Journal: bioRxiv

Article Title: Principles of in situ protein sequencing: expansion microscopy-adapted Edman degradation and amino acid recognition

doi: 10.64898/2026.01.29.702630

Figure Lengend Snippet: (A) Assay to assess efficiency of Edman reagent conjugation on synthetic peptides in ExMre gels. Synthetic peptide AGGAGGLLGGSRGGK{acr} (abbreviated as A15-peptide; K{acr} denotes an acryloyl functional group on the lysine side chain); amino acids of the peptide chain are depicted as light grey beads, with arginine in the dark grey bead, as site of trypsin cleavage. Peptides are embedded into ExMre gels during free-radical polymerization of the swellable gel, and separate gels are subjected to various in-gel Edman conditions. All ExMre gels are then immersed in trypsin for digestion overnight to acquire the fragment upstream to the trypsin cleavage site at arginine, for further analysis. Supernatant is analyzed with liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QToF MS, abbreviated LC/QToF). (B) In-gel Edman chemistry with formamide as conjugation solvent and PITC as the Edman reagent. Bar graphs representing the relative abundance (arbitrary units, a.u., with all samples processed with spiked-in control compounds; see Methods and Supplementary Figure 12) of different peptide ion species detected on the LC/QToF. Bar graphs were obtained from measuring the area under the curve (AUC) of the chromatogram of various species (extracted based on the exact mass, see Methods for details, and Source Data for raw traces). The separate conditions were solvent only (“formamide”), PITC to solvent (1:1000 ratio PITC:solvent) for 1 hour at 50 °C (“formamide with PITC”), TFA for 30 min at 50 °C (“formamide, then TFA”), PITC to solvent (1:1000 ratio PITC:solvent) for 1 hour at 50 °C followed by TFA for 30 min at 50 °C (“formamide with PITC, then TFA”). The relative abundance of the ion species: (i) non-modified peptide (AGGAGGLLGGSR), (ii) peptide conjugated to PITC, phenylthiocarbamyl (PTC)-peptide (PTC-AGGAGGLLGGSR), and (iii) peptide with cleaved N-terminal amino acid (GGAGGLLGGSR), were reported throughout the in-gel Edman degradation process in the various conditions (dots, individual experiments; blue bar, mean; error bar, standard deviation, n=3 separate gelation solutions). (C) In-gel Edman chemistry as in B, but with DMSO as solvent. (D) In-gel Edman chemistry as in C, but with 23:77 of DMSO:0.1 M sodium bicarbonate pH 8.5 as conjugation solution with FITC (5.9 mM) as Edman reagent. The peptide conjugated to FITC is fluorescein-thiocarbamyl (FTC)-peptide ( FTC -AGGAGGLLGGSR). (E) Top/flat surface size of ExMre gels throughout the Edman degradation process. Normalized top/flat surface size at each step for ExMre gels with (i) formamide conjugation with PITC, (ii) DMSO conjugation with PITC, or (iii) 5.9 mM FITC in 23:77 DMSO:0.1 M NaHCO3 pH8.5 (thick dashed black line, top/flat surface size of the gel after washes in 1 M Tris pH 9.5 (1 mL x 3) before Edman degradation; error bar, standard deviation; black dots, individual experiments; n=3 separate gelation solutions). (F) Assay to assess efficiency of Edman degradation on synthetic peptides in ExMre gels. Synthetic peptide: K{N3}GGAGGLLGGSRGGK{acr} (abbreviated K{N3}15-peptide, where K{N3} is 6-azido-lysine), and amino acids of the peptide chain are depicted as light grey beads, with arginine, “R”, with the dark grey bead, as site of trypsin cleavage. The peptides are embedded into the ExMre gels during free-radical polymerization of the first gel, and separate gels are subjected to various Edman conditions. Read-out is performed using strain-promoted alkyne-azide cycloaddition (SPAAC) with dibenzocyclooctyne AlexaFluor 488 (DBCO-AF488) on the embedded peptides followed by analysis of the fluorescence. Note: sloped intensity profiles were likely due to excitation light attenuation in deeper layers of highly fluorescent gels. (G) ExM gels containing K{N 3 }15mer-peptide at 1 mM were cast in a gelation chamber, expanded and re-embedded to reach ∼2.7X expansion factor at a final peptide concentration of ∼50 μM. The separate conditions were DMSO only (“DMSO”), PITC to DMSO (1:1000 ratio PITC:DMSO; “PITC:DMSO”), DMSO followed by TFA, PITC to DMSO (1:1000 ratio PITC:DMSO) followed by TFA, and the same condition but with 20 ug/mL trypsin (“PITC:DMSO+TFA+trypsin”). After SPAAC with 20 μg/mL DBCO-AF488 in PBS, bulk gel fluorescence was imaged using a confocal microscope with a 10 μm Z-step. Analysis was performed on raw images. (i) A representative raw image of the fluorescence intensity is depicted for each gel condition, taking the 20th slice of the Z-stack for each (∼200 μm deep into the gel). Scale bar is 500 μm. (ii) The fluorescence intensity of the ExMre gels in different conditions was compared throughout the gel thickness (∼500 μm) (line, mean; shaded area, standard deviation; n=3 separate gelation solutions). (iii) Average fluorescence intensity of ExMre gels in the different conditions across the whole volume imaged (colored bar, mean; black dots, individual experiments; error bar, standard deviation, n=3 separate gelation solutions). (H) (i) Independent assay for in-gel Edman degradation, with phenylthiohydantoin (PTH)-F detection of Edman degraded synthetic peptide: FGGAGRGLGK{acr} (abbreviated “F 1 peptide”) embedded in ExMre gels, as in (A). Separate gels were subjected to various Edman conditions. The conditions included TFA only for 30 min at 50 °C, and PITC to DMSO (1:1000 ratio PITC:DMSO) for 1 hour at 50 °C followed by TFA for 30 min at 50 °C. Subsequently, TFA was removed from the gels and they were immersed in 50 μL of 1:1 acetonitrile to water and agitated. Read-out was then performed by injecting the supernatant into LC/QToF using LC Method (see Methods) . (ii) Results acquired as in (i). Analysis of PTH-F abundance was performed using PTH-F exact mass, 282.0827 ± 0.0056 Da (see Methods for Edman degradation and PTH detection for details). (blue bar, mean; error bar, standard deviation; black dots, individual experiments; n=3 separate gelation solutions).

Article Snippet: 20 μg/mL of DBCO-AF488 (Lumiprobe, #218F0) was prepared, from a stock of 10 mg/mL fluorophore in DMF stored at -20 °C, in 1X PBS (500 μL per tube).

Techniques: Conjugation Assay, Functional Assay, Liquid Chromatography, Mass Spectrometry, Solvent, Control, Modification, Standard Deviation, Fluