10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

{0068} In determining a sequence of amino acids in analyte 212 in anchored analyte
226, with reference to FIG. 10, N-terminal amino acid 220 can be converted to inert residue
232. As used herein, “inert residue” refers to an amino acid that does not bind to amino acid-
specific binder 200. The inert residue can be subsequently removed to expose the new
penultimate residue 234 such that every new amino acid in analyte 212 can be sequentially
subjected to binder complex 228 for sequencing.

[0069] With reference to FIG. 11, selectively binding binder complex 228 to
analyte 212, not in anchored analyte 226, forms isolation complex 244, e.g.. to isolate analyte
212, to determine an identity of amino acid 209 in analyte 212 of isolation complex 244 and
the like, Isolation complex 244 can be isolated from a heterogeneous composition containing
analyte 212 using properties of isolation complex 244 such as the molecular weight. A
difference in molecular weight between the isolation complex and undesired components in
the composition must be great enough so that isolation complex 244 can be separated from
other constituents in the composition by dialysis, chromatography. and the like.

[0070] Amino acid-specific binder 200 can be made in various ways. A process for
making amino acid-specific binder 200 can include selecting a sequence for amino acid-
specific binder 200 and expressing and purifying amine acid-specific binder 200 from an
organism or by recombinant formation. A protein can be purified from the organism with a
purification technique. Purification can include ion-exchange on a column that includes a
cation- exchanger column or anion-exchanger column (e.g. diethylaminomethyl (DEAE)
column), a mixed-mode ion exchanger (e.g., hydroxyapatite), or column that separates proteins
based on hydrophobicity. A protein can be purified by size exclusion chromatography (e.g..
gel-filiration) or in a density gradient (such as glycerol). Purification can be performed with
binding to a different column that can include a specific chemical characterization of each

protein. For recombinant expression in Lsch

chia eoli, purification can be facilitated using

a tag such as histidine, maltose binding protein (MBP). glutathione S-transferase (GST). and

the like. A gene can be cloned into a pET15b vector with an additional HisG-tag at an N-
terminus of the protein, followed by a tobacco etch virus (TEV) protease cleavage site
(MGHHHHHHENLYFQG (Sequence ID No. 13)}, using the Neo! and Xhol restriction sites
and expressed in BL21 ©. coli cells. Expression from pET vector is induced with 06.5 mM
IPTG when optical density at 600 nm (ODooo) reaches 1.0 absorbance units and further
incubated for 6 hours at 37°C or 16 hours at 15°C. Cells are harvested by centrifugation at
5000 g for 20 minutes, and cell pellets can be frozen. Frozen cell pellets are resuspended in a

lysis buffer (¢.g., 100 mM Tris-HCl, pH 8.0,

mM NaCl, 25 mM imidazole, or SO mM

19


WO 2022/236190 PCT/US2022/028400

18. The process of embodiment 17, further comprising:
subjecting the taggant of the isolation complex to a stimulus:
producing, by the taggant of the isolation complex, a taggant signal in response to the
stimulus;
5 detecting the taggant signal: and

identifying the N-terminal amino acid based on the taggant signal

19. The process of embodiment 17, wherein the stimulus comprises a photon: and
the taggant signal comprises fluorescence emitted from the taggant.
10
20. The process of embodiment 11, further comprising:
contacting the amino acid-specific binder with an adjunct to form a binder complex

ptior to contacting the amino acid-specific binder with the analyte.

40


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

removing the N-terminal amino acid from the anchored analyte so that a penultimate
residue becomes the N-terminal amino acid of the anchored analyte:

contacting the N-terminal amino acid of the anchored analyte with the binder complex:

selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid is the binding
amino acid to form the tagged complex:

subjecting the taggant of the tagged complex to the stimulus;

producing, by the taggant of the tagged complex, the taggant signal in response to the
stimulus;

detecting the taggant signal; and

identifying the N-terminal amino acid based on the taggant signal.

8. The process of embodiment 5, further comprising:

converting the N-terminal amino acid to an inert residue:

converting a penultimate residue to be the N-terminal amino acid when the inert residue
is produced:

contacting the N-terminal amino acid of the anchored analyte with the binder complex:

selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid is the binding
amino acid to form the tagged complex:

subjecting the taggant of the tagged complex to the stimulus;

producing, by the taggant of the tagged complex, the taggant signal in response to the
stimulus;

detecting the taggant signal; and

identifying the N-terminal amino acid based on the taggant signal.

9. The process of embodiment 8, wherein converting the N-terminal amino acid to the
inert residue comprises chemically changing the N-terminal amino acid prior to

producing the inert residue.

10, The process of embodiment 9, wherein chemically changing the N-terrninal amino
acid prior to producing the inert residue comprises phosphorylating a free amine of the

N-terminal amino acid.


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

QVHYEGQAIV WVGPQVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 3);
ASVVPQERQQDTRKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNKTSDRAWELTN
QOVHYEGOAIV WVGPQEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D No, 4);
ASVVPQERQQVTRKHY PNYKVIVLNDDFENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYKGQAIV WVGPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 5):
ASVVPQERQOV ARKHYPNYK VIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTIN
QVHYEGQAIV WVGPQEQAELY HEOLLRAGLTMAPLEPE (Sequence ID No. 6);
ASVVPQERQODTRKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYKGQAIV WVGPOVQAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 7):
ASVVPQERQQDTRKHYPNYKVIVLNDDFNTERHV AACLMK YIPNMTSDRAWELTN
OVHYEGOAIVWVGPOVQAELYHEOLLRAGLTMAPLEPG (Sequence ID No. 8):
ASVVPQEGQQVTRKHY PNYK VIVLNDDENTFQHVV ACLKKYIPNMISDRAWELTNO
VHYGGRAIV WVGPQEQAELNHEQLLRAGLTMAPLEPE (Sequence 1D No. 9); or
ASVVPQERQOV ARKHY PNYK VIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGGAIV WVGPQVQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10}

7. The process of claim 5, further comprising:

removing the N-terminal amino acid from the anchored analyte so that a penultimate
residue becomes the N-terminal amino acid of the anchored analyte:

contacting the N-terminal amino acid of the anchored analyte with the binder complex:

selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid is the binding
amino acid to form the tagged complex:

subjecting the taggant of the tagged complex to the stimulus:

producing. by the taggant of the tagged complex, the taggant signal in response to the
stimulus:

detecting the taggant signal; and

identifying the N-terminal amino acid based on the taggant signal

8, The process of claim 5, further comprising:
converting the N-terminal amine acid to an mert residue:
converting a penultimate residue to be the N-terminal amino acid when the inert residue

is produced;

45


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

binding amino acid 210, e.g.. of analyte 212), and the like. Further, adjunct 236 can be attached
to amino acid-specific binder 200 either directly, indirectly, or a combination thereof, With
reference to FIG. 3, when adjunct 236 is directly attached to amino acid-specific binder 200,
direct attachment occurs in an absence of an intervening member between adjunct 236 and
amino acid-specific binder 200 as shown in panel A. When adjunet 236 is indirectly attached
to amino acid-specific binder 200 as shown in panel B, indirect attachment occurs in a presence
of the intervening member 246 between adjunct 236 and amino acid-specific binder 200.
Accordingly, with reference to FIG. 2, amino acid-specific binder 200 selectively binds to
binding amino acid 210 of analyte 212 but does not bind to non-binding amino acid 211

[0046] In binder complex 228, with reference to FIG. 3, adjunct 236 can determine
a position or identity of amino acid-specific binder 200 and determine if amino acid-specific
binder 200 is bound to analyte 212. Adjunct 236 can be taggant 214, protein 238, substrate
240, chemical modifier 242, or a combination thereof, e.g, as shown in FIG. 4. In an
embodiment, adjunct 236 includes taggant 214. In an embodiment. adjunct 236 includes a
substrate such that analyte 212 can be immobilized when in contact with amino acid-specific
binder 200. Binder complex 228 can include an arbitrary number of amino acid-specific binder
200 and adjunct 236 that can be connectedly attached in an arbitrary arrangement as shown in
FIG. 5

{0047} Protein 238 can include a protein to facilitate expression or purification of
amino-acid specific binder 200 such as a protein with a functional group that can be
immobilized on a resin, an antibody, Protein A, Protein G, a peptide of six histidine residues,
Glutathione S-transferase. maltose binding protein, biotin, or streptavidin. Moreover, protein
238 can include a protein with a reactive property such as enzymatic activity, a protease
cleavage site, or fluorescence that can be stimulated to produce a signal and can be green
fluorescent protein, horseradish peroxidase, luciferase, and the like. Moreover. protein 238 can
include proteins with a selected molecular weight, isoelectric point, or functional group that

can facilitate separation of binding complex 238, ¢.g., by dialysis, chromatography, or gradient

centrifugation. Exemplary proteins 238 include an immunoglobulin, a high molecular weight

protein CHMWP), DNA-binding protein, oligosaccharide binding protein, and the like. In an

embodiment, protein 238 is biotinylated and can be attached to a substrate through interaction
with streptavidin.

[0048] Substrate 240 can include magnetic beads, fluorescent beads, silica

coverslips, or microplates to attach amino acid-specific binder 200 to the substrate surface and

can be a functionalized glass slide. Moreover, the substrate can be used for localization of


WO 2022/236190

&)

PCT/US2022/028400
6/20
A mB 2 © a)
yy
is Pa re 206 208 a
7 ©
ae ra 0 Par
242, 248, 280
0) a
22, 288, 250

9
i H i H q
4 NN 3
Y Y ~~
2A 288 20K
FIG.6

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

4/20
A) om 8 28

n4 ae B a

0) 228

ne
eS
oS

2m 20) shu

FIG. 4

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

10/20

os
he
eS

OA 2H INR ANC AMD 224

a p22
2 at ait
22
| Convert 2094
8 2
SN 22) 0K 24 2080 A
28 Kon

Na

FIG, 10

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

14/20

FIG, 14

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

2/20

25 20 210
B 20h
ve 208
Se 0 Q Ni
25 i) OK
FIG. 2

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
os

WO 2022/236190 PCT/US2022/028400

amino acid 210 to form tagged complex 230 by incubating anchored analyte 226 and binder
complex 228 in a reaction buffer for a time (e.g., from 5 sec to 30 min) for the binding reaction
to occur based on a binding affinity of amino acid-specific binder 200 under a set of binding
conditions (e.g., in LX PBS at 30°C). When N-terminal amino acid 220 includes binding amino

acid 210, the binding reaction occurs, and the tagged complex forms.

[0085] The process for selectively identifying N-terminal amino acid 220, also can
include subjecting taggant 214 of tagged complex 230 to stimulus 218. In an embodiment,
tagged complex 230 is exposed to a selected wavelength and intensity of light to excite the
fluorophore. In an embodiment, subjecting taggant 214 of tagged complex 230 to stimulus 218
includes adding a chromogenic substrate. Table 1 lists a taggant, stimulant, signal, and

detection for adjuncts shown in Table 2.

[0086} The process for selectively identifying N-terminal amino acid 220 also can

include producing, by taggant 214 of tagged complex 230, taggant signal 216 in response to

stimulus 218. In an embodiment, taggant 214 is a fluorophore that emit light as taggant
response at an emission wavelength after being stimulated by an excitation wavelength as the
stimulus. In an embodiment, chromogente substrate produces a chromogenic signal as when
contacted by HRP as taggant 214.

{0087} The process for selectively identifying N-terminal amino acid 220 also can
include detecting taggant signal 216 by methods listed in Table 2 for each taggant. In an
embodiment, detection can involve a microscope with a CCD camera and selected filters in an
optical system that detects a wavelength of emitted light. In an embodiment. a
spectrophotometer measures absorbance at a selected wavelength to detect a chromogenic

substrate. In an embodiment, a scintillation counter measures radioactivity of a radiolabeled

complex.
Table 2
Adjunct Isolation Technique Separation Property
High molecular weight Dialysis Size
protein
High molecular weight Ultracentrifugation Size
protein
Substrate Physical Separation binding analytes are anchored



10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

What is claimed is:

i, A binder complex for selectively identifying an amino acid, the binder complex
comprising:

an amino acid-specific binder, and
an adjunct attached to the amino acid-specifie binder,
wherein the amino acid-specific binder binds selectively to a binding amino acid. and
the amino acid-specific binder comprises an amino acid sequence comprising:

a first amino acid sequence comprising X1-GQQVT-X2-QHV V-X3-K-X4-
MI-XS-~GGR-X6-E-X7-Y-X8-E;

a second amino acid sequence comprising X1-RQQDT-X2-RHVA-X3-M-X4-
MT-X5-EGQ-X6-E-X7-¥-X8-E;

a third amino acid sequence comprising X1-RQQVA-X2-RHVA-X3-M-X4-
MT-X5-EGQ-X6-V-X7-Y-X8-G;

a fourth amino acid sequence comprising XI1-RQQDT-X2-RHVA-X3-M-X4-
KT-XS-EGQ-X6-E-X7-Y-X8-E;

a fifth amino acid sequence comprising X1-RQQVT-X2-RHVA-X3-M-X4-
MT-X5-KGOQ-X6-E-X7-N-X8-E:

a sixth amino acid sequence comprising X1-RQQVA-X2-RHVA-X3-M-X4-
MT-X5-EGQ-X6-E-X7-¥-X8-E;

aseventh amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-
X4-MT-XS-KGQ-X6-V-X7-Y-X8-G: or

an eighth amino acid sequence comprising X1-RQQDT-X2-RHVA-X3-M-
X4-MT-XS-EGQ-X6-V-X7-Y-X8-G;

wherein:

X1 comprises an amino acid sequence comprising ASVVPQE (Sequence ID
No. 20):

X2 comprises an amino acid sequence comprising
RKHYPNYKVIVLNDDENTF (Sequence ID No. 21):

X3 comprises an amino acid sequence comprising ACL (Sequence ID No. 22):

X4 comprises an amino acid sequence comprising KYIPN (Sequence ID No.
23);

X5 comprises an amino acid sequence comprising SDRAWELTNOVHY
{Sequence ID No. 24},

41


INTERNATIONAL SEARCH REPORT

Information on patent family members

International application No

PCT/US2022/028400
Patent document Publication Patent family Publication
cited in search report date member(s) date
WO 2020102741 Al 22-05-2020 AU 2019380606 Al 27-05-2021
BR 112021008098 A2 10-08-2021
cA 3117889 Al 22-05-2020
cN 113287020 A 20-08-2021
EP 3881078 Al 22-09-2021
JP 2022507516 A 18-01-2022
KR 20210091243 A 21-07-2021
Tw 202032125 A 01-09-2020
us 2020209249 Al 02-07-2020
us 2020209253 Al 02-07-2020
us 2020209254 Al 02-07-2020
us 2020209255 Al 02-07-2020
us 2020209256 Al 02-07-2020
us 2020209257 Al 02-07-2020
us 2020219590 Al 09-07-2020
wo 2020102741 Al 22-05-2020

US 2020385432 AL 10-12-2020 US 2020148727 A1 14-05-2020
us 2020385432 Al 10-12-2020

Form PCTASA/210 (patent family annex) (April 2005)


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

ASV VPQERQODTRKHYPNYKVIVLNDDFNTFRHV AACLMRKYIPNMTSDRAWELTN
QVHYEGOAIV WV GPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 8):
aninth amino acid sequence comprising

ASV VPQEGQQV TREHY PNY KVIVLNDDENTFOHV VACLKRY IPNMISDRAWELTNO
VHYGGRAIV WV GPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 9):

atenth amino acid sequence comprising, or

ASV VPQERQOV ARKHY PNY K VIVLNDDFNTFRHV AACLMK YIPNMTSDRAWELTN
QVHYEGOATY WV GPOV QAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10):

or an eleventh amino acid sequence with a homology of at least 30% compared to an amino

acid sequence comprising the first amino acid sequence, the second amino acid sequence, the
third amino acid sequence, the fourth amino acid sequence. the fifth amino acid sequence, the
sixth amino acid sequence, the seventh amino acid sequence. the eighth amino acid sequence,
the ninth amino acid sequence, or the tenth amino acid sequence.

fO006} Disclosed is a process for selectively identifying an N-terminal amino acid,
the process comprising: providing an analyte; contacting a C-terminal end of the analyte with
an anchor, anchoring the C-terminal end to the anchor to form an anchored analyte: contacting
an N-terminal amino acid of the anchored analyte with a binder complex, the binder complex
comprising: an amino acid-specific binder: and a taggant attached to the amino acid-specific
binder: selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid is a binding amino
acid to form a tagged complex; subjecting the taggant of the tagged complex to a stimulus:
producing, by the taggant of the tagged complex, a taggant signal in response to the stimulus:
detecting the taggant signal: and identifving the N-ierminal amino acid based on the taggant
signal, wherein the amino acid-specific binder binds selectively to the binding amino acid, and
the amino acid-specific binder comprises:
a first amino acid sequence comprising
ASV VPQEGOOVTREHY PNYEVIVUNDDENTFORV V ACLEK YVIPNMISDRAW
VHY GGRATY WV GPQEQAELYHEOLLRAGLTMAPL

3 (Sequence ID No. 1}:

a second amino acid sequence comprising
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHY EGOATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D No. 2):
athird amino acid sequence comprising

ASVVPOERGOV ARKHYPNYKVIVLNDDFNTFRHV AACLMRYIPNMTSDRAWELIN
QVHYEGQATV WVGPOVOAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 3);


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid comprises a
binding amino acid to form a tagged complex;

subjecting the taggant of the tagged complex to a stimulus;

producing, by the taggant of the tagged complex, a taggant signal in response to the
stimulus:

detecting the taggant signal; and

identifying the N-terminal amino acid based on the taggant signal,

wherein the amino acid-specific binder binds selectively to the binding amino acid, and

the amino acid-specific binder comprises an amino acid sequence comprising:

a first amino acid sequence comprising X1~GQQVT-X2-QHVV-X3-K-X4-MI-X5-
GGR-X6-E-X7-Y-X8-E;
asecond amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-MT-X5-

EGQ-X6-E-X7-Y-N8-E:

a third amino acid sequence comprising X1-RQQVA-X2-RHVA-X3-}
EGO-X6-V-X7-Y-X8-G;

a fourth amino acid sequence comprising X1-RQQDT-X2-RAV A-X3-M-X4-KT-X5-
EGQ-X6-E-X7-Y-X8-E:

a fifth amino acid sequence comprising X1-RQQVT-X2-RHVA-X3-M-X4-MT-XS-
KGQ-X6-E-X7-N-X8-E;

a sixth amino acid sequence comprising X1-RQQVA-X2-RHVA-X3.
EGQ-X6-E-X7-Y-X8-E,

a seventh amino acid sequence comprising X1-ROQDT-X2-RHVA-X3-M-X4-MT-
X5-KGQ-X6-V-X7-Y-X8-G: or

an eighth amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-MT-
KS-EGQ-X6-V-X7-Y-X8-G:

-X4-MT-X5-

-X4-MT-X5-

wherein:

XI comprises an amino acid sequence comprising ASVVPQE (Sequence ID No. 20);

X2 comprises an amino acid sequence comprising RKHYPNYKVIVLNDDENTF
(Sequence ID No. 21:

X3 comprises an amino acid sequence comprising ACL (Sequence ID No. 22);

4 comprises an amino acid sequence comprising KYIPN (Sequence ID No. 23);

X5 comprises an amino acid sequence comprising SDRAWELTNQVHY (Sequence ID
No. 24):


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

sodium phosphate, 300 mM NaCl, or 20 mM Hepes, pH 8.0, 150 mM KCI) and sonicated on a
500 W sonicator with a C1334 probe at 20% amplitude for a time (e.g. 4 seconds on, 20
seconds off, for 90 minutes) that provides a selected total time (e.g., 15 minutes) of sonication.
The lysate is centrifuged (e.g.. at 20,000 g for 40 minutes) and then incubated (e.g.. for one

hour) that can include a chelating fast flow sepharose resin coated with nickel and pre-

equilibrated in lysis buffer. The mixture is centrifuged (e.g., at 1000 g for 10 minutes) and
supematant removed. and the resin resuspended in lysis buffer that can be used to form a
column. The column is washed with lysis buffer, wash buffer (e.g. lysis buffer with
imidazole), and eluted with elution buffer. Protein that is eluted is subjected to dialysis into
lvsis buffer. Protein is removed from dialysis tubing and centrifuged, and the supernatant
concentration measured by Bradford assay against a BSA standard curve. The protein is loaded
onto a size exclusion chromatography column pre-equilibrated in lysis buffer. Fractions are
collected from the size exclusion chromatography column and monitored at 280 nm, wherein
absorption peaks are compared with a standard and analyzed by electrophoresis such as SDS-
PAGE. Fractions are combined, concentrated by centrifugation with a molecular weight cutoff,
such as 10 kDa, centrifuged, and measured by Bradford assay to prepare amino acid-specific
binder 200.

[0071] In an embodiment, making binder complex 228 includes expressing a fusion
protein of amino acid-specific binder 200 and adjunct protein 238 in an organism and purifying
the fusion protein from the organism. In an embodiment, making binder complex 238 includes
expressing a tagged variant of amino acid-specific binder such that it can be labeled with biotin
during expression. The biotin contacts amino acid-specific binder 200 with substrate 240. In

an embodiment, making binder complex 238 includes incubating the amino acid-specific

binder 200 with an amine reactive chemical moiety such as NHS-ester HRP or taggant such as
a fluorophore such as an NHS-ester fluorescein so that the amino-acid specific binder 200
lysine residues are linked to the fluorophore or chemical moiety

[0072] Amino acid-specific binder 200 has numerous advantageous and
unexpected benefits and uses. In an embodiment, with reference to FIG. 7 and FIG. 9, a process
for selectively identifying N-terminal amino acid 220 includes providing analyte 212 including
protein 250, peptide 248, amino acid 209, or a combination thereof; contacting C-terminal end
224 of analyte 212 with anchor 222: anchoring C-terminal end 224 to anchor 222 to form

anchored analyte 226; contacting N-terminal amino acid 220 of anchored analyte 226 with

binder complex 228, binder complex 228 include: amino acid-specific binder 200; and taggant

244 attached to amino acid-specific binder 200; selectively binding amino acid-specific binder

20


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

incubating amino acid-specific binder with the analyte or analyte within a mixture in a reaction
buffer for a time for binding to occur if the analyte includes binding amino acid 210. The
process includes selectively binding amino acid-specific binder 200 to N-terminal amino acid
20 of analyte 212 when N-terminal amino acid 220 includes binding amino acid 210 to form
isolation complex 244 by incubating them in a reaction buffer for a time for binding to occur.
The process also includes separating, ¢.9., by dialysis, isolation complex 244 from a fuid in
which isolation complex 244 is disposed to selectively isolate analyte 212.
fG099} In the process for selectively isolating analyte 212. separating isolation
complex 244 from the fluid can include separating isolation complex 244 based on a size of
isolation complex 244 relative to a size of other constituents in fluid by dialysis in which the
isolation complex is too large to move through a pore but other constituents that the complex
is being isolated from communicate through the pore. In an embodiment, the isolation complex
is an immunoglobulin fusion, and analyte 212 is phenylalanine. The complex can be isolated
by dialysis through. e.g.,a 10 KDa molecular weight cutoff membrane. Separating can include
precipitating isolation complex 244 from the fluid, ultracentrifuging in a glycerol gradient and
separating the gradient fractions, and the like.
fOOLOO] Amino acid-specific binder 200 and processes disclosed herein have
mumerous beneficial uses, including protein sequencing. peptide fingerprinting. and isolating
amino acid analytes. Advantageously, amino acid-specific binder 200 overcomes limitations
or technical deficiencies of conventional articles such as the sefectivity or specificity for a
particular amino acid over similar amino acids. Additionally, amino acid-specific binder 200
has higher affinity combined with high specificity than conventional approaches. As such,
amino acid-specific binder 200 can discriminate amino acids for sequencing. Amino acid-

specific binder 200 is specific for an N-terminal amino acid rather than an intemal residue

containing the same amino acid sidechain. Accordingly. ammo acid-specific binder 200

determines the identity of the amino acid and a position of the amino acid in a peptide or
protein. Additionally, amino acid-specific binder 200 binds a binding amino acid that is not
part of a protein or peptide. To circumvent an inability to determine a position of an amino
acid (N-terminal, internal, or C-terminal), conventional sequencing or fingerprinting methods
rely on a loss of signal if the amino acid being detected is removed from the peptide and
interpret a loss of signal at a particular iteration as an indication of position or register within
the peptide. A detection scheme that relies on loss of signal is limited when another factor
{such as quenching of a signaling fluorophore) also results in a loss of signal. Amino acid-

specific binder 200 and tagged complex described herein overcome this limitation by adding a

26


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

affinity of amino acid-specific binder 200 under a set of a binding conditions (e.g., in LX PBS
at 30°C). When N-terminal amino acid 220 includes binding amino acid 210, the binding
reaction occurs,

{0078} With reference to taggants and stimulants, signal. and detection listed in

Table 1, in the process for selectively identifying N-terminal amino acid 220

bjecting
taggant 214 of tagged complex 230 to stimulus 218 includes exposing tagged complex 230 on
a fluorescent microscope that provides a select wavelength of light as a stimulant to produce
taggant response, wherein an LED can produce excitation at 628 nm as a stimulus

{0079} In the process for selectively identifying N-terminal amino acid 220,

producing, by taggant 214 of tagged complex 230, taggant signal 216 in response to stimulus

218 includes, e.g., producing a fluorescent photon.

[0080} in the process for selectively identifving N-terminal amino acid 220,
detecting taggant signal 216 includes detecting emission with a microscope that includes a
detector that detects a selected wavelength of emission, e.g., 692 nm fluorescence.

fo08E] In the process for selectively identifying N-terminal amino acid 220,
identifying N-terminal amino acid 220 based on taggant signal 216 includes analyzing the
signal response and interpreting the response based on the experimental design associated with
the tagged binder complex 228. In an embodiment, the taggant is a fluorophore with a selected
wavelength of emission response that provides a signal for detection through fluorescence
intensity at a selected wavelength of the response to identity binding amino acid 210.

[0082] With reference to FIG. 9 and FIG. 10, the process for selectively identifying
N-terminal amino acid 220, also can include removing N-terminal amino acid 220 from
anchored analyte 226 so that penultimate residue 234 becomes N-terminal amino acid 220 of
anchored analyte 226 by Edman degradation.

[0083] The process for selectively identifying N-terminal amino acid 220, also can
include contacting N-terminal amino acid 220 of anchored analyte 226 with binder complex
228 by incubating anchored analyte 226 and binder complex 228 in a reaction buffer for a time
{e.g., from 5 see to 30 min) for the binding reaction to occur based on a binding affinity of
amino acid-specific binder 200 under a set of a binding conditions (e.g., in 1X PBS at 30°C)
When N-terminal amino acid 220 includes non-binding amino acid 211, the binding reaction
does not occur,

[0084] The process for selectively identifying N-terminal amino acid 220, also can
include selectively binding amino acid-specific binder 200 of binder complex 228 to N-

terminal amino acid 220 of anchored analyte 226 when N-terminal amino acid 220 is binding

22


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

X6 comprises an amino acid sequence comprising AIVWVGPQ (Sequence ID No. 25),
X7 comprises an amino acid sequence comprising QAEL (Sequence ID No. 26), and
X8 comprises an amino acid sequence comprising HEQLLRAGLTMAPLEP
(Sequence ID No. 27).

such that a total percentage amount of substitutions and deletions to X1, X2,
X3, X4, X5, X6, X7, and X8 is from 0% to fess than 30%, exclusive of
PSV VPQEROQV TREHY PNY KVIVLNDDENTFEQHYV AACLMKYIPNMTSDRAW
ELTNOVHYEGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID
No. 28)

6. The process of embodiment 5, wherein the amino acid sequence comprises:
ASV VPQEGOQVTRKHY PNYRVIVLNDDFNTFQHV VACLKKYIPNMISDRAWELTNOQ
VHYGGRATV WV GPQEQAELYHEOLLRAGLTMAPLEPE (Sequence 1D No. 1)
ASV VPQERQODTRKEHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATYV WV GPOE! ELYHEQLLRAGLTMAPLEPE (Sequence ID No. 2);
ASVVPQERQOV ARKHY PNYEVIVLNDDFNTFRHV AACLMEY IPNMTSDRAWELTN
QVHYEGOAIVWVGPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 3):
ASV VPQEROQQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNKTSDRAWELTN
QOVHYEGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID } 4):
ASVVPOERQOVTRKHYPNYKVIVLNDDFENTFRHV AACLMRYIPNMTSDRAWELTN
QVHYRGOATV WV GPQOEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 5):
ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOEQGAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 6)
ASVVPQEROQQDTREHY PNY KVIVLNDDENTERHV AACLMKYIPNMTSDRAWELTN
OVHY KGQATV WV GPQVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 7):
ASVVPQERGODTRAHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVAYEGOATY WV GPQVOAELYHEQLLRAGLTMAPLEPG (Sequence HD No, 8):
ASV VPQEGOOVTREHY PNYEVIVUNDDENTFORV V ACLEK YVIPNMISDRAW ‘NQ
VHY GGRAIV WVGPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 9): or
ASVVPQEROOV ARKHY PNYKVIVLNDDFNTFRHVAACLMEYIPNMTSDRAWELTN
OVHYEGOAIV WV GPOV QAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10).

7. The process of embodiment 5, further comprising


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

biphenylalanine or azidophenylalanine. Accordingly, binding amino acids 210 that are non-
naturally occurring amino acids include 5-bromo-iryptophan, homophenylalanine,
homophenylalanine methyl ester hydrochloride, and the like,

[0059] As used herein, “modified non-naturally occurring amino acid” refers to a
non-naturaily occurring amino acid that bas been modified. Exemplary modifications include
such as methylation, phosphorylation, glycosylation, deamination, oxidation, or selenocysteine
formation. Accordingly. binding amino acids 210 that are modified non-naturally occurring
amino acids include 5-bromo-try ptophan, homophenylalanine, homopenylalanine methyl ester
hydrochloride, and the like.

[0060] — Amino acid-specific binder 200 does not bind to non-binding amino acid
211. Non-binding amino acid 211 can be a naturally occurring or non-naturally occurring
amino acid exclusive of binding amino acid 210. Exemplary non-binding amino acids 211
include arginine, alanine, serine, threonine, proline, aspartic acid, asparagine, glutamine,
glutamic acid. Since amino acid-specific binder 200 does not bind te non-binding amino acid
21 but does selectively bind io binding amino acid 210, non-binding amino acid 211 is
determined as not belonging to the group of binding amino acids 210 selectively bound by
amino acid-specific binder 200. Accordingly, while binding of amino acid-specific binder 200
to binding amino acid 210 can be used to isolate binding amino acid 210 from other components
in a fluid, identify binding amino acid 210 as a particular species of amino acid (e.g., Leu, Phe,
Trp, Tyr}, and the like, not binding non-binding amino acid 211 can be used separate non-
binding amino acid 211 from binding amino acid 210 and. by negative implication, determine
a set of possible identities for binding amino acid 210

[0061] Peptide 248 can include a plurality of amino acids, including binding amino
acid 210, non-binding amino acid 211, or a combination thereof, Moreover, amino acids in
peptide 248 are arranged to include N-terminal amino acid 220 and C-terminal end 224.
Peptide 248 can be naturally occurring or can be a portion of a longer peptide or protein
Exemplary peptides 248 include a peptide from a proteolytic or tryptic digest of an isolated
protein or protein found in blood or serum. Binding of amino acid-specific binder 200 to
binding amino acid 210 can be used to isolate binding amino acid 210 from other components
ina fluid, identify binding amino acid 210 as a particular species of amino acid (e.g., Leu, Phe,
Trp, Ty), and the like

[0062] Protein 250 can include a plurality of amino acids, including binding anuno
acid 210, non-binding amino acid 211, or a combination thereof. Moreover, amino acids in

protein 250 are arranged to include N-terminal amino acid 220 and C-terminal end 224. Protein

17


WO 2022/236190 PCT/US2022/028400

pela Sm

Fluorescent <3
tiny

FIG. 12

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
oh

30

WO 2022/236190 PCT/US2022/028400

{0027} FIG. 19 shows alignment of Thermosynechoceccus elongaius CipS2
{Sequence ID No. 11), Agrobacteriiun tumefaciens CipS2 (Sequence ED. No. 12), and an
engineered NAAB-Phe protein sequences. 1. elongatus CipS2 bas 29% sequence identity to
A, tumefaciens ClpS2 and 31% sequence identity to the engineered NAAB-Phe. Positions of
library-selected mutations for 7. e/ongatus CipS2 are indicated by pointers. Sequence features
used to distinguish phylum of CipS variant are boxed: and

[0028] FIG. 20 shows the association curve of 7. efongatus CipS2 with the Leu
peptide variant and the association curve with the same Leu peptide for a composite protein

sequence which incorporates the mutants from the library generated from T. elongatus ClpS2.

DETAILED DESCRIPTION

{0029} A detailed description of one or more embodiments is presented herein by
way of exemplification and not limitation.

{0030} Tt has been discovered that an amino acid-specific binder herein selectively
binds to a binding amino acid selected from a group of specific amino acids. Indeed, the amino

acid-specific binder overcomes a central challenge in single-molecule protein se

ition, detection ¢ sific

ver, the amino acid-specific binder
Rule Pail

mine acid (NAA) on a peptide chain,

peplide sequence.

overcomes Jack of selectivity involved with an N

ior protein

natively recogniz

ks selectivity and affinity § pide sequencing, Beneficially and

& peptide or protein. Advantageously, the amino

acid-specific binder determines a sequence or fingerprint of amino acids in a peptide or protein
when used iteratively

[003 Ef Amino acid-specific binder 200 selectively binds to binding amino acid 210
in analyte 212. In an embodiment, amino acid-specific binder 200 is a first amino acid
sequence including X1-GQOVT-X2-QHVV-X3-K-X4-MI-N5-GGR-X6-E-X7-Y-X8-E: a
second amino acid sequence including X1-RQQDT-X2-RHVA-X3-M-X4-MT-X5-EGQ-X6-
E-X7-Y-X8-E; a third amino acid sequence including X1-RQQVA-X2-RHV A-X3-M-X4-MT-
X5-EGQ-X6-V-X7-Y-X8-G; a fourth amino acid sequence including X1-ROQDT-X2-RHVA-
X3-M-X4-K T-X5-EGQ-X6-E-X7-Y-N8-E: a fifth amino acid sequence including X1-



10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

a sixth amino acid sequence comprising X1-RQQVA-X2-RHVA-X.

MT-X5-EGQ-X6-E-X7-Y-X8-E;

aseventh amino acid sequence comprising X1-RQQDT-X2-RAVA-
MT-X5-KGQ-X6-V-X7-Y-X8-G: or

an eighth amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-
MT-X5-EGQ-X6-V-X7-Y-X8-G;

wherein:

X1 comprises an amino acid sequence comprising ASVVPQE (Sequence ID
No. 20),

X2 comprises an amino acid sequence comprising
REHYPNYKVIVLNDDENTE (Sequence ID No. 21;

X3 comprises an amino acid sequence comprising ACL (Sequence ID No. 22):

X4 comprises an amino acid sequence comprising KYIPN (Sequence 1D No.
23);

X5 comprises an amino acid sequence comprising SDRAWELTNQVHY
{Sequence 1D No. 24);

X6 comprises an amino acid sequence comprising AIVWVGPOQ (Sequence [D

X7 comprises an amino acid sequence comprising QAEL (Sequence ID No. 26):
and

X8 comprises an amino acid sequence comprising HEQLLRAGLTMAPLEP
{Sequence ID No. 27),

such that a total percentage amount of substitutions and deletions to X1, X2,
X3, X4, XS, X6, X7, and X8& is from 0% to less than 30%, exclusive of
PSVVPQOERQOVTRKHY PNYKVIVLNDDENTFOHV AACLMKY IPNMTSDRAW
ELTNQVHYEGQALV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID
No. 28).

2. The binder complex of embodiment 1, wherein the amino acid sequence comprises:
ASVVPQEGQOV TREHYPNYKVIVLNDDENTFQHVV ACLKRYIPNMISDRAWELTNO
VHYGGRAIVWVGPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 1k
ASV VPQEROQDTRRHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QOVHYEGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 2)
ASVVPOERQOV ARKHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELIN

32


WO 2022/236190 PCT/US2022/028400

19/20

FIG, 19

SUBSTITUTE SHEET (RULE 26)


INTERNATIONAL SEARCH REPORT

International application No

PCT/US2022/028400
A. CLASSIFICATION OF SUBJECT MATTER
INV. C0O7K14/405 G01N33/68 CO7K14/195 G01N33/58
ADD.

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

cO7K GO1N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

EPO-Internal, Sequence Search

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category’ | Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
x WO 2020/102741 Al (QUANTUM SI INC [US]) 1-4
22 May 2020 (2020-05-22)
Y paragraph [0325]; example 5; sequence 12 5-20
Y US 2020/385432 Al (TULLMAN JENNIFER A [US] 5-20

the whole document

ET AL) 10 December 2020 (2020-12-10)

im Further documents are listed in the continuation of Box C.

* Special categories of cited documents :

"A" document defining the general state of the art which is not considered
to be of particular relevance

“E" earlier application or patent but published on or after the international
filing date

“L” document which may throw doubts on priority claim(s) or which is
cited to establish the publication date of another citation or other
Special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or other
means

"P" document published prior to the international filing date but later than
the priority date claimed

“T" later document published atter the international filing date or priority
date and not in conflict with the application but cited to understand
the principle or theory underlying the invention

“X" document of particular relevance:; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive
step when the document is taken alone

“Y" document of particular relevance:; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art

“&" document member of the same patent family

Date of the actual completion of the international search

26 September 2022

Date of mailing of the international search report

11/10/2022

Name and mailing address of the ISA/
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040,
Fax: (+31-70) 340-3016

Authorized officer

Wiame, Ilse

Form PCT/ASA/210 (second sheet) (April 2005)



International application No.

INTERNATIONAL SEARCH REPORT
i s c ° PCT/US2022/028400

Box No.1 Nucleotide and/or amino acid sequence(s) (Continuation of item 1.c of the first sheet)

1. With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the international search was
carried out on the basis of a sequence listing:

forming part of the international application as filed:

in the form of an Annex C/ST.25 text file.

im on paper or in the form of an image file.

b. furnished together with the international application under PCT Rule 13ter.1(a) for the purposes of international search
only in the form of an Annex C/ST.285 text file.

furnished subsequent to the international filing date for the purposes of international search only:

in the form of an Annex C/ST.25 text file (Rule 13ter.1(a)).

im on paper or in the form of an image file (Rule 13ter.1(b) and Administrative Instructions, Section 713).

2. im In addition, in the case that more than one version or copy of a sequence listing has been filed or furnished, the required
statements that the information in the subsequent or additional copies is identical to that forming part of the application as
filed or does not go beyond the application as filed, as appropriate, were furnished.

3. Additional comments:

Form PCT/SA/210 (continuation of first sheet (1)) (January 2015)


10

IS

20

ho
oh

30

WO 2022/236190 PCT/US2022/028400

and Taq DNA polymerase. The PCR product was used for homologous recombination, as
described below. The libraries cach contained at least 1 million naive members, and greater
than 50% of the colonies contained at least one mutation from the ten colonies sequenced.

fOOLLL] With regard to Saccharomyces cerevisiae (yeast) transformation, EBY 100

strain 5.

jae were transformed with pCTCON2 plasmids containing the wild-type CipS

genes using the Frozen EX Yeast Transformation TI kit and subsequently grown on selective

media as the pCTCON2 plasmid harbors the ability to synthesize tryptophan. Synthetic
dextrose media supplemented with casamino acids lacking tryptophan (SD-CAA) and
containing 100 pg/mL ampicillin was used to grow all the yeast used in this study. Protein
surface expression was induced by resuspending the cells in synthetic galactose media
supplemented with casamino acids lacking tryptophan (SG-CAA).

[OOLI2] With regard to site-saturation library creation/homologous recombination in
yeast, EBY100 S. cerevisiae cells were grown overnight to an ODeoo of 3 in YPD media at
30°C. This was used to inoculate a 100 mL culture of YPD to ODooo 0.3. After 5 hours, when
cells had grown to ODeuo 1.0, the cells were transferred to 50 mL conical tubes and centrifuged
at 3000 g for 3 min at 4°C. The cell pellet was washed twice with 50 mL ice cold sterile water
and then washed once with 50 mL ice cold electroporation buffer (1M sorbitol/ImM CaCh).
The cells were conditioned for electroporation by resuspending the cell pellet in 20 mL 0.1M
LiAc, 10 mM DTT and shaking for 30 min at 30 degrees. The cells were centrifuged as above
and washed with 25 mL per tube of electroporation buffer before being resuspended in 200 pL.
of electroporation buffer to reach a final volume of about | mL. Cefls were kept on ice until
electroporation,

fO0113} For electroporation, 400 tL of competent cells prepared as above were
incubated with the vector and insert, in a 1:3 ratio, and kept on ice for 5 minutes. The vector
used was the pCTCON2 plasmid described above containing the clpS gene of interest and
digested within the clpS gene with the restriction enzymes Nhel and BamHI. The insert used
was the error prone PCR library obtained as described above. The cells were then transferred
to a 0.2 em electroporation cuvette and electroporated on the pre-set yeast settings (1.5 kV, 25
uF). The cells were transferred to a tube containing 4 mL of YPD media and 4 mL of | M
sorbitol and incubated at 30 °C for 1 hour, 225 rpm. The cells were then centrifuged and
resuspended in SD-CAA media and dilutions were plated to calculate library size. and the rest
was grown in a flask containing 250 mL of SD-CAA media and passaged once before

selections or sorting.

29


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

a first amino acid sequence comprising
ASVVPQEGQOVTREHYPNYKVIVLNDDENTFOHVV ACLKERYIPNMISDRAWELTNO
VHY GGRAIVWY GPOEQAELYHEQLLRAGLTMAPLE]

2 (Sequence 1D No. PD:
asecond amino acid sequence comprising
ASVVPGERQODTRKHYPNYKVIVLNDDFNTFRHV AACLMRYIPNMTSDRAWELTN
QOVHYEGQATV WVGPQEQAELYHEOLLRAGLTMAPLEPE (Sequence ID No. 2);

a third amino acid sequence comprising

ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOV QOAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 3),

a fourth amino acid sequence comprising

ASV VPQEROQQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNKTSDRAWELTN
QVHYEGQAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 4):

a fifth amino acid sequence comprising

ASV VPQERQOVTRKHYPNYKVIVLNDDENTFRHV AACLMRKYIPNMTSDRAWELTN
QVHYKGOAIV WV GPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 5};

a sixth amino acid sequence comprising

ASVVPQEROOV ARKHYPNYEVIV DDFNTFRAV AACLMEYIPNMTSDRAWELTN
OVHYEGOAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 6):

a seventh amino acid sequence comprising
ASVVPGERQODTRKHYPNYKVIVLNDDFNTFRHV AACLMRYIPNMTSDRAWELTN
QVHYKGOAIV WVGPOVQAELYHEOLLRAGLTMAPLEPG (Sequence ID No. 7);

an eighth amino acid sequence comprising
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OQVHYEGOAIV WVGPOV QAELY HEOLLRAGLTMAPLEPG (Sequence ID No. 8)

aninth amino acid sequence comprising

ASV VPQEGOQVTREHY PNYRVIVLNDDFNTFQHV VACLKKYIPNMISDRAWELTNOQ
VHYGGRATYV WV GPQEQAELNHEOLLRAGLTMAPL
atenth amino acid sequence comprising, or

ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOV OAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10):

or an eleventh amino acid sequence with a homology of at least 30% compared to an amino

3 (Sequence 1D No. 9);

acid sequence comiprising the first amino acid sequence, the second amino acid sequence, the

third amino acid sequence, the fourth amino acid sequence. the fifth amino acid sequence, the

6


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

a fourth amino acid sequence comprising
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNKTSDRAWELTN
OVHY EGOATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D No. 4):

a fifth amino acid sequence comprising
ASVVPOERQOVTRKHYPNYKVIVLNDDFENTFRHV AACLMRYIPNMTSDRAWELTN
QVHYER GOAT WV GPOEGAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 5):
asixth amino acid sequence comprising

ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOEQGAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 6);

a seventh amino acid sequence comprising

ASV VPQEROQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHYKGOATV WV GPOV QGAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 7};

an eighth amino acid sequence comprising

ASV VPQERQODTRKEHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATYV WV GPOVQAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 8),

aninth amino acid sequence comprising

ASVVPQEGQQYV TREAYPNYKVIVLNDDENTFOHV VACLKEY IPNMISDRAWELTNO
JHY GGRAIW WV GPOEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 9}:

atenth amino acid sequence comprising, or

ASVVPOERQOV ARKHYPNYKVIVLNDDENTFRHV AACLMK YIPNMTSDRAWELIN

QVHYEGOATV WV GPQV GAELNHEQLLRAGLTMAPLEPE (Sequence 1D No. 10):

or an eleventh amino acid sequence with a homology of at least 30% compared to an amino

acid sequence comprising the first amino acid sequence, the second amino acid sequence, the

third amino acid sequence, the fourth amino acid sequence, the fifth amino acid sequence, the

sixth amino acid sequence, the seventh ammo acid sequence. the eighth amino acid sequence,

the ninth amino acid sequence, or the tenth amino acid sequence.

[0007] Disclosed is a process for selectively isolating an analyte, the process
comprising: contacting an amino acid-specific binder with an analyte comprising a protein, a
peptide, an amino acid, or a combination comprising at least one of foregoing: selectively
binding the amino acid-specific binder to the N-terminal amino acid of the analyte when the
N-terminal amino acid is a binding amino acid to form an isolation complex: separating the
isolation complex from a fluid in which the isolation complex is disposed to selectively
isolating the analyte, wherein the amino acid-specific binder binds selectively to the binding

amino acid, and the amino acid-specific binder comprises:


10

IS

20

No
Or

30

WO 2022/236190 PCT/US2022/028400

250 can be obtained from a mixture of proteins as found within a blood or serum sample. In an
embodiment, protein 250 includes serum proteins

[0063] In some embodiments, with reference to FIG, 7, analyte 212 forms anchored
analyte 226 in combination with anchor 222. Anchor 222 can include a substrate containing a
surface on which to immobilize the analyte such that it can be sequestered or measured. Anchor
222 can be a resin, glass slide, magnetic bead. Exemplary anchor 222 includes a streptavidin
coated sensor, microplate. and the like. In an embodiment, anchor 222 includes a streptavidin
coated microplate, and intervening member 264 includes biotin.

[0064] Exemplary anchored analyte 226 includes a peptide analyte 212 anchored
via the lysine sidechain to an NHS-ester coated glass slide and the like. It is contemplated that
attachment of analyte 212 to anchor 222 can include a covalent bond, an ionic bond,
electrostatic interaction (e.g., a m-cation interaction, dipole-dipole interaction, a multi-pole
interaction, and the like), intercalation, a clathrate arrangement (e.g.. with analyte 212 partially
or wholly trapped in anchor 222 or vice-versa, such that N-terminal amino acid 220 or binding
amino acid 210 is exposed to amino acid-specific binder 200 for selectively binding), and the
like. Further, analyte 212 can be attached to anchor 222 either directly, indirectly, or a
combination thereof. When analyte 212 is directly attached to anchor 222, direct attachment
occurs in an absence of an intervening member between analyte 212 and anchor 222. When
analyte 212 is indirectly attached to anchor 222, indirect attachment occurs in a presence of the
intervening member 246 between analyte 212 and anchor 222.

{0065} in an embodiment, anchored analyte 226 includes a peptide analyte 212
anchored via the lysine sidechain to an NHS-ester coated glass slide and the like

[0066] With reference to FIG. 8, selectively binding binder complex 228 to
anchored analyte 226 forms tagged complex 230, e.g., to determine an identity of amino acid
209 in analyte 212 of anchored analyte 226. When analyte 212 is protein 250, amino acids in
protein 250 can be sequenced using binder complex 228.

[0067] In determining a sequence of amino acids in analyte 212 in anchored analyte
226, with reference to FIG. 9, N-terminal amino acid 220 can be removed by chemical
modification to expose the penultimate residue 234 as the new N-terminal amino acid 220
{panel D). Subsequent removal to expose the next penultimate residue 234 as the new N-
terminal amino acid 220 can be repeated such that every new amino acid in analyte 212 can be

sequentially subjected to binder complex 228 for sequencing.


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

{0018} FIG. 10 shows an anchored analyte in panel A: in panel B, the anchored
analyte shown in panel A after production of an inert residue from an N-terminal amino acid:
and in panel C, the anchored analyte shown in panel B after production of another inert residue,

fOOL9} FIG. 11 shows, in panel A, an isolated complex that mcludes an amino acid-

cific binder selectively bound to an analyte: in panel B, an isolated complex that includes

an amino acid-specific binder selectively bound an N-terminal amino acid that is a binding
anmuno acid in an analyte: and in panel C. an isolated complex that includes an amino acid-
specific binder of a binder complex selectively bound to an N-terminal amino acid that is a
binding amino acid in an analyte:

[0020] FIG. 12 shows formation of a tagged complex and detection of a taggant
signal;

[0021] FIG. 13 shows formation of a tagged complex and detection of a taggant
signal;

£0022 FIG. 14 shows a fluorescent labeling for detection of peptide binding during
flow cytometry, wherein myc tag 260 is detected with fluorescent label taggant 214 on anti-
mye antibody 256. Peptide 248 is detected using streptavidin-PE 258 that binds biotin 264
attached to C-terminus 224 of peptide 248. It should be appreciated that there are other tags
besides myc and different fluorophores:

{0023} FIG. 15 shows an expected flow cytometry result for yeast that displays a
non-binding protein in quadrant 1 (QU), veast that binds the peptide in Q2, yeast that does not
display the proteim in Q3, and yeast that exhibits non-specific binding to the peptide in Q4:

f0024} FIG. 16 shows a graph of fluorescent taggant fluorescence versus
phycoerythrin (PE) fuorescence for ow cytometry plots displaying PE fluorescence seen in
a naive library against a leucine (Leu) peptide, wherein Q2 correspond to cells carried on to a
next round of sorting after outgrowth:

{0025} FIG. 17 shows a graph of fluorescent taggant fluorescence versus PE
fluorescence for flow cytometry plots displaying increased PE fluorescence seen in cach round
after the data shown in FIG. 15 and an additional 3 rounds of selection of a library against a
Leu peptide:

[0026] FIG. 18 shows the number of cells counted in Q2, as determined via

fluorescent taggant fluorescence versus PE fluorescence for flow cytometry plots.

showing the

magnitude of difference in binding against Phe. Tyr, Trp, and a variety of Leu peptides between

anaive library and a library resulting from three rounds of selection:


WO 2022/236190 PCT/US2022/028400

Protein with different Precipitation Solubility or
solubility Molecular weight
Protein with different Isoelectric Gradient tsoelectric point

isoelectric point

Protein with different Density Gradient Density

densities

fO088] The process for selectively identifying N-terminal amino acid 220, also can
include identifying N-terminal amino acid 220 based on taggant signal 216 by analyzing the
signal response and interpreting the response based on tagged binder complex 228. When
taggant 214 is a fluorophore, the intensity and wavelength of the taggant response identifies a
binding amino acid 210 due to a higher signal than non-binding amino acid 211.

[0089} With reference to FIG. 10, instead of or in combination with removing N-
terminal amino acid 220, the process for selectively identifying N-terminal amino acid 220 also
can include converting N-terminal amino acid 220 to inert residue 232 by performing a partial
Edman degradation reaction so that phenylsothiocyanate (PETC) remains attached to the N-
terminal amino acid such that a binding reaction does not occur,

[0090] The process for selectively identifying N-terminal amino acid 220, also can
include converting penultimate residue 234 to N-terminal amino acid 220 when inert residue
232 is removed by continuing the Edman degradation reaction to remove the PITC

{0091} The process for selectively identifying N-terminal amino acid 220, also can
include contacting N-terminal amino acid 220 of anchored analyte 226 with binder complex
228 by incubating anchored analyte 226 and binder complex 228 in a reaction buffer for a time
{e.g., fromm 5 sec to 30 min) for the binding reaction to occur based on a binding affinity of
amino acid-specific binder 200 under a set of a binding conditions (e.g., in LX PBS at 30°C).
When N-terminal amino acid 220 includes binding amino acid 210, the binding reaction occurs,
and the tagged complex forms.

[0092] The process for selectively identifying N-terminal amino acid 220 also can
include selectively binding amino acid-specific binder 200 of binder complex 228 to N-
terminal amino acid 220 of anchored analyte 226 when N-terminal amino acid 220 is binding
amino acid 210 to form tagged complex 230 by incubating anchored analyte 226 and binder
complex 228 in a reaction buffer for a time (e.g., from 5 sec to 30 min) for the binding reaction

to occur based on a binding affinity of amino acid-specific binder 200 under a set of binding

24


=
<
J
a
=
=)
aA
a
gS
a
a
—
a
S

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

=

7

(19) World Intellectual Property
Organization
International Bureau

(10) International Publication Number

(43) International Publication Date wo 2022/ 236190 Al
10 November 2022 (10.11.2022) WIPO! PCT

6p CO ads ened eneR Ta/9s (2006.01) Published: ternational search report (Are. 2103)
GOIN 33/68 (2006.01) GOIN 33/58 (2006.01) with international search report (Art, 21(3)

(21) International Application Number:
PCT/US2022/028400

(22) International Filing Date:
09 May 2022 (09.05.2022)

(25) Filing Language: English
(26) Publication Language: English
(30) Priority Data:

63/185,483 07 May 2021 (07.05.2021) US

(71) Applicant: GOVERNMENT OF THE UNITED
STATES OF AMERICA, AS REPRESENTED BY THE
SECRETARY OF COMMERCE [US/US]; 100 Bureau
Drive, Gaithersburg, Maryland 20899 (US).

Inventors: KELMAN, Zvi; 31 Treworthy Road, Gaithers-
burg, 20878 (UM). MARINO, John Paul; 113 Lexing-
ton Drive, Silver Spring, Maryland (US). CALLAHAN,
Nicholas William; 421 W Side Drive, Apt. #104, Gaithers-
burg, Maryland 20878 (US).

Agent: JOHNSON, Spencer J.; 100 Bureau Drive,
Gaithersburg, Maryland 20899 (US).

(72)

(4)

(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY. BZ,
CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH. GM, GT, HN,
HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP. KE, KG, KH,
KN, KP. KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA,
MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
NO, NZ, OM, PA, PE, PG, PH, PL, PT. QA, RO. RS, RU,
RW, SA, SC, SD, SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM,
TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM,
ZW.

(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
MC, MK, MT, NL, NO, PL, PT, RO, RS, SE. SI, SK, SM,
TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
KM. ML, MR, NE, SN, TD, TG).

before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments (Rule 48.2(h))

with sequence listing part of description (Rule 5.2(a))

(64) Title: AMINO ACID-SPECIFIC BINDER AND SELECTIVELY IDENTIFYING AN AMINO ACID

(87) Abstract: N-terminal amino acid binding (NAAB) reagents are a tool for parallel, high-throughput proteomics. A fluorescently-la-
beled NAAB allows immobilized peptides to be identified by their N-terminal residues using single-molecule fluorescent microscopy,
which enables novel proteomic analysis, like fluorescence-based next-generation protein sequence. Amino acid-specific binders are
presented which bind to leucine at the N-terminus of peptides/polypeptides/proteins with high affinity and specificity, allowing detec-

tion of an N-terminal leucine with high confidence.


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

a fourth amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-KT-X5-
EGQ-X6-E-X7-Y-X8-E,

a fifth amino acid sequence comprising X1-RQOQVT-X2-RHVé
KGQ-X6-E-X7-N-X8-E:

a sixth amino acid sequence comprising X1-RQQVA-K2-RHV A-X3-M-X4-MT-X5-
EGQ-X6-E-X7-Y-X8-E;

a seventh amino acid sequence comprising X1-RQQDT-X2-RHVA-X3-M-N4-MT-
X5-KGQ-X6-V-X7-Y-X8-G: or

an eighth amino acid sequence comprising XI-ROQDT-X2-RHV A-X3-M-X4-MT-
XS-EGO-X6-V-X7-¥-X8-G;

3-M-X4-MT-X5~-

3

wherein:

XI comprises an amino acid sequence comprising ASVVPQE (Sequence ID No. 20).

X2 comprises an amino acid sequence comprising RKHYPNYKVIVLNDDENTE
(Sequence ID No. 21);

X3 comprises an amino acid sequence comprising ACL (Sequence ID No. 22):

X4 comprises an amino acid sequence comprising KYIPN (Sequence ID No. 23):
TNOVHY (Sequence ID

XS comprises an amino acid sequence comprising SDRAWEL
No. 24):

X6 comprises an amino acid sequence comprising ATVWVGPQ (Sequence ID No. 25);

X7 comprises an amino acid sequence comprising QAEL (Sequence ID No. 26); and

X8 comprises an amino acid sequence comprising HEQLLRAGLTMAPLEP
{Sequence ID No. 27),

such that a total percentage amount of substitutions and deletions to X1, X2,

X3, X4, XS, X6, X7, and X8& is from 0% to less than 30%, exclusive of

PSVVPQOERQOVTRKHY PNYKVIVLNDDENTFOHV AACLMKY IPNMTSDRAW

ELTNQVHYEGQALV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID

No. 28).

12. The process of claim 11, wherein the amino acid sequence comprises:
ASVVPQEGOOVTREKHYPNYKVIVLNDDENTFOHVV ACLEKYIPNMISDRAW
ELTNQV HY GGRAIV W VGPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID
No. D:

ASV VPQGERQODTRKHYPNYKVIVLNDDFNTFRHV AACLMRY IPNMTSDRAW
ELTNOVHY EGOAW WV GPOEOAEL YHEQLLRAGLTMAPLEPE (Sequence ID

47


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

ASV VPQEGQOVTRAHY PNYKVIVLNDDFNTFOHV V ACLKKYIPNMISDRAWELTNQ
VHY GGRATV WV GPQEQAELNHEQLLRAGLTMAPL (Sequence ID No. 9):
ASV VPQEROQOV ARKHYPNYERVIVLNDDENTFRAV AACLMEY IPNMTSDRAWELTN

QOVHY EGOAIV WV GPQV QAELNHEQLLRAGLTMAPLEPE (Sequence 1D No. 10): or an

amino acid sequence with a homology of at least 30% compared fo an amino acid sequence
comprising the amino acid sequence with Sequence ID No. f, Sequence ID No. 2, Sequence
ID No. 3, Sequence ID No. 4, Sequence ID No. 5, Sequence ID No. 6, Sequence ID No. 7,
Sequence ID No. 8, Sequence ID No. 9, or Sequence ID No. 10. Amino acid-specific binder
200 binds selectively to binding amino acid 210 selected from the group consisting of
isoleucine, leucine, phenylalanine, tryptophan, tvrosine, and valine; and chemically modified
amino acids phenylalanine. tryptophan, tyrosine. isoleucine, leucine, and valine. Accordingly,
with reference to FIG. 1, amino acid-specific binder 200 selectively binds to binding amino
acid 210 of analyte 212 but does not bind to non-binding amino acid 211.

