LC-MS methodsProteinsLC-MS analysis of protein samples was executed using an SQ Detector 2 mass spectrometer connected to an Acquity UPLC system with an Axquity UPLC BEH300 C4 column (1.7 µm, 2.1 mm × 50 mm). The eluents used were water with 0.1% (v/v) formic acid (solvent A) and 71% (v/v) acetonitrile in water with 0.075% (v/v) formic acid (solvent B). The flow rate was kept constant at 0.2 mL/min and the gradient was programmed as follows: from a starting point of a 72:28 ratio of A:B, the gradient moved to 71.2% B over 12 min, then moved to 100% B over 1 min, remained at 100% B for 3 min, then moved back to the starting ratio of 72:28 A:B (72:28) over 0.5 min, and remained at this ratio for 3 min. The electrospray source was set at 3.0 kV capillary and 30 V cone voltage. Nitrogen was used as the desolvation gas at 800 L/h. Mass spectra were reconstructed from the measured ion series using the MaxEnt algorithm within MassLynx software (Waters).PeptidesLC-MS analysis was performed for peptides as described above for proteins. The only significant changes were the column type and solvent gradient. An Acquity UPLC BEH300 C18 column (1.7 µm, 2.1 mm × 50 mm) was used and the gradient (with the same eluent system) was as follows: 2 min at 100% solvent A, a gradient moving steadily to reach 100% solvent B at 11 min, remaining at 100% solvent B until 16 min, and then moving back to 100% solvent A at 16.1 min until the end of the run at 20 min.Peptide reactionsAll peptides were purchased from GenScript (Supplementary Information). Peptides (100 µM) were reduced with DTT (200 µM) for 1 h at room temperature and then incubated with the indicated compound (200 µM) in PBS (pH 7.4) for 2 h at room temperature. The reaction was monitored and analysed by LC-MS.D1/D2-peptide fusionsPlasmid constructionTemplate plasmid pCantab6 D1/D2 (10 µg, supplied by AstraZeneca) was mixed with 10x CutSmart® buffer (5 µL), HindIII-HF®/NotI-HF® restriction enzymes (2.5 µL each) and sterile water (total reaction volume = 50 µL). Samples were incubated for 2 h at 37 °C then analysed via agarose gel electrophoresis to confirm complete digestion. The digested plasmid was excised from the gel, purified with a QIAquick® Gel extraction Kit and quantified using a Lunatic UV/Vis spectrometer (λ = 260 nm, Unchained Labs).Hybrid plasmids P1-FLAG, P1-TEV, P1-Xa, P2-FLAG, P3-FLAG and P4-FLAG were constructed via Gibson Assembly® following the standard NEB protocol and using the strings specified in the Supplementary Information, transformed into chemically competent BL21 (DE3) E. coli and plated O/N at 37 °C on 2xTYAG agar. The next day, plasmid DNA was isolated from individual colonies (5 per construct) using a Plasmid Plus Mini Kit (Qiagen) and sequenced (Source BioScience).Hybrid plasmids P5-FLAG and P6 were kindly gifted by Dr Carole Urbache (AstraZeneca).Protein production and purification2xTYAG media (10 mL) was inoculated with a single transformed BL21 (DE3) colony and incubated O/N at 30 °C and 280 RPM. The starter culture was diluted (50x in 2xTYAG) and incubated at 25 °C and 280 RPM until OD600nm = 0.6. Expression was induced by the addition of IPTG (50 µL, 1.0 M). After 16 h at 25 °C and 250 RPM, the cultures were centrifuged for 20 mins at 4 °C and 2500 × g. The cell pellet was resuspended in TES buffer (1 mL), incubated at room temperature for 15 min, added to cold 1:5 TES buffer: sterile water (1.5 mL) and left on ice for 30 min. The periplasmic fraction was clarified via centrifugation at 4 °C and 2500 × g for 30 min. Fusion proteins P1-FLAG, P1-TEV, P1-Xa and P6 were purified using His SpinTrap™ columns (GE Healthcare). Fusion proteins P2-FLAG, P3-FLAG, P4-FLAG and P5-FLAG were purified using