide comprising a first light-emitting label to a second double-stranded oligonucleotide comprising a second light-emitting label, wherein the first double-stranded oligonucleotide comprises a first binding moiety; contacting the ligated double-stranded oligonucleotide with a multivalent protein that binds to the first binding moiety to form a complex comprising the ligated double-stranded oligonucleotide and the multivalent protein; contacting the complex with a reaction component comprising a second binding moiety, wherein the multivalent protein of the complex binds to the second binding moiety to form a luminescently labeled reaction component.

181. 181. The method of claim 180, wherein the first double-stranded oligonucleotide comprises two or more first luminescent labels.

182. 182. The method of claim 180 or 181, wherein the second double-stranded oligonucleotide comprises two or more second luminescent labels.

183. 183. The method of any one of claims 180 to 182, wherein the first luminescent label is different from the second luminescent label.

184. 183. The method of any one of claims 180 to 182, wherein the first luminescent label is the same as the second luminescent label.

185. 185. The method of any one of claims 180 to 184, wherein the first and second luminescent labels comprise cyanine dyes.

186. 186. The method of any one of claims 180-185, wherein the first and second luminescent labels are each independently selected from the group consisting of Cy®3, Cy®3B, ATRho6G, C530NS, and iFluor®570.

187. 187. The method of any one of claims 180 to 186, wherein the first or second double-stranded oligonucleotide comprises a third luminescent label different from the first and second luminescent labels.

188. 188. The method of claim 187, wherein each of the first luminescent label and the second luminescent label is Cy®3, and the third luminescent label is selected from the group consisting of Cy®3B, ATRho6G, C530NS, and iFluor®570.

189. 189. The method of claim 188, wherein the third luminescent label is iFluor® 570.

190. Prior to the ligation, contacting the first double-stranded oligonucleotide with the second double-stranded oligonucleotide under hybridization conditions; the first double-stranded oligonucleotide comprises a first overhang; the second double-stranded oligonucleotide comprises a second overhang complementary to the first overhang; 190. The method of any one of claims 180-189, wherein the hybridization conditions are sufficient to hybridize the first overhang of the first double-stranded oligonucleotide to the second overhang of the second double-stranded oligonucleotide.

191. 191. The method of any one of claims 180 to 190, wherein the first and second luminescent labels of the luminescently labeled reaction components are separated from each other by a distance of at least 10 nm.

192. 192. The method of any one of claims 180 to 191, wherein the first double-stranded oligonucleotide comprises one or more isoguanine and / or isocytosine nucleotides, and the second double-stranded oligonucleotide does not comprise one or more isoguanine and / or isocytosine nucleotides.

193. 193. The method of any one of claims 180 to 192, wherein the second double-stranded oligonucleotide comprises one or more isoguanine and / or isocytosine nucleotides, and the first double-stranded oligonucleotide does not comprise one or more isoguanine and / or isocytosine nucleotides.

194. 194. The method of any one of claims 180 to 193, wherein the first or second double-stranded oligonucleotide comprises at least one diaminopurine nucleotide.

195. 195. The method of any one of claims 180 to 194, wherein the first and second binding moieties are a first biotin moiety and a second biotin moiety, respectively.

196. 196. The method of claim 195, wherein at least one of the first and second biotin moieties is a bis-biotin moiety.

197. 197. The method of any one of claims 180 to 196, wherein the multivalent protein is an avidin protein.

198. 198. The method of claim 197, wherein the avidin protein comprises streptavidin.

199. 200. The method of any one of claims 180-198, wherein the reaction component comprises an amino acid recognition molecule.

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