[0033] In an embodiment, the amino acid sequence includes:
ASVVPQEGQQV TREKHYPNYKVIVLNDDENTFOHV VY ACLEKYIPNMISDRAWELTNO
VHYGGRATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. I:
ASVVPQEROQQDTREHY PNY KVIVLNDDENTERHV AACLMKYIPNMTSDRAWELTN
OVHYEGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 2):
ASVVPOERGOV ARKHY PNYKVIVLNDDFNTFRHV AACLMRYIPNMTSDRAWELIN
QVHYEGQAIV WVGPOVOAELYHEQLLRAGLIMAPLEPG (Sequence ID No. 3):
ASV VPQERQODTRKHYPNYKVIVLNDDENTFRHV AACLMRKYIPNRTSDRAWELTN
QVHYEGOATV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 4):
ASVVPQERQOVTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHY KGQATYV WV GPQEQAELNHEOLLRAGLTMAPLEP
ASV VPQEROOV ARKHYPNYKVIVLNDDENTFRHV AACLMEY IPNMTSDRAWE!
QOVHYEGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID }
ASVVPQERQODTREAYPNYRVIVLNDDENTFRHVAACLMEY IPNMTSDRAW
QVAYRGOATV WV GPOVQAELYHEQLLRAGLTMAPLEPG (Sequence 1D No. 7):
ASV VPQERQODTRKHYPNYKVIVLNDDFNTFRHYV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WVGPOVOAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 8):
ASVVPQEGQQYV TREAYPNYKVIVLNDDENTFOHV VACLKEY IPNMISDRAWELTNO

JHY GGRATW W VGPOEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 9): or
ASVVPOERGOV ARKHYPNYKVIVLNDDFNTFRHV AACLMRYIPNMTSDRAWELIN
QVHYEGQATV WVGPOVOAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10)

(Sequence ID No. 5)

Bt


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

ROQVT-X2-RHV A-N3-M-X4-MT-X5-KGQ-N6-E-X7-N-NX8-E; a sixth amino acid sequence
including X1-RQQVA-X2-RHVA-X3-M-X4-MT-X5-EGOQ-X6-E-X7-Y-X8-E:
amino acid sequence including X1-RQQDT-X2-RHV A-X3-M-X4-MT-XS-KGQ-X6-V-X7-
Y-X8-G; or an eighth amino acid sequence including X1-RQQDT-X2-RHV A-X3-M-X4-MT-

XS-EGQ-X6-V-X7-Y-X8-G, wherein: X1 includes an amino acid sequence including

a seventh

ASVVPQE (Sequence ID No. 20). X2 includes an amino acid sequence including
RKHYPNYKVIVLNDDFNTF : X3 includes an amino acid sequence including ACL
{Sequence ID No. 21}; X4 includes an amino acid sequence including KYIPN (Sequence ID
No. 22); XS includes an amino acid sequence including SDRAWELTNOVHY (Sequence ID
No. 23); X6 includes an amino acid sequence including AFVWVGPQ (Sequence ID No. 24);
X7 includes an amino acid sequence including QAEL (Sequence ID No. 25); and X8 includes
an amino acid sequence including HEQLLRAGLTMAPLEP (Sequence ID No. 26), such that
a total percentage amount of substitutions and deletions to AI, X2, X3, X4, X5, X6, X7, and
XS is from O% to less than 30%, exclusive of
PSV VPQERQQOV TRKHYPNYKVIVLNDDFENTFOQHV AACLMRKY IPNMTSDRAWELTN
QVHYEGOATV WV GPOEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 27).

[0032] In an embodiment, amino acid-specific binder 200 is a protein that includes
an amino acid sequence that is
ASVVPQEGOQV TREHY PNY KVIVLNDDFENTFOHV V ACLKKY IPNMISDRAWELTNO
VHYGGRAIV WVGPQEQAELYHEOLLRAGLTMAPLEPE {Sequence [ID No. 1):
ASV VPQERQODTRKEHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 2):
ASVVPQEROOV ARKHY PNYKVIVLNDDFNTFRHVAACLMEYIPNMTSDRAWELTN
QVHYEGOAIVWVGPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 3):
ASV VPQEROQQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNKTSDRAWELTN
QOVHYEGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID } 4):
ASVVPOERQOVTREAYPNYRVIVLNDDENTFRHVAACLMREY IPNMTSDRAW N
QVHYRGOAL WV GPOEQAELNHEQLLRAGL TMAPLE! (Sequence ID No, 5}
ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 6)
ASVVPQEROQQDTREHY PNY KVIVLNDDENTERHV AACLMKYIPNMTSDRAWELTN
OVHY KGQATV WV GPQVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 7):
ASVVPQERGODTRAHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGQAIV WVGPOVOAELYHEQLLRAGLIMAPLEPG (Sequence ID No. 8):

10


WO 2022/236190
A
ann
ay
F
8)
at
iC} 28

PCT/US2022/028400

11/20

m9 uM
/ a

QA BND

FIG. 11

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

3. The binder complex of claim 1, wherein the adjunct comprises a taggant, a protein,

a substrate, or a chemical modifier.

4. The binder complex of claim 3, wherein the taggant comprises:
a fluorescent moiety, an electrochemical moiety, or a combination comprising at least
one of the foregoing moieties, and the taggant produces a taggant signal in response to receiving

a stimulus.

8. A proc

for selectively identifying an N-terminal amino acid, the process
comprising:

providing an analyte comprising a protein, a peptide. an amino acid, or a combination
comprising at least one of foregoing:

contact!

:a C-terminal end of the analyte with an anchor:
anchoring the C-terminal end to the anchor to form an anchored analyte:
contacting an N-terminal amino acid of the anchored analyte with a binder complex,
the binder complex comprising
an amino acid-specific binder; and
a taggant attached to the amino acid-specific binder;
selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid comprises a
binding amino acid to form a tagged complex;
subjecting the taggant of the tagged complex to a stimulus:
producing, by the taggant of the tagged complex, a taggant signal in response to the
stimulus:
detecting the taggant signal: and
identifying the N-terminal amino acid based on the taggant signal,
wherein the amino acid-specific binder binds selectively to the binding amino acid, and
the amino acid-specific binder comprises an amino acid sequence comprising:
a first amino acid sequence comprising X1-GQQVT-X2-QHVV-X3-K-X4-MI-X5-
GGR-X6-E-X7-Y-X8-E;

asecond amino acid sequence comprising X1-RQQDT-X2-RHVA-X3-M-X4-MT-X5-
EGO-X6-E-X7-Y-X8-E;
a third amino acid sequence comprising X1-RQQVA-X2-RHVA-X3-M-X4-MT-XS-

EGQ-X6-V-X7-Y-X8-G,


10

20

No
Or

30

WO 2022/236190 PCT/US2022/028400

fOOLI7| With regard io peptides, all peptides are named by indicating the first two
residues, with the full sequence available in Table 3. All of the peptides had the same sequence
for the C-terminal end of the peptide, XXAVECK (Sequence ID No. 18), where the N-terminal
amino acids are varied. The peptides also contained a biotin linked via the lyse side chain on
the C-terminal residue. X1G2 peptides started from a lyophilized form. Peptides were
resuspended in IX DPBS and diluted to the appropriate concentration into the experiment
buffer. The peptides were synthesized in-house on a 20 pmoi scale on a peptide synthesizer
with amino acid reagents and biotin resin. Synthesis resulted in a peptide with an ethylene
diarnine spacer and then the biotin moiety. The peptide was then cleaved from the resin using
3 mL of trifluoroacetic acid (TFA), phenol, water, and triisopropylsilane (TIPS) in an 88:5:5:2
ratio. The peptide was subsequently rinsed with ice cold ether, pelleted by centrifugation at
4500 g for 10 min at 4°C and decanted three times. It was then dried under nitrogen overnight
at room temperature and subsequently lyophilized and stored at -20°C until resuspension in the

assay buffer.
Embodimenis include:

1. A binder complex for selectively identifying an amino acid, the binder complex
comprising:

an amino acid-specific binder: and
an adjunct attached to the amino acid-specific binder,
wherein the amino acid-specific binder binds selectively to a binding amino acid, and
the amino acid-specific binder comprises an amino acid sequence comprising:

a first amino acid sequence comprising X1-GQQVT-X2-QHVV-X3-K-X4-MI-
XS8-GGR-X6-E-X7-¥-X8-E:

asecond amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-
MT-X5-EGQ-X6-E-X7-¥-X8-E:

a third amino acid sequence comprising X1-RQQVA-X2-RHV A-X3-M-X4-
MT-X5-EGOQ-X6-V-X7-Y-X8-G;

a fourth amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-
KT-X5-EGO-X6-E-X7-Y-X8-E;

a fifih amino acid sequence comprising X1-RQQVT-X2-RHVA-X.
MT-X5-K.GOQ-X6-E-X7-N-X

~X4-



WO 2022/236190 PCT/US2022/028400

No. 2}:
ASVVPOERQOV ARBHYPNYKVIVLNDDFENTFRHV AACLMKYIPNMTSDRA
WELTNOVHY EGOATV WVGPOVQAELYHEQLLRAGLTMAPLEPG (Sequence
ID No. 3):
5 ASV VPQERQOQDTREHY PNYKVIVLNDDFENTFRHV AACLMKYIEPNKTSDRAW
ELTNOVHY EGOAW WV GPOEOAEL YHEQLLRAGLTMAPLEPE (Sequence ID
No. 4):
ASV VPQEROQQVTRKHY PNY KVIVLNDDFNTFRHV AACLMKYIPNMTSDRAW
ELTNOVHY RGQAIV W VGPOQEQAELNHEOLLRAGLIMAPLEPE (Sequence ID
10 No. 5);
ASVVPOERQOVARKHYPNYKVIVLNDDFENTFRHV AACLMKYIPNMTSDRA
WELTNOVHY EGQAITV W VGPGEQAEL YHEQLLRAGL TMAPLEPE (Sequence
ID No. 6);
ASV VPQERQQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAW
15 ELTNOVHYKGQAIV WVGPQVQAELYHEQLLRAGLTMAPLEPG (Sequence ID
No. 7),
ASVVPOERQODTRKAY PNYERVIVLNDDENTFRHV AACLMEY IPNMTSDRAW
ELTNQVHYEGQALIV WV GPQOV GAEL YHEQLLRAGLTMAPLEPG (Sequence ID
No. 8);
20 ASVVPQEGQOVTREHYPNYKVIVLNDDFENTFOHVV ACLERYIPNMISDRAW
ELTNOVHY GGRAIV WV GPQEOAELNHEQLLRAGLTMAPLEPE (Sequence ID
No. 9}; or
ASVVPOERQOV ARBHYPNYKVIVLNDDFENTFRHV AACLMKYIPNMTSDRA
WELTNOVHY EGOATV WVGPOV QAELNHEQLLRAGLTMAPLEPE (Sequence
25 ID No. £0).

13. The process of claim 11, wherein separating the isolation complex from the fluid

in which the isolation complex is disposed comprises:

separating the isolation complex based on a size of the isolation complex relative to a
30 size of other constituents in the fluid;

precipitating the isolation cornplex from the fluid:

centrifuging: or

a combination comprising at least one of the foregoing separations.


10

30

WO 2022/236190 PCT/US2022/028400

fOOLE4] With regard to library selection, fluorescence-activated cell sorting (FACS)
was performed using slightly modified protocols from the 2003 Pacific Northwest National
Lab Yeast Display ScFV Antibody Library User's Manual and 2004 Methods in Molecular
Biology Flow Cytometry Protocols. Yeast displaying a library of mutant ClpS proteins were
grown in SD-CAA media ovemight at 30°C until the ODooo was approximately 4.0. The yeast
was used to inoculate a fresh culture at an ODsoo of 1.0 in a mixture of 80 % SG-CAA/20 %
SD-CAA and incubated for 24 hours at 20°C. Approximately 109 yeast were washed and
resuspended in 1 mL of Dulbecco’s phosphate buffered saline containing 0.5% bovine serum
albumin (PBS/BS A) (DPBS) containing 100 1M biotinylated peptide at room temperature for
1 hour. The yeast was pelleted by centrifugation at 3000 g for 2 minutes. The cells were then
incubated with streptavidin- R-phycoerythrin (PE), and anti-myc AF647 for 30 minutes on ice
A typical reaction contained 1 mL of cells (containing approximately 107 cells), peptide at a
concentration of 100 nM, and 2S wu. of a master mix containing 2 pL of an anti-myc antibody,
4 ul of the SAPE (streptavidin, R-phycoerythrin conjugate - | mg/mL) and 19 uL of PBS/BSA
for each sample.

[OOLIS} Cells were washed with PBS/BSA, sorted using a FACS Sony SH800
cytometer, and collected in | mL of SD-CAA media. The threshold for collection was based
on background levels of anti-~myc AFG4?7 and streptavidin-R-PE by cells in the absence of
peptide. Cells which showed greater than background binding for both metrics in the presence
of peptide are grouped in Q2. Q2 Cells were grown out and used to repeat the selection
procedure. The number of cells that bound the peptide improved with each round. After three

rounds of selection, not only is the count of Q2 cells higher for peptides with leucine, but it did

not markedly change for other peptides shown to also bind to 7. e/ongatus CipS2.

fOOLIG}] Celis from the library after three rounds of sorting were lysed by shaking
with glass beads. The DNA from the lysate was purified by phenol-chloroform extraction and
ethanol precipitation, then the available ClpS genes were amplified using the primers from the
previous error-prone PCR. The linear PCR product was sent for Ilumina deep sequencing. This
produced ~83,000 sequence reads with 8467 unique amino acid sequences. Based on these
sequences, mutational hotspots in the sequence of 7. elongatus ClpS2 were identified. For each
hotspot, the most statistically prevalent amino acid was identified. The combination of these
most prevalent hotspot mutants is predicted to produce a CipS variant with improved leucine

binding activity and selectivity


WO 2022/236190 PCT/US2022/028400

18/20
OD=4 ant
‘e000 wee
Ny
(0000 =
B :
= ‘00
2 i
= i =
|
hs x .S ce Sy ck ech
SE GS CEEECLE ES
Ris SS Pwr’ gw W'S
alalve wort}
FIG. 18

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
oh

30

WO 2022/236190 PCT/US2022/028400

sixth amino acid sequence, the seventh amino acid sequence, the eighth amino acid sequence,

the ninth amino acid sequence, or the tenth amino acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] — The following description should not be considered limiting in any wav
With reference to the accompanying drawings, like elements are numbered alike

[0009} FIG. 1 shows an amino acid-specific binder selectively bound to an amino
acid that is a binding amino acid of an analyte in panel A, and panel B shows a non-binding
amuno acid unbound to an amino acid-specific binder;

fOOLO} FIG. 2 shows an amino acid-specific binder of a binder complex selectively
bound to a binding amino acid of an analyte in panel A, and panel B shows a non-binding
amino acid unbound to an amino acid-specific binder of a binder complex;

FOOL L} FIG. 3 shows a binder complex in an absence of an intervening member in
panel A and inclusion of an intervening member in panel B:

[0012] FIG. 4 shows a binder complex that includes an amino acid-specific binder
attached to a taggant in panel A, a protein in panel B, a chemical modifier in panel C. and a
substrate in panel D;

{0013} FIG. 5 shows a binder complex that includes a plurality of amino acid-
specific binders attached to an adjunct in panel A and panel B and attached to a plurality of
adjuncts in panel C:

[0014] FIG. 6 shows an analyte that includes a binding amino acid in panel A, a
non-binding amino acid in panel B, a binding amino acid and non-binding amino acid in panel
C. and an analyte that is a peptide or protein that includes a plurality of amino acids in panel D
and panel E;

fO015} FIG. 7 shows an anchored analyte in an absence of an intervening member
in panel A and inclusion of an intervening member in panel B,

[0016] FIG. 8 shows an anchored analyte in panel A: and a tagged complex in panel

[0017] FIG. 9 shows an anchored analyte in panel A: in panel B, the anchored
analyte shown in panel A after removal of an N-terminal amino acid: in panel C. the anchored
analyte shown in panel B after removal of an N-terminal amino acid: and in panel D, a tagged

complex with production of a taggant signal:


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

200 of binder complex 228 to N-terminal amino acid 220 of anchored analyte 226 when N-
terminal amino acid 220 includes binding amino acid 210 to form tagged complex 230:
subjecting taggant 214 of tagged complex 230 to stimulus 218; producing, by taggant 214 of
tagged complex 230, taggant signal 216 in response to stimulus 218: detecting taggant signal
216: and identifying N-terminal amino acid 220 based on taggant signal 216, wherein amino
acid-specific binder 200 binds selectively to binding amino acid 210.