Pierce™ Anti-DYKDDDDK Affinity Resin (Thermo Fisher Scientific). Protein concentration was quantified using a Lunatic UV/Vis spectrometer (λ = 280 nm, Unchained Labs).SDS-PAGEPurified protein (2 µg) was mixed with 4x Novex™ Sharp Pre-stained Protein Standard (2.5 µL, Thermo Fisher Scientific), TCEP (1 µL, 500 mM) and sterile water (total reaction volume = 10 µL). The samples were heated at 96 °C for 2 min, then loaded into the wells of a NuPAGE 4-12% Bis-Tris gel (Invitrogen) alongside a SeeBlue™ Plus2 Pre-stained Protein Standard (10 µL, Thermo Fisher Scientific). Electrophoresis was run with 1xNuPAGE™ MES SDS Running Buffer (Thermo Fisher Scientific) at 200 V for 30 min. Protein bands were visualised using InstantBlue™ Coomassie Protein Stain (Abcam) and photographed using a Chemidoc™ MP imaging system (Biorad) fitted with a white tray (coomassie blue protein gel setting).CCA cyclization of D1/D2-peptide fusionsFLAG-capped proteins (25 µg) were combined with reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 8.0) to a total reaction volume of 20 µL. Enterokinase light chain enzyme (NEB) was added (1 µL) and the reaction was incubated for 16 h at 25 °C. Samples were then buffer exchanged into PBS (pH 7.4) using Amicon Ultra 0.5 spin columns following the manufacturer’s instructions.Enterokinase-cleaved proteins (10 µM) were reduced with DTT (1 mM) in PBS (pH 7.4) for 30 min at 37 °C in a total volume of 95 µL. Linker CCA (250 µM) in DMSO (5% total volume) was then added and the reaction was left to incubate for 2 h at room temperature. All reactions were monitored and analysed by LC-MS.Phage production, cyclization and infectivity studiesProduction of Ph1-FLAG phage particlesPlasmid F2-FLAG (section S0) was transformed into a chemically competent Mix and Go! TG1 E. coli (Zymo) and plated O/N at 37 °C on 2xTYAG agar. The next day, 2xTYAG (25 mL) was inoculated with a single transformed TG1 colony and the culture was incubated at 37 °C and 280 RPM until OD600nm = 0.5–1.0. Helper phage was added (2.5 µL) and the mixture was incubated at 37 °C and 150 RPM for 1 h. The cultures were then centrifuged at 3500 RPM for 15 min at room temperature. The supernatant was decanted, and the cell pellet was resuspended in 2xTYAK media (25 mL).Following O/N incubation at 25 °C and 280 RPM, the culture was centrifuged at 8000 RPM and 4 °C for 20 min. The supernatant was collected in a pre-chilled bottle, and phage particles were precipitated by adding 2.5 M sodium chloride with 20% (v/v) PEG (7.5 mL) and storing it on ice for 2 h.The supernatant was transferred to a clean centrifuge pot and the phage particles were pelleted by centrifugation at 8000 RPM and 4 °C for 20 min. The supernatant was decanted and the phage pellet was resuspended in PBS (2 mL). Finally, the phage stock was centrifuged at 11,000 RPM and 4 °C for 10 min and the supernatant was stored at 4 °C.Modification of Ph1-FLAG and Ph1 phage particlesPh1-FLAG phage particles (~ 1011) were combined with reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 8.0) to a total reaction volume of 100 µL. Enterokinase light chain enzyme (NEB) was added (2 µL) and the reaction was incubated for 16 h at 25 °C. Enterokinase-cleaved Ph1-FLAG (Ph1) phage particles were buffer exchanged into PBS (pH 7.4) using Zeba™ Spin Desalting Columns (7 K MWCO, 0.5 mL) following the manufacturer’s instructions.Ph1-FLAG and Ph1 phage particles (~ 1011) were reduced with DTT (1 mM) for 30 min at 37 °C in a total volume of 100 µL. Linker CCA (final concentration = 150 µM) in DMSO (5% total volume) was added and the reaction was left to incubate for 2 h at room temperature. CPO-PEG2-biotin (final concentration = 150 µM) in DMSO (5% total