{0073} In the process for selectively identifying N-terminal amino acid 220,
providing analyte 212 includes purifying or extracting the analyte 212 from a mixture of
components that may interfere with subsequent reactions. Exemplary purifications include
high performance liquid chromatography (HPLC) or precipitation with ammonium sulfate. A
protein can also be digested using a protease such as trypsin to create multiple peptides which
can serve as analytes 212. An immobilized trypsin can be used to create multiple peptides by
digestion of a protein or serum sample and purification of the peptides from the trypsin.

[0074] In the process for selectively identifying N-terminal amino acid 220,
contacting C-terminal end 224 of analyte 212 with anchor 222 includes incubating or flowing
the C-terminal end 224 of analyte 212 over the anchor 222.

[0075] In the process for selectively identifying N-terminal amino acid 220,
anchoring C-terminal end 224 to anchor 222 to form anchored analyte 226 includes incubating
the C-terminal end 224 with anchor 222 under reaction conditions to covalently link the two.
Exemplary reactions would include performing an N-hydroxysuccinimide (NHS)-ester
reaction to link the C-terminal amino acid sidechain lysine within analyte 212 with anchor 222
that is modified with an NHS-ester to produce an amide bond.

{0076} In the process for selectively identifying N-terminal amino acid 220,
contacting N-terminal amino acid 220 of anchored analyte 226 with binder complex 228
includes incubating anchored analyte 226 and binder complex 228 in a reaction buffer for a

time (e.

from 5 sec to 30 min) for the binding reaction to occur based on a binding affinity
of amino acid-specific binder 200 under a set of binding conditions (e.g., in 1X PBS at 30°C).
When N-terminal amino acid 220 is non-binding amino acid 211, the binding reaction does not
occur,

{0077 In the process for selectively identifying N-terminal amino acid 220,
selectively binding amino acid-specific binder 200 of binder complex 228 to N-terminal amino
acid 220 of anchored analyte 226 when N-terminal amino acid 220 includes binding amino
acid 210 includes incubating anchored analyte 226 and binder complex 228 in a reaction buffer

for a time (e.g., from S see to 30 min) for the binding reaction to occur based on a binding

21


WO 2022/236190 PCT/US2022/028400

a» 9/20
a
Nek Bh 8 ae 90 aH
;
m0 n
!
a ee
Ree erm!
2
Remove 2094
, 2
a 2
SNe AGA 106198 200-2 80 24
Cure Na
oom
Reece ee
212
1 U6
Om
SN me OM
a 0 sc 2 9c
M
26 4p
0) ny ene
| 200.
ne a 100 2B 2 Dh
whe D-Qe IL a
4 it vo Ot
nag eye!
216 9
FIG.9

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

16/20

PEA-Compensated

FIG. 16

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

X5 comprises an amino acid sequence comprising SDRAWELTNOVHY (Sequence
ID No. 24}:

‘X6 comprises an amino acid sequence comprising AIVWVGPQ (Sequence ED No.
25),

X7 comprises an amino acid sequence comprising QAEL (Sequence ID No. 26); and

X8& comprises an amino acid sequence comprising HEQLLRAGLTMAPLEP
(Sequence ID No. 27),

such that a total percentage amount of substitutions and deletions to X1, X2,

X3, X4, X5, X6, X7, and X8 is from 0% to less than 30%, exclusive of

PSV VPOERQOVTREHY PNYRVIVLNDDENTFORV AACLMKYIPNMTSDRAW

ELTNQVHYEGQAIV WV GPQEQAEL YHEOLLRAGLTMAPLEPE (Sequence ID

No. 28).

12. The process of embodiment 11, wherein the amino acid sequence comprises:
ASV VPQEGQQV TREHY PNY KVIVLNDDFENTFOHV V ACLKKYIPNMISDRAW
ELTNOVHY GGRATV WVGPOQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID
No. Dy

ASV VPOERQODTRKHY PNYKVIVLNDDFENTFRHV AACLMEY IPNMTSDRAW
ELTNQVHYEGQALV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID
No. 2);

ASV VPQERQOV ARKHY PNYEBVIVLNDDENTFRAVAACLMEYIPNMTSDRA
WELTNQVHYEGOAIV W VGPOV QAELY HEQLLRAGLTMAPLEPG (Sequence
ID No. 3):

ASV VPOERQODTRKHAY PNYEVIVLNDDENTFRHV AACLMEY IPNETSDRAW
ELTNQVHYEGQAIV WV GPQEQAEL YHEOLLRAGLTMAPLEPE (Sequence ID

No. 4);
ASVVPQERQQVTREHYPNYKVIVLNDD RHV AACLMKYIPNMTSDRAW
ELTNOVHY KGQATY W VGPQEQAELNHEOQLLRAGLTMAPLEPE (Sequence ID

No. S}:

ASVVPQERGQOV ARKHYPNYKVIVLNDDFNTFRHYV AACLMKYIPNMTSDRA
WELTNOVHY EGOATYV WV GPOQEQAELYHEQLLRAGLTMAPLEPE (Sequence
ID No. 6):

ASV VPQERQODTREHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAW
ELTNQVHYKGQAIV WVGPOV QAELYHEQLLRAGLTMAPLEPG (Sequence ID


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

Size 6

f

No. 7}:

ASVVPQERQODTREKHY PNYRVIVLNDDFENTFRHV AACLMEYIPNMTSDRAW
ELTNQOVHYEGOATV WV GPOFV OAELYHEQLLRAGLTMAPLEPG (Sequence ID
No. 8);

ASVVPQEGQOVTREHYPNYKVIVLNDDFENTFOHVV ACLERYIPNMISDRAW
ELTNOVHY GGRAIV W VGPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID
No. 9}: oF

ASV VPQEROQV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRA
WELTNOVHYVEGOAI WV GPOVQAELNHEQLLRAGLTMAPLEPE (Sequence
1D No. £0)

13. The process of embodiment 11, wherein separating the isolation complex from the
fluid in which the isolation complex is disposed comprises:

separating the isolation complex based on a size of the isolation complex relative to a
other constituents in the fluid:

precipitating the isolation complex from the fluid:

centrifuging: or

acombination comprising at least one of the foregoing separations.

14. The process of embodiment Li, wherein the amino acid-specific binder is amember

of a binder complex.

15. The process of embodiment 14, wherein the binder complex comprises:
the amino acid-specific binder; and

an adjunct attached to the amino acid-specific binder.

a substrate, or a chemical modifier.

17. The process of embodiment 16, wherein the taggant comprises

a fluorescent moiety, an electrochemical moiety, or a combination comprising at least

one of the foregoing moieties, and

the taggant produces a taggant signal in response to receiving a stimulus.


WO 2022/236190

5/20

a4
{fh
iy 2

B

2008

in
7

pred
Bs
fees

FIG.5

SUBSTITUTE SHEET (RULE 26)

PCT/US2022/028400


10

30

WO 2022/236190 PCT/US2022/028400

new amino acid-specific binder in each iteration of the sequencing process. By tagging amino
acid-specific binder 200 rather than tagging the analyte directly, embodiments herein provide
greater signal control and a more universal approach than conventional technology
Embodiments herein produce or detect different types of signals and include a plurality of
detection schemes that provide enhanced flexibility in types of substrates and different contexts
in which analyte are manipulated.

fOO101L} Amino acid-specific binder 200 and processes herein unexpectedly isolate
analytes from a composition that includes the analyte disposed in a fluid that also includes other

constituents

such as amino acids, proteins, and the like. Due to the ability to anchor either the
analyte or the amino acid-specific binder, the reagent can be used in many different separation
processes in addition to sequencing processes.

{00102} The articles and processes herein are illustrated further by the following

Example, which is non-limiting

AMPLE

{00103} Enhanced N-terminal Amino Acid Binding

fOOLO4] Using available scientific literature, it was found that Synechoceccus
elongatus ClpS demonstrates a preference for binding to peptides with an N-terminal feucine.
The distinct sequence features of S. elongatus ClpS were discerned by comparison to other
well-characterized ClpS variants and, using the publicly available Pfam database, a list of ClpS
variants with sequence features like those of S. elongatus CipS was compiled.
Thermosynechococcus elongatus CipS2 was selected from this list as the variant of greatest
experimental interest, due to ease of handling proteins from thermophilic organisms.

fOOLOS| With regard to plasmid construction, wild-type 7. elongaius CipS2 gene was
synthesized by GeneArt and cloned into the pETI5b vector with an additional His6-tag at the
N-terminus of the protein, followed by a Tobacco Etch virus (TEV) protease cleavage site,
using the Ncol and Xhol restriction sites

[00106] With regard to protein purification, the wild-type 7. elongatus CipS2
proteins were expressed in BL21 Escherichia coli cells. Expression was induced with 0.5 mM

IPTG when the ODeoo reached 1.0 and incubated for 16-18 hours at 20 °C. The cells were

harvested by centrifugation at 5000 g for 20 minutes, and the cell pellets frozen for future use.
{00107} Frozen cell pellets were resuspended in phosphate-buffered saline (pH 7.2}
and sonicated with a probe at 20 % amplitude for 4 seconds on, 20 seconds off, for 90 minutes,

which results in 15 minutes total sonication time. Sonicated material was heated to 60°C for

27


WO 2022/236190 PCT/US2022/028400
8/20
a) 22 / / 6
a 0 2 OD
2 hm

8)

FIG.&

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

17/20

ar

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

ASV VPQERQODTRKHYPNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOAIV WV GPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 8):
aninth amino acid sequence comprising

ASV VPQEGQQV TREHY PNY KVIVLNDDENTFOHV VACLKRY IPNMISDRAWELTNO
VHYGGRAIV WV GPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 9):

atenth amino acid sequence comprising, or

ASV VPQERQOV ARKHYPNYKVE DDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATY WV GPOV QAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10}.

[0005] Disclosed is a binder complex for selectively identifying an amino acid, the
binder complex comprising: an amino acid-specific binder, and an adjunct attached to the
amino acid-specific binder, wherein the amino acid-specific binder binds selectively to a
binding amino acid, and the amino acid-specific binder comprises:

a first amino acid sequence comprising

ASV VPQEGOOVTRKHY PNYKVIVLNDDFENTFOHV V ACLKEKYIPNMISDRAWELTNQ
VHY GGRAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 1):

a second amino acid sequence comprising

ASVVPQEROQQDTREHY PNY KVIVLNDDENTERHV AACLMKYIPNMTSDRAWELTN
OVHYEGOAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 2):

a third amino acid sequence comprising

ASVVPOERQOV ARKHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELIN
QVHYEGOATV WV GPOV OAELYHEQLLRAGL TMAPLEPG (Sequence ID No. 3);

a fourth amino acid sequence comprising
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNKTSDRAWELTN
OVHY EGOATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D No. 4):

a fifth amino acid sequence comprising

ASVVPQERGOVTRKAHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVAYRGOATY WV GPOEQAELNHEQLLRAGL TMAPLEPE (Sequence 1D No. 5):
asixth amino acid sequence comprising

ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYEPNMTSDRAWELTN
QVHYEGOATV WV GPOEQGAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 6);

a seventh amino acid sequence comprising

ASV VPQEROQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHYKGOATV WV GPOV QGAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 7};

an eighth amino acid sequence comprising


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

contacting the N-terminal amino acid of the anchored analyte with the binder complex:

selectively binding the amino acid-specific binder of the binder complex to the N-
terminal amino acid of the anchored analyte when the N-terminal amino acid is the binding
amino acid to form the tagged complex:

subjecting the taggant of the tagged complex to the stimulus:

producing, by the taggant of the tagged complex, the taggant signal in response to the
stimulus:

detecting the taggant signal: and

nal.

identifying the N-terminal amino acid based on the taggant sig

9. The process of claim 8, wherein converting the N-termimal amino acid to the inert
residue comprises chemically changing the N-terminal amino acid prior to producing

the inert residue.

10. The process of claim 9, wherein chemically changing the N-terminal amino acid
prior to producing the inert residue comprises phosphorylating a free amine of the N-

terminal amino acid,

11. A process for selectively isolating an analyte. the process comprising:

contacting an amino acid-specific binder with an analyie comprising a protein, a
peptide, an amino acid, or a combination comprising at least one of foregoing:

selectively binding the amino acid-specific binder to the N-terminal amino acid of the
analyte when the N-terminal amino acid comprises a binding amino acid to form an isolation
complex:

separating the isolation complex from a fluid in which the isolation complex is disposed
to selectively isolating the analyte,

wherein the amino acid-specific binder binds selectively to the binding amino acid, and

the amino acid-specific binder comprises an amino acid sequence comprising:

a first amino acid sequence comprising X1-GQQVT-X2-QHVV-X3-K-X4-ML-N5-

GGR-X6-E-X7-Y-X8-E;

asecond amino acid sequence comprising X1-RQOQDT-X2-RHVA-X3-M-X4-MT-X5-
EGO-X6-E-X7-Y-X8-E;
a third amino acid sequence comprising X1-RQQVA-X2-RHVA-X3-M-X4-MT-XS-

EGQ-X6-V-X7-Y-X8-G,

46


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

a fourth amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-KT-X5-
EGQ-X6-E-X7-Y-X8-E,

a fifth amino acid sequence comprising X1-RQQVT-X2-RHVé
KGQ-X6-E-X7-N-X8-E:

a sixth amino acid sequence comprising X1-RQQVA-K2-RHV A-X3-M-X4-MT-X5-
EGQ-X6-E-X7-Y-X8-E;

a seventh amino acid sequence comprising X1-RQQDT-X2-RHVA-X3-M-N4-MT-
X5-KGQ-X6-V-X7-Y-X8-G: or

an eighth amino acid sequence comprising XI-ROQDT-X2-RHV A-X3-M-X4-MT-
XS-EGO-X6-V-X7-¥-X8-G;

3-M-X4-MT-X5~-

3

wherein:

XI comprises an amino acid sequence comprising ASVVPQE (Sequence ID No. 20).