volume) was then added and the reaction was left to incubate for a further 2 h at room temperature. All reactions were monitored and analysed by phage ELISA.Positive control: Ph1-FLAG and Ph1 phage particles (~ 1011) was modified with iodoacetyl-PEG2-biotin (final concentration = 1 mM) following a literature procedure2.Negative control: Ph1-FLAG and Ph1 phage particles (~ 1011) were treated with DMSO (5% total volume) instead of linker CCA then CPO-PEG2-biotin as described above.Phage ELISAEnterokinase cleavage ELISA: Biotinylated antigen = Biotinylated Anti-His Tag Antibody (Acro Biosystems), assay buffer = PBS, blocking buffer = 3% (w/v) mPBS. CCA cyclization ELISA: Biotinylated antigen = none required, assay buffer = N/A, blocking buffer = 3% (w/v) mPBS.All incubation steps were performed at room temperature unless otherwise stated. A Pierce™ streptavidin-coated 96 well plate (Thermo Fisher Scientific) was treated O/N at 4 °C with biotinylated antigen (50 µL/well, 10 ng/µL in assay buffer). The next day, the plate was washed x3 with PBS to remove unbound antigen and incubated for 1 h with blocking buffer (300 µL/ well). At the same time, phage cultures (100 µL, ~ 1010) were incubated for 1 h with blocking buffer (400 µL).The 96-well plate was washed x3 with PBS and subsequently incubated with blocked phage (50 µL/ well) for 1 h. The plate was washed x3 with PBST, and bound phage was detected via the addition of anti-M13 HRP conjugated secondary antibody (50 µL/ well, 1/5000 dilution in blocking buffer, Abcam). After 1 h, the plate was washed x3 with PBST. HRP was developed using TMB substrate (50 µL/ well, Thermo Fisher Scientific), quenched after 5 min with H2SO4 (50 µL/ well, 0.5 M) and visualised at λ = 280 nm using an EnVision Microplate Reader (PerkinElmer). Data was processed using SoftMax® Pro Software v7.Infectivity studiesCCA: Ph1 phage particles (~ 1011) were reduced with DTT (1 mM) for 30 min at 37 °C in a total volume of 95 µL. Linker CCA (final concentration = 150 µM) in DMSO (5% total volume) was added and the reaction was left to incubate for 2 h at room temperature. DBX: Ph1 phage particles (~ 1011) were reduced with TCEP (1 mM) for 30 min at 37 °C in a total volume of 95 µL. Reduced Ph1 phage particles were buffer exchanged into NH4HCO3 (20 mM, pH 8.0) using Zeba™ Spin Desalting Columns (7 K MWCO, 0.5 mL) following the manufacturer’s instructions. Linker DBX (final concentration = 150 µM) in DMSO (5% total volume) was added and the reaction was left to incubate for 2 h at room temperature.For each sample, two 100-fold dilutions in 2xTY were prepared. An aliquot (10 μL) of the 10−4 dilution sample was added to TG1 E. coli (990 µL) grown to OD600nm = 0.6. After 1 h of infection at 37 °C and 150 RPM, four further 10-fold dilutions in 2xTY were prepared (10−7 – 10−10). Aliquots (100 µL) of dilution samples 10−7 – 10−10 were plated O/N on 2xTYAG agar at 37 °C. The next day, phage titres were calculated according to the equation.Cfu/mL = [No. of colonies] x [Dilution factor] x [1/Volume plated (mL)].Library construction and cyclizationKunkel mutagenesis
Preparation of dU-ssDNA
Stop template ST1, kindly provided by Dr Carole Urbache (AstraZeneca), was transformed into chemically competent CJ236 E. coli (Supplementary Information). 2xTYAGC (5 mL) inoculated with a single transformed CJ236 colony was incubated at 37 °C and 300 RPM until OD600nm = 0.8 – 1. Wild-type M13KO7 helper phage (provided by AstraZeneca) was added to a multiplicity of infection of 10 (1 µL, 3 × 1013 phage/mL stock) and the mixture was incubated without shaking for 10 min at 37 °C. An aliquot (1 mL) of the culture was diluted in 2xTYAK (30 mL) supplemented with uridine (0.25 µg/mL).
Following O/N incubation at 37 °C and 300 RPM, the cells were harvested by centrifugation for 10 min at 2 °C and 15,000 RPM. The supernatant was transferred to a fresh tube and 2.5 M sodium chloride with 20% (v/v) PEG (9 mL) was added. Phage particles were collected by incubating at room temperature for 10 min and centrifugation for 10 min at 2 °C and 10,000 RPM. The supernatant was decanted and the phage pellet was resuspended in PBS (0.5 mL) and centrifuged for 5 min at room temperature and 13,000 RPM. The supernatant was transferred to a clean Eppendorf and the dU-ssDNA template was purified using an E.Z.N.A.® M13 DNA Mini Kit.
Phosphorylation of mutagenic oligonucleotides
Mutagenic oligonucleotide O1 (0.7 µg) was mixed with 10x TM buffer (2 µL), ATP (2 µL, 10 mM), DTT (1 µL, 100 mM) and sterile water (total reaction volume = 18 µL). T4 polynucleotide kinase (2 µL, 10 U/µL) was then added and the mixture was incubated for 1 h at 37 °C.
Annealing of mutagenic oligonucleotide to template at a ratio of 3:1
To the phosphorylated oligo (20 µL) was added ST1 dU-ssDNA (20 ug), 10x TM buffer (25 µL) and sterile water (total reaction volume = 250 µL). The mixture was split equally between two PCR tubes and incubated at 90 °C for 2 min, 50 °C for 3 min and 20 °C for 5 min.
Mutagenesis reaction
To the annealed oligo/ template (250 µL) was added ATP (10 µL, 10 mM), dNTPs (10 µL, 25 mM), DTT (15 µL, 100 mM), T4 DNA ligase (5 µL, 6 U/µL) and T7 DNA polymerase (3 µL, 10 U/µL). The mixture was split evenly between two reaction wells per library (21.5 µL/well) and incubated for 3 h at 20 °C.
The reaction product was affinity-purified into water (50 µL) using a Roche High Pure DNA purification kit and transformed into electrocompetent HB2151 E. coli. For this, an aliquot of cells (1.6 mL) was added to an Eppendorf alongside the mutated plasmid (500 ng). The cell/ DNA mixture was equally portioned into four prechilled 0.2 mm Gene Pulser/MicroPulser Electroporation Cuvettes (Invitrogen). Each electroporation pulse was executed at 2500 V, 15 µF, and 335 R. Immediately after electroporation, 2xTYG (1 mL, prewarmed to 37 °C) was added to each cuvette and the cell suspension was transferred to a 50 mL Falcon tube (one per library), rinsing with 2xTYG (1 mL). Following 1 h of incubation at 37 °C and 150 RPM, the cells were pelleted by centrifugation for 10 min at room temperature and 3200 RPM, resuspended in 2xTYG (1 mL) before plating O/N on a 2xTYAG bioassay plate at 37 °C.
The next day, colonies were picked and prepared for sequencing via colony PCR. The library was scraped from the bioassay plate into 2xTY media (5 mL) mixed with 50% (v/v) glycerol (2.5 mL), portioned into 500 µL aliquots and stored at − 80 °C.