X2 comprises an amino acid sequence comprising RKHYPNYKVIVLNDDENTE
(Sequence ID No. 21);

X3 comprises an amino acid sequence comprising ACL (Sequence ID No. 22):

X4 comprises an amino acid sequence comprising KYIPN (Sequence ID No. 23):
TNOVHY (Sequence ID

XS comprises an amino acid sequence comprising SDRAWEL
No. 24):

X6 comprises an amino acid sequence comprising ATVWVGPQ (Sequence ID No. 25);

X7 comprises an amino acid sequence comprising QAEL (Sequence ID No. 26); and

X8 comprises an amino acid sequence comprising HEQLLRAGLTMAPLEP
{Sequence ID No. 27),

such that a total percentage amount of substitutions and deletions to X1, X2,

X3, X4, XS, X6, X7, and X8& is from 0% to less than 30%, exclusive of

PSVVPQOERQOVTRKHY PNYKVIVLNDDENTFOHV AACLMKY IPNMTSDRAW

ELTNQVHYEGQALV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID

No. 28).

6. The process of claim 5, wherein the amino acid sequence comprises.
ASVVPQEGQOV TREHYPNYKVIVLNDDENTFQHVV ACLKRYIPNMISDRAWELTNO
VHYGGRAIV WV GPOEQAELYHEQLLRAGL TMAPLEPE (Sequence [D No. 1):

ASV VPQEROQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGQAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 2),
ASVVPOERQOV ARKHYPNYKVIVLNDDENTFRHV AACLMK YIPNMTSDRAWELIN

44


WO 2022/236190 PCT/US2022/028400

20/20

BLI Response

Oo an Mi { iC ig
Clos Concentration

@ feCip82 © NAAB Leu (predieted)

FIG. 20

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
os

WO 2022/236190 PCT/US2022/028400

amino acid-specific binder 200 by providing separation either by size or magnetism or physical
movement of the substrate. The substrate can also be used to detect a taggant signal such as
with fluorescent microscopy and can be a functionalized surface that is optically clear
Exemplary substrates 240 include NHS-ester functionalized glass slides, streptavidin coated
magnetic beads or microplates, a nickel coated resin, and the like. In an embodiment, substrate
240 includes a nickel coated resin.

[0049] Chemical modifier 242 can include a reactive species that can be used in a
non-covalent binding reaction or a cross-linking reaction or can be used to amplify a signal.
Exemplary chemical modifiers 242include click-chemistry compatible moieties, N-
hydroxysuccinimide esters, biotin, maleimide. hydrazide, carbodiirnide compounds for

carboxylic acid cross-linking, photocatalysts, or electrocatalysts. In an embodiment, chemical

modifier 242 includes an azide

f00S0} Exemplary taggant 214 are listed in Table 1 and can include a fluorescent
moiety that can include embedded a fluorophore disposed in a shell, an electrochemical moiety,
chemiluminescent moiety, Forster resonance energy transfer (FRET) pair. catalytic enzyme,
chemical modification, or a combination comprising at least one of the foregoing moieties, that
transduce or amplify stimulus 218 to a measurable response as taggant signal 216 for detecting
a presence of amino acid-specific binder 200. In an embodiment, taggant 214 is a fluorophore
(e.g. a fluorophore commercially available as ALEXAFLUOR such as ALEXAFLUORG647
and the like) that includes conjugated electrons to produce fluorescence upon stimufation by
stimulant 218. Exemplary taggants 214 include horseradish peroxidase, fluorescein,
rhodamine, and the like. In an embodiment. taggant 214 includes a fluorescently labelled dye
{e.g., a dye such as commercially available as ATTOS32). Taggani 214 produces taggant

signal 216 in response to being subjected to stimulus 218

Table 1

Faggant Complex Stimulant Signal Detection
formation
method

Fluorophore NHS-ester fysine | Photon Photon intensity or
sidechain wavelength

Chemiluminescence Luciferase fusion | ATP Photon intensity

Electrochemiluminescence | Fusion with Electrode photon PMT
Ru(Bpy)3 potential

4


WO 2022/236190 PCT/US2022/028400

7/20

ne
RS

8) a
246

ne
RS

FIG. 7

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190 PCT/US2022/028400

(A
Nas Ceavage
ence ne
aye 30D
a, 0
2308 x

Flores
A

i er

Cleavage

FIG, 13

SUBSTITUTE SHEET (RULE 26)


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

chemical moiety, nucleic acid, and the like. Moreover, intervening member 246 can be
chemtcally inert such that it does not interfere with binding or signaling. Exemplary
intervening members 264 include a poly-glycine or serine peptide, a polyethylene glycol
(PEG), a glycan, an oligonucleotide, and the like. In an embodiment, intervening member 264
includes a GSGG peptide.

[0054] Amino acids 209 include binding amino acid 210 and analyte 212 as shown
in FIG. 6. Here, in analyte 212. peptide 248 and protein 250 include a plurality of amino acids
209 (e.g. 209A, 209B, ..., 209k) interconnected and terminating with N-terminal amino acid
220 that bas free amine 252 and penultimate residue 234 and terminating with C-terminal end
224.

[0055] Amino acid-specific binder 200 selectively binds to binding amino acid 210
of analyte 212. Analyte 212 can include binding amino acid 210, non-binding amino acid 211,
peptide 248, protein 250, or a combination thereof. Exemplary analytes 212 include proteins,
peptides, free amino acids, and the like. In an embodiment. analyte 212 includes a protein that
is cleaved using trypsin to produce a mixture of analytes 212 including binding amino acids
210 and non-binding amino acids 211.

[0056] — Amino acid-specific binder 200 selectively binds to binding amino acid 210.
Binding amino acid 210 can include certain naturally occurring amino acids, modified naturally
occurring amino acids, non-naturally occurring amino acids, or modified non-naturally
occurring amino acids. Selective binding of amino acid-specific binder 200 to binding amino
acid 210 isolates binding amino acid 210 from other components in a fluid, identifies binding
amino acid 210 as a particular species of amino acid (e.g.. Leu. Phe, Trp, Tyr), and the like.

{0057} As used herein, “naturally occurring amino acid” refers to the 20 naturally
occurring amino acids, Binding amino acids 210 that are naturally occurring amino acids are
selected from group consisting of phenylalanine, tryptophan, tyrosine, leucine, isoleucine, and

valine. As used herein, “modified naturally occurring amino acid” refers to naturally occurring

amino acids in which a sidechain has been modified. Exemplary modifications include
methylation. phosphorylation. glycosylation, deamination, oxidation. or selenocysteine
formation. Accordingly, binding amino acids 210 that are modified naturally occurring amino
acids include phosphotyrosine, N-acetylated valine, kynurenine and the like.

[0058] As used herein, “non-naturally occurring amino acid” refers to amino acids
that are not naturally incorporated into peptide or protein polymers but can be synthetically
incorporated into a polypeptide. Exemplary non-naturally occurring amino acids are D- amino

acids, homo-amino acids, and amino acids with a non-natural sidechain such as

16


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

an eighth amino acid sequence comprising
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHYEGOAIV WV GPOV QAELY HEQOLLRAGLTMAPLEPG (Sequence ID No. 8)
aninth amino acid sequence comprising

ASVVPQEGOOVTREHY PNYBVIVLNDDENTFOHVV ACLEKYIPNMISDRAWELTNOQ
VHY GGRAIV WV GPQEQAELNHEOLLRAGLTMAPLEPE (Sequence ID No. 9};

a tenth amino acid sequence comprising, or

ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOVOAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10).

[0004] Disclosed is an amino acid-specific binder for selectively binding to an

amino acid in an analyte, the amino acid-specific binder comprising an amino acid sequence
with a homology of at least 30% compared to an amino acid sequence comprising:

a first amino acid sequence comprising

ASV VPQEGOOVTRKHY PNYKVIVLNDDFENTFOHV V ACLEKYIPNMISDRAWELTNQ
VHY GGRAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 1):

a second amino acid sequence comprising

ASVVPQEROQQDTREHY PNY KVIVLNDDENTERHV AACLMKYIPNMTSDRAWELTN
OVHYEGOAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 2):

a third amino acid sequence comprising

ASVVPOERQOV ARKHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELIN
QVHYEGOATV WV GPQOV GAELYHEQLLRAGL TMAPLEPG (Sequence ID No. 3);

a fourth amino acid sequence comprising
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNKTSDRAWELTN
OVHY EGOATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D No. 4):

a fifth amino acid sequence comprising

ASVVPQERGOVTRKAHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVAYR GOAT WV GPOEQAELNHEQLLRAGL TMAPLEPE (Sequence 1D No. 5):
asixth amino acid sequence comprising

ASV VPQERQOV ARKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATV WV GPOEQGAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 6);

a seventh amino acid sequence comprising

ASV VPQEROQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHYKGOATV WV GPOV QGAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 7};

an eighth amino acid sequence comprising

nN


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

11. A process for selectively isolating an analyte, the process comprising:

contacting an amino acid-specific binder with an analyte comprising a protein, a
peptide, an amino acid, or a combination comprising at least one of foregoing:

selectively binding the amino acid-specific binder to the N-terminal amino acid of the
analyte when the N-terminal amino acid comprises a binding amino acid to form an isolation
complex;

separating the isolation complex from a fluid in which the isolation complex is
disposed to selectively isolating the analyte.

wherein the amino acid-specific binder binds selectively to the binding amino acid,
and

the amino acid-specific binder comprises an amino acid sequence comprising:

a first amino acid sequence comprising X1-GQQVT-X2-QHVV-X3-K-X4-ML-NX5-
GGR-XG6-E-X7-Y-X8-E;

asecond amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-MT-
X5-EGQ-X6-E-X7-Y¥-X8-E:

a third amino acid sequence comprising X1-RQOV A-X2-RHV A-X3-M-X4-MT-XS-
EGQ-X6-V-X7-Y-X8-G;

a fourth anuno acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-KT-X5-
EGQ-X6-E-X7-Y-X8-E;

a fifth amino acid sequence comprising X1-RQQVT-X2-RHV A-X3-M-X4-MT-X5-
KGO-X6-E-X7-N-X8-E:

asixth amino acid sequence comprising X1-RQQVA-X2-RHV A-X3-M-X4-MT-X5-
EGQ-X6-E-X7-Y-X8-E,

aseventh amino acid sequence comprising X1-RQQDT-X2-RHV A-X3-M-X4-MT-
X5-KGQ-X6-V-X7-¥-X8-G: or

an eighth amino acid sequence comprising X1-RQQDT-X2-RHVA-X3-M-X4-MT-
XS-EGQ-) J-XT-Y¥-X8-G;

wherein:

XI comprises an amino acid sequence comprising ASVVPQE (Sequence ID No. 20);

X2 comprises an amino acid sequence comprising RKHYPNYKVIVLNDDENTF
{Sequence ID No. 21;

3 comprises an amino acid sequence comprising ACL (Sequence ID No. 22);

X4 comprises an amino acid sequence comprising KYIPN (Sequence ID No. 23);


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

conditions (e.g., in 1X PBS at 30°C). When N-terminal amino acid 220 includes binding amino
acid 210, the binding reaction occurs and tagged complex 230 forms.

[0093] The process for selectively identifying N-terminal amino acid 220, also can
include subjecting taggant 214 of tagged complex 230 to stimulus 218 by exposing tagged
complex 230 to a selected wavelength and intensity of light to excite the fluorophore. In an
embodiment, subjecting taggant 214 of tagged complex 230 to stimulus 218 includes adding
chromogenic substrate.

fO094] The process for selectively identifying N-terminal amino acid 220 also can
include producing, by taggant 214 of tagged complex 230, taggant signal 216 in response to
stimulus 218, e.g., by a method listed in Table 1. In an embodiment, detection can include
detecting taggant response with a microscope including a CCD camera and filters in an optical
system to detect a wavelength of emitted light. In an embodiment, a spectrophotometer
measures absorbance at a selected wavelength to detect a chromogenic substrate, In an
embodiment, a scintillation counter measures radioactivity of a radiolabeled complex.

fO095} The process for selectively identifying N-terminal amino acid 220, also can
include detecting taggant signal 216. The process for selectively identifying N-terminal amino
acid 220, also can include identifying N-terminal amino acid 220 based on taggant signal 216
In the process, converting N-terminal amino acid 220 to inert residue 232 can include
chemically changing N-terminal amino acid 220 prior to producing inert residue 232.

{0096} In an embodiment, with reference to FIG. 12 and FIG. 13, a process for
sequencing protein 250 includes providing sample 208; extracting protein 250 from sample
208; enzymatically digesting protein 250 to provide a plurality of peptides 248 (e.g.. 248A,
248D); forming anchored analyte 226 by immobilizing peptides 248 on anchor 222: producing
tagged complex 230: detecting taggant signal 216 from taggant 214; removing binder complex
228 by washing; cleaving N-terminal amino acid 220 so that penultimate residue 234 becomes
anew N-terminal amino acid 220; and repeating so that multiple repetitions of steps occur,
detecting taggant signal 216 from taggant 214; and

including producing tagged complex 2.

removing binder complex 228 by washing: cleaving N-terminal amino acid 220 so that
penultimate residue 234 becomes a new N-terminal amino acid 220 io sequence protein 250.
{0097} In the process, chemically changing N-terminal amino acid 220 prior to
producing inert residue 232 can include phosphorylating free amine 252 of N-terminal amino
acid 220
{0098} in an embodiment, with reference to FIG. 11, a process for selectively

isolating analyte 212 includes contacting amino acid-specific binder 200 with analyte 212 by

25


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

AMINO ACID-SPECIFIC BINDER AND SELECTIVELY IDENTIFYING AN AMINO
ACID

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with United Siated Government support from the
National Institute of Standards and Technology (NIST), an agency of the United Stated
Department of Commerce. The Government has certain rights to this invention.
CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial
No. 63/185,483 (filed May 7, 2021), which is herein incorporated by reference in its entirety
BRIEF DESCRIPTION

{0003} Disclosed is an amino acid-specific binder for selectively binding to an
amino acid in an analyte, the amino acid-specific binder comprising:
a first amuno acid sequence comprising
ASVVPQEGQQOV TREHYPNYKVIVLNDDENTFOHV V ACLKKYIPNMISDRAWELTNO
VHY GGRAT WV GPOQEOAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 1:
a second amino acid sequence comprising
ASV VPQEROQDTREHY PNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGQAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 2):
a third amino acid sequence comprising
ASV VPQERQOV ARKHY PNYK VIVLNDDFENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOATYV WV GPOVQAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 3),
a fourth amino acid sequence comprising
ASVVPQEROQQDTREHY PNYKVIVLNDDENTERHV AACLMKYIPNK TSDRAW
OVHYEGOAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No. 4):

a fifth amino acid sequence comprising
ASVVPOERQOVTREAYPNYRVIVLNDDENTFRHVAACLMREY IPNMTSDRAW
QVAYRGOATY WV GPOEQAELNHEQLLRAGL TMAPLEPE (Sequence 1D No. 5):

a sixth amino acid sequence comprising

ASVVPQERQOV ARKHY PNYEVIVLNDDFNTFRHV AACLMEYIPNMTSDRAWELTN
OVHY EGOATV WV GPOEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D No. 6):
aseventh amino acid sequence comprising

ASVVPQERGODTRAHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYER GOAT WV GPOVQAELY HEQLLRAGLTMAPLEPG (Sequence ID No. 7),


10

Or

20

ho
os

one of the for

WO 2022/236190 PCT/US2022/028400

14. The process of claim 11, wherein the amino acid-specific binder is a member of a

binder complex.