Phage library rescueL1 HB2151 glycerol backup stock was added to 2xTYAG media (400 mL) to an OD600nm ~ 0.1. The bacterial culture was incubated at 37 °C and 280 RPM until OD600nm = 0.5–1.0. Helper phage was added (80 µL) and the mixture was incubated at 37 °C and 150 RPM for 1 h. The cultures were pelleted by centrifugation for 15 min at room temperature and 3500 RPM and resuspended in 2xTYAK media (400 mL).Following O/N incubation at 25 °C and 280 RPM, the culture was centrifuged at 8000 RPM and 4 °C for 20 min. The supernatant was collected in a pre-chilled bottle, and phage particles were precipitated by adding 2.5 M sodium chloride with 20% (v/v) PEG (120 mL) and stored on ice for 1 h.The supernatant was transferred to a clean centrifuge pot and the phage particles were pelleted by centrifugation at 8000 RPM and 4 °C for 20 min. The supernatant was decanted, the phage pellet was resuspended in TE buffer (10 mL) and 2.5 M sodium chloride with 20% (v/v) PEG (3 mL) was added.Following a 1 h precipitation on ice, the phage stock was centrifuged at 11,000 RPM and 4 °C for 15 min. The supernatant was decanted and the phage pellet was resuspended in TE buffer (3 mL). Finally, the phage stock was centrifuged at 11,000 RPM and 4 °C for 10 min and the supernatant was stored at 4 °C.Phage display selectionsStreptavidin selection
Negative selection and CCA cyclization
Streptavidin-coated magnetic Dynabeads™ (50 μL/ selection, prewashed with 1 mL PBS) were blocked with 3% (w/v) mPBS (1 mL) for 1 h on a rotary mixer at room temperature and 20 RPM. L1 phage particles (~ 1011) were reduced with DTT (1 mM) for 30 min at 37 °C in PBS (total reaction volume = 100 µL). The beads were pelleted on a magnetic stand and reduced L1 phage particles (~ 1011) were added. After 1 h on a rotary mixer at room temperature and 20 RPM, the beads were pelleted and the supernatant was transferred to a fresh Eppendorf. Linker CCA (final concentration = 150 µM) in DMSO (5% total volume) was added and the reaction was left to incubate for 2 h at room temperature.
Positive selection
L1-Red: L1 phage particles (~ 1011) were reduced with DTT (1 mM) for 30 min at 37 °C in PBS (total reaction volume = 100 µL).
L1-CCA: Output phage from the negative streptavidin selection above.
L1-Red/ L1-CCA input phage (~ 1011) and streptavidin-coated magnetic Dynabeads™ (50 μL/ selection, prewashed with 1 mL PBS) were incubated with 3% (w/v) mPBS (volume made up to 500 μL for phage, 1 mL for beads) for 1 h on a rotary mixer at room temperature and 20 RPM. The beads were pelleted on a magnetic stand and resuspended in 3% (w/v) mPBS (100 μL). An aliquot of the blocked beads (50 μL) was added to the blocked phage. The mixture was incubated for 1 h on a rotary mixer at room temperature and 20 RPM.
Using a Kingfisher mL apparatus, the beads were washed x5 with PBST (1 mL) and eluted into trypsin solution (10 µg/ mL trypsin in 0.1 M pH 7.0 sodium phosphate buffer, 200 µL). Trypsin digest was performed for 30 min at 37 °C and 600 RPM, the beads were pelleted and the supernatant was added to OD600nm = 0.6 TG1 E. coli (800 µL). After 1 h at 37 °C and 250 RPM, output titres were performed and the remaining cells were plated O/N on a 2xTYAG bioassay plate at 37 °C. The next day, the cells were harvested from the plate, as described below.
αvβ3 selection
Negative selection and CCA cyclization
Assay buffer = HEPES (pH 7.0, 50 mM), NaCl (150 mM), MnCl (0.1 mM)
Blocking buffer = HEPES (pH 7.0, 50 mM), NaCl (150 mM), MnCl (0.1 mM) with 2% (v/v) BSA.
Streptavidin-coated magnetic Dynabeads™ (50 μL/ selection, prewashed with 1 mL PBS) were blocked with 3% (w/v) mPBS (1 mL) for 1 h on a rotary mixer at room temperature and 20 RPM. L1 phage particles (~ 1011) were reduced with DTT (1 mM) for 30 min at 37 °C in assay buffer (total reaction volume = 100 µL). Biotinylated αvβ3 (50 nM, Acro Biosystems) was added and the mixture was incubated for 1 h on a rotary mixer at room temperature and 20 RPM. The beads were pelleted on a magnetic stand and the reduced L1 phage particle/ biotinylated αvβ3 mixture was added. After 1 h on a rotary mixer at room temperature and 20 RPM, the beads were pelleted and the supernatant was transferred to a fresh Eppendorf.
The supernatant was buffer exchanged into PBS (pH 7.4) using Zeba™ Spin Desalting Columns (7 K MWCO, 0.5 mL) following the manufacturer’s instructions. Linker CCA (final concentration = 150 µM) in DMSO (5% total volume) was added and the reaction was left to incubate for 2 h at room temperature.
Positive selection
L1-Red: L1 phage particles (~ 1011) were reduced with DTT (1 mM) for 30 min at 37 °C in PBS (total reaction volume = 100 µL).
L1-Dis: L1 phage particles with no further modification.
L1-CCA: Output phage from the negative streptavidin selection above.
L1-Red/ L1-DiS/ L1-CCA input phage (~ 1011) and streptavidin-coated magnetic Dynabeads™ (50 μL/ selection, prewashed with 1 mL PBS) were incubated with blocking buffer (volume made up to 500 μL for phage, 1 mL for beads) for 1 h on a rotary mixer at room temperature and 20 RPM. The beads were pelleted on a magnetic stand and resuspended in a blocking buffer (100 μL). To deselect against streptavidin, an aliquot of the blocked beads (50 μL) was added to the blocked phage. After 1 h on a rotary mixer at room temperature and 20 RPM, the beads were pelleted and the supernatant was transferred to a fresh Eppendorf. Biotinylated antigen was added at the concentration required. After 1 h rotating on an end-over-end rotor, the remaining aliquot of blocked beads (50 µL) was added and the mixture was equilibrated for 5 min on an orbital shaker at 37 °C and 300 RPM.
Using a Kingfisher mL apparatus, the beads were washed x5 with PBST (1 mL) and eluted into trypsin solution (10 µg/ mL trypsin in 0.1 M pH 7.0 sodium phosphate buffer, 200 µL). Trypsin digest was performed for 30 min at 37 °C and 600 RPM, the beads were pelleted and the supernatant was added to OD600nm = 0.6 TG1 E. coli (800 µL). After 1 h at 37 °C and 250 RPM, output titres were performed and the remaining cells were plated O/N on a 2xTYAG bioassay plate at 37 °C. The next day, the cells were harvested from the plate, as described below.