15. The process of claim 14. wherein the binder complex comprises:
the amino acid-specific binder: and

an adjunct attached to the amino acid-specifie binder.

16. The process of claim 15, wherein the adjunct comprises a taggant. a protein, a

substrate, or a chemical modifier.

17. The process of claim 16. wherein the taggant comprises:
a fluorescent moiety, an electrochemical moiety, or a combination comprising at least

ing moieties, and

the taggant produces a taggant signal in response to receiving a stimulus.

18. The process of claim 17, further comprising
subjecting the taggant of the isolation complex to a stimulus:

producing. by the taggant of the isolation complex. a taggant signal in response to the

stimulus:

detecting the taggant signal: and

identifying the N-terminal amino acid based on the taggant signal

19. The process of claim 17, wherein the stimulus comprises a photon: and

the taggant signal comprises fluorescence emitted from the taggant.

20. The process of claim 11, further comprising:

contacting the amino acid-specific binder with an adjunct to form a binder complex

prior to contacting the arnino acid-specific binder with the analyte.

49


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

QVHYEGOAIVWVGPOVOQAELYHEQLLRAGLTMAPLEPG (Sequence ID No, 3):
ASVVPQERQQDTRKHY PNYKVIVLNDDFNTFRHV AACLMKYIPNKTSDRAWELTN
QVHYEGOAIVWVGPQEQAELYHEQLLRAGLTMAPLEPE (Sequence 1D 4);
ASVVPQERQQVTRKHY PNY KVIVLNDDFENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYKGQAIVWVGPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 5);
ASVVPQERQOV ARKHYPNYK VIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGOAIVWVGPQEQAELYHEOLLRAGLTMAPLEPE (Sequence ID No. 6):
ASV VPQERQODTRKHY PNY KVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYKGQAIVWVGPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 7):
ASVVPQERQQDTRKHY PNY KVIVLNDDFNTERHV AACLMK YIPNMTSDRAWELTN
OVHYEGOAIVWVGPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 8):
ASVVPQEGQQVTRKHY PNYK VIVLNDDENTFQHVV ACLKKYIPNMISDRAWELTNO
VHYGGRAIV WVGPQEQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 9): or
ASVVPQERQOV ARKHY PNYK VIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
QVHYEGGAIV WVGPQVQAELNHEQLLRAGLTMAPLEPE (Sequence ID No. 10}

3. The binder complex of embodiment 1, wherein the adjunct comprises a taggant, a

protein, a substrate. or a chemical modifier.

4. The binder complex of embodiment 3, wherein the taggant comprises:
a fluorescent moiety. an electrochemical moiety, or a combination comprising at least
one of the foregoing moieties, and the taggant produces a taggant signal in response to receiving

a stimulus.

5. A process for selectively identifying an N-terminal amino acid. the process
comprising:

providing an analyte comprising a protein, a peptide, an amino acid, or a combination
comprising at least one of foregoing:

contacting a C-terminal end of the analyte with an anchor:

anchoring the C-terminal end to the anchor to form an anchored analyte:

contacting an N-terminal amino acid of the anchored analyte with a binder complex,
the binder complex comprising:

an amino acid-specific binder, and

a taggant attached to the amino acid-specific binder;


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

X6 comprises an amino acid sequence comprising AIVWVGPQ (Sequence ID
No, 25),

X7 comprises an amino acid sequence comprising QAEL (Sequence [1D No.
26); and

X8 comprises an amino acid sequence comprising HEQLLRAGLTMAPLEP
{Sequence ID No. 27),

such that a total percentage amount of substitutions and deletions to X1, X2,
X3, X4, X5, X6, X7, and X8 is from 0% te less than 30%, exclusive of
PSV VPOERGQOVTRKHY PNYRVIVLNDDENTFOHV AACLMKYVIPNMTSDRAW
ELTNOVHYEGQATV WY GPQEQAEL YHEOLLRAGLTMAPLEPE (Sequence ID
No. 28}.

2. The binder complex of claim 1, wherein the amino acid sequence comprises:
ASV VPQEGOOVTRKHY PNYKVIVLNDDFENTFOHV V ACLEKYIPNMISDRAWELTNQ
VHY GGRAIV WV GPQEQAEL YHEQLLRAGLTMAPLEPE (Sequence 1D No. 1):
ASVVPQERQODTREHYPNYKVIVLNDDENTFRHV AACLMKYIPNMTSDRAWELTN
OVHYEGOATV WV GPOEQAEL YHEQLLRAGLTMAPLEPE (Sequence ID No, 2);
ASV VPQEROOV ARKHYPNYEVIVLNDDENTFRHV AACLMEY IPNMTSDRAWELTN
QVHYEGQAIV WV GPQV QAELY HEQGLLRAGL TMAPLEPG (Sequence ID No. 3);
ASVVPGERQODTRKHYPNYKVIVLNDDFNTFRHV AACLMRYIPNKTSDRAWELTN
QVHYEGQAIV WV GPQEQAELYHEOLLRAGLTMAPLEPE (Sequence ID No. 4);
ASV VPQERQOVTRKHYPNYKVIVLNDDFNTFRHYV AACLMKYIPNMTSDRAWELTN
QVHYKGQALV WV GPQEQ AELNHEQLLRAGLTMAPLEPE (Sequence ID No. 5):
ASVVPQEROOV ARKHY PNYERVIVLNDDENTFRAV AACLMEY IPNMTSDRAWELTN
OVHY EGOAIV WV GPQEQAELYHEQLLRAGLTMAPLEPE (Sequence ID No. 6):
ASVVPQERGODTRAHY PNYKVIVLNDDFNTFRHV AACLMKYIPNMTSDRAWELTN
QVHYRGOATV WV GPOVQAELYHEQLLRAGLTMAPLEPG (Sequence ID No. 7);
ASVVPQERQODTREAYPNYRVIVLNDDENTFRHVAACLMEY IPNMTSDRAW
QVHYEGOATYV WV GPQV QAEL YHEQLLRAGLTMAPLEPG (Sequence ID No. 8):
ASVVPQEGQOVTREHYPNYKVIVLNDDENTFOHVV ACLKERYIPNMISDRAWELTNO
VHYGGRAIV WV GPQEQAELNHEQLLRAGLTMAPLEPE (Sequence [D No. 9): or
ASV VPQEROOV ARKHYPNYEVIVLNDDENTFRHV AACLMEY IPNMTSDRAWELTN
QOVHYEGOQAIV WV GPQOV OAELNHEGLLRAGLTMAPLEPE (Sequence ID No. 10).

42


10

Or

20

ho
os

30

WO 2022/236190 PCT/US2022/028400

sixty minutes, then cooled on ice for fifteen minutes. The lysate was centrifuged at 20000 g for
36 minutes and then incubated for one hour with chelating fast flow sepharose resin coated
with nickel and pre-equilibrated in lysis buffer. The resin was separated from the lysate by
collection on a gravity-flow column. The column was then washed with 10 column volumes
(CVs) of PBS, and then 5 CVs of wash buffer (PBS + 50 mM imidazole,}, before eluting with
5 CVs of elution buffer (PBS + 250 mM imidazole). The eluted protein was then loaded onto
a §200 26/60 size exclusion chromatography column pre-equilibrated in 2CV of 20 mM
HEPES. pH &. 5 mL Fractions were collected and tracked at 280 nm; peaks were compared
with a gel filtration standard and further analyzed by SDS-PAGE. Fractions were combined,
concentrated by ultra-centrifugation with a 10 kDa MWCO, centrifuged for 40 minutes at
20,000 g. and measured by the Nanodrop assay.

{OO108} The initial binding activity of 7. e/ongatus ClpS2 was characterized using
bio-layer interferometry (BLI). In BLI. a functionalized fiber-optic probe is placed in a solution
of analyte and the accumulation of analyte on the functional group of the probe is measured by
changes in reflected light within the probe. A BLI probe functionalized with streptavidin is
allowed to accumulate biotinylated peptide. Probes with peptide are then placed in solutions of

CipS of varying concentration and allowed to equilibrate. BLI response is then graphed to

concentration to establish a binding curve. Dilutions of 7 e/ongatus ClpS2 were made in 20
mM HEPES pH 8, 150 mM KCl, 0.1% BSA, and 0.08% Tween-20 at concentrations of 0.312,

0.625, 1.25, 2.5, S, 10, 20, and 40 uM. Comparison acro:

four peptides that vary only by their
N-terminal amino acid show a strong preference for binding leucine.

fOOLO9] The gene was cloned into the pCTCON2 vector for yeast display by
amplifying the gene by polymerase chain reaction (PCR} with primers to add the Nhel and
BamHI sites, and then ligating this to the vector such that the gene is located to the C-terminal
end of the aga2-encoding gene, separated by a factor XA cleavage site, an HA-tag and a
{GGGGS (Sequence ID No. 14)}x3 spacer. There is also a C-terminal mye-tag before the stop
codon. A vector was also created that contains no gene insert, but rather a Sacll site and a Spef
site, so that the vector can be linearized at this site for homologous recombination.

fOOLLO] With regard to random mutagenesis library creation, to create the random
mutagenesis libraries, error-prone PCR was used. The gene encoding the CipS protein of
interest was amplified using the HA-tag for (CCATACGACGTTCCAGACTAC (Sequence ID
No. 15) and T7 (TAATACGACTCACTATAGGG (Sequence ID No. 16) primers in a reaction
containing 0.2 mM dATP, i mM dCTP. 0.2 mM dGTP, i mM dTTP, 10 mM MeCh, and 0.5
mM MaCh, 1X Taq reaction buffer (20 mM Tris-HCI, pH 8.4, 50 mM KCI) without MgCh,

28


10

Or

20

ho
os

WO 2022/236190 PCT/US2022/028400

{0034} According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. | and binds selectively to leucine.

[0035] According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence [D No. 2 and binds selectively to jeucine.

{0036} According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. 3 and binds selectively to leucine

[0037] According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. 4 and binds selectively to leucine.

[0038] According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. 5 and binds selectively to leucine.

{0039} According to an embodiment. amino acid-specific binder 200 is a protein
with Sequence ID No. 6 and binds selectively to leucine.

[0040] According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. 7 and binds selectively to leucine.

{0041} According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. 8 and binds selectively to leucine.

[0042] According to an embodiment, amino acid-specific binder 200 is a protein
with Sequence [D No. 9 and binds selectively to leucine.

{0043} According io an embodiment, amino acid-specific binder 200 is a protein
with Sequence ID No. 10 and binds selectively to leucine.

[0044] According to an embodiment, amino acid-specific binder 200 is a protein
with a sequence homology of at least 30% compared to an amino acid sequence selected from
ithe group consisting essentially of the amino acid sequence with Sequence ID No. |, Sequence
ID No. 2, Sequence ID No. 3, Sequence ID No. 4, Sequence ID No. 5, Sequence ID No. 6,
Sequence ID No. 7, Sequence ID No. 8, Sequence ID No. 9, and Sequence ID No. 10 and binds
selectively to leucine

[0045] Binder complex 228 selectively identifies an amino acid. fn an
embodiment, binder complex 228 includes amino acid-specific binder 200 and adjunct 236
attached to amino acid-specific binder 200. Attachment of adjunct 236 to amino acid-specific
binder 200 can include a covalent bond, an ionic bond, electrostatic interaction (e.g., a R-cation
interaction, dipole-dipole interaction, a multi-pole interaction, and the like), intercalation. a
clathrate arrangement (e.g., with adjunct 236 partially or wholly trapped in amino acid-specific

binder 200 or vice-versa, such that amino acid-specific binder 200 can still selectively bind to

12


WO 2022/236190 PCT/US2022/028400

15/20

6

Alexa 647

FIG. 18

SUBSTITUTE SHEET (RULE 26)


WO 2022/236190

4/20
A
29
? — Ny
m
B)
9
Y QO Noy
moon
FG]

SUBSTITUTE SHEET (RULE 26)

PCT/US2022/028400


WO 2022/236190

3/20

a »

de

2a

8) ee

26 246 a

FIG.3

SUBSTITUTE SHEET (RULE 26)

PCT/US2022/028400


WO 2022/236190 PCT/US2022/028400

FRET pair a fluorophore on | Photon Photon Intensity or
amino acid- wavelength
specific binder
200, a
fluorophore on
analyte 212. or
fluorophores on
amino acid-

specific binder

260

Catalytic enzyme Horseradish Addition of | Absorbance | Spectrophotometer
peroxidase chromogenic | ata
fusion substrate wavelength

Radioactive clement S§-metbionine, | None Radioactivity | Scintillation
“=p. counting or radio
phosphorylation, image

oF iia
labeling of
amino acid

binder 200

fO0S1] Stimulus 218 can include light emitted from a lamp, laser, LED, or a
chromogenic subsirate such as tetramethylbenzidine (TMB). Exemplary stimulus 218 includes

laser light such as 30 mW, 488 nm laser light. In an embodiment, stimulus 218 is a photon,

rom a light source such as a laser, flash lamp, and the like. In an embodiment, stimulus
218 is a redox potential pulse.

[0052] — Taggant signal 216 can have a temporal duration suitable for detection by
an electrical aniplifier, photodetector, scintillator, camera, and the like. In an embodiment,
taggant signal 216 is fluorescence emission that is detected, ¢.g., by a detector such as a
microscope that transmits the fluorescence to a CCD camera, wherein the location of emission
can be correlated with the intensity of the signal.

[0053} In binder complex 228, with regard to indirect attachment of adjunct 236 to
amino acid-specific binder 200, intervening member 246 can include a linker to connect
adjunct 236 to amino acid-specific binder 200 but that does not provide additional functionality

other than linking the two together. Intervening member 246 can be a protein, peptide,

5