Selection rescueInfected bacteria were scraped from the 2xTYAG bioassay plate with glycerol-medium (10 mL, 2xTY media mixed with 50% v/v glycerol in a 2:1 ratio) using a disposable plastic spreader. The cell suspension was transferred into a 50 mL Falcon tube and placed on an end-over-end rotor for 10 min to fully resuspend the bacterial plate scrape. An aliquot of the plate scrape (1 mL) was stored at − 80 °C as a backup stock.2xTYAG media (25 mL) was inoculated with a sufficient quantity of the bacterial plate scrape to reach OD600nm = 0.1. The bacterial culture was incubated at 37 °C and 280 RPM until OD600nm = 0.5-1.0. M13K07trp helper phage (2.5 µL, 3 × 1013 cfu/mL stock) were added and the culture was incubated for 1 h at 37 °C and 150 RPM. The cells were centrifuged at 3200 xg for 10 min and resuspended in 2xTYAK media (25 mL).Following O/N incubation at 25 °C and 280 RPM, the culture was centrifuged at 8000 RPM and 4 °C for 20 min. The supernatant was collected in a pre-chilled bottle, and phage particles were precipitated by adding 2.5 M sodium chloride with 20% (v/v) PEG (7.5 mL) and storing it on ice for 2 h.The supernatant was transferred to a clean centrifuge pot and the phage particles were pelleted by centrifugation at 8000 RPM and 4 °C for 20 min. The supernatant was decanted and the phage pellet was resuspended in PBS (2 mL). Finally, the phage stock was centrifuged at 11,000 RPM and 4 °C for 10 min and the supernatant was stored at 4 °C.Input and output titres2xTY media (50 mL) was inoculated with a fresh single TG1 colony and incubated at 37 °C and 280 RPM until OD600nm = 0.6.Input titresA serial dilution of the input phage stock was performed as follows:
(1)
10 µL input phage + 990 µL 2xTY media(1:100)102
(2)
10 µL (1) + 990 µL 2xTY media(1:100)104
(3)
10 µL (2) + 990 µL OD600nm = 0.6 TG1(1:100)106
Sample (3) was incubated for 1 h at 37 deg and 150 RPM then diluted as below:
(4)
100 µL (3) + 900 µL 2xTY media(1:10)107
(5)
100 µL (4) + 900 µL 2xTY media(1:10)108
(6)
100 µL (5) + 900 µL 2xTY media(1:10)109
(7)
100 µL (6) + 900 µL 2xTY media(1:10)1010
An aliquot of dilution samples 107 – 109 (100 µL) was plated O/N on 2xTYAG agar at 37 deg. The next day, input titres were calculated according to the equation:Cfu/mL = [No. of colonies] x [Dilution factor] x [1/Volume plated (mL)].Output titresA serial dilution of the output phage stock was performed as follows:
(1)
10 µL output phage + 90 µL 2xTY media(1:10)101
(2)
10 µL output phage + 990 µL 2xTY media(1:100)102
(3)
100 µL (2) + 900 µL 2xTY media (1:10)103
(4)
100 µL (3) + 900 µL 2xTY media(1:10)104
An aliquot of dilution samples 101 – 104 (100 µL) was plated O/N on 2xTYAG agar at 37 deg. The next day, output titres were calculated according to the equation:Cfu/mL = [No. of colonies] x [Dilution factor] x [1/Volume plated (mL)].Peptide hit validationPhage ELISAIndividual phage constructs were rescued, precipitated, reduced with DTT and cyclized with linker CCA as described previously (section 0).
Streptavidin-binding ELISA:
Biotinylated antigen = none required, assay buffer = N/A, blocking buffer = 3% (w/v) mPBS.
αvβ3-binding ELISA:
Biotinylated antigen = biotinylated αvβ3 (Acro Biosystems), assay buffer = HEPES (pH 7.0, 50 mM), NaCl (150 mM), MnCl (0.1 mM), blocking buffer = HEPES (pH 7.0, 50 mM), NaCl (150 mM), MnCl (0.1 mM) with 2% (v/v) BSA.
The phage ELISA was performed as described previously (section 00).
Construction of D1/D2-pep fusions Str1/2/3 and αvβ3-1/2/3Primers were designed for each construct using NEBaseChanger™ software to introduce the desired mutation. Site-directed mutagenesis was performed using an NEB Q5® Site-Directed Mutagenesis Kit following the manufacturer’s instructions.Fusion proteins were expressed (section 00), purified using His SpinTrap™ columns (GE Healthcare) and cyclized (section 00) as described previously.OctetD1/D2 fusion samples were run using a Basic Kinetic Experiment setup on an Octet® RED384 from ForteBio in combination with streptavidin (SA) sensors (ForteBio). Prior to measurement, the biosensor tips were rehydrated for at least 10 min in 200 μL of assay buffer supplemented with 0.1% BSA and 0.02% Tween20 (BuffA). Tilted bottom 384-well plates were used with 50 μL solution per well, which was centrifuged at 1000 RPM for 2 min before measurements were performed. Tips underwent the following steps at 25 °C with 500 Hz: Baseline (BuffA) (60 s), Loading (Antigen at 50 nM) (50 s), Baseline (BuffA) (60 s), Association (D1/D2 fusion proteins ranging from 2 µM to 15.6 nM) (300 s), Dissociation (BuffA) (900 s). Experimental runs were limited to 1 h to minimise evaporation. Raw sensorgrams were processed in R by first subtracting the background from a reference sample (antigen-loaded, no D1/D2 fusion).αvβ3 assay buffer: HEPES (pH 7.0, 50 mM), NaCl (150 mM), MnCl (0.1 mM).Binding kinetic analysis of synthetic peptidesThe affinity of peptides Str1 and Str1-CCA to streptavidin was measured using the Biacore 8 K (GE Healthcare) at 25 °C.For cyclization, peptide Str1 (5 mg, GL Biochem) was dissolved in PBS (20 mL) and treated with DTT (1.2 eq.) for 1 h at RT then linker CCA (2 eq.) for 3 h at RT. The reaction was purified by RP-HPLC and characterised by LC-MS to confirm the correct identity of the cyclized product, Str1-CCA. Str1 = 1351 Da and Str1-CCA = 1601 Da.Streptavidin was covalently immobilised to a CM5 chip surface (Cytiva, BR100530, lot 10340325) using standard amine coupling techniques at a concentration of 16 µg/ml in 10 mM sodium acetate pH 4.5.The peptides were serially diluted (4.1 – 9000 nM) in HBS-EP + buffer pH 7.4 and flowed over the chip at 30 µL/min, with 180 s association and 600 s dissociation. Multiple buffer-only injections were made under the same conditions to allow for double reference subtraction of the final sensorgram sets, which were analysed using Biacore 8 K Evaluation Software. Each experiment was repeated three times, which gave consistent results. Representative results are presented in the paper.Computational modellingModelled structures of peptides Str7-Lin, Str7-DiS and Str7-CCA were constructed using Molecular Operating Environment software (MOE, v 2020.09). Full energy minimisations were run for all peptides using the MMF94x force field available in MOE until the RMSD gradient values were below 0.001. The streptavidin (1DF8) PDB structure was checked for missing atoms and residues using the MOE software before carrying out the docking studies.The prepared streptavidin structure and lowest energy Str7-CCA peptide conformation were imported into MOE software. Minimising contacts for hydrogen, the structures were subjected to an AMBER94 energy minimisation protocol. The docking energy calculation was carried out within a user-specified three-dimensional docking box (3D docking box) using the simulated annealing method under the MMFF94 force field. The energy grids for docking were generated as grid-based potential fields by the MOE-Dock programme, to reduce the calculation time. Multiple docking poses were generated and analysed to identify the most favourable binding interactions.Bioinformatic analysisThe sequences used for each method are depicted in Tables S1 and S2 for Pep and Str peptides, respectively.Similarity calculations were performed using Python scripts (python version 3.6.13) that are available at https://github.com/ana-laura476/peptide_similarity. Peptide sequences were converted into SMILES strings using the NovoPro peptide SMILES converter PepSMI (https://www.novoprolabs.com/tools/convert-peptide-to-smiles-string. MAP4 fingerprints were calculated using the script available at https://github.com/reymond-group/map4, and the Jaccard distance was obtained with the get_distance() function from tmap/Minhash library, version 1.0.6. Since this last function gives dissimilarity values (0 for identical, 1 for highly dissimilar), it was transformed by applying 1 – Jaccard distance giving a similarity value (1 for identical, 0 for highly dissimilar) more easily interpretable and comparable. Using Scikit-learn’s TfidfVectorizer() function, we counted the occurrence of each amino acid and then employed the Scikit-learn’s cosine_similarity() function to compare text similarities of the written peptides sequences (Scikit-learn version 0.24.2 was used).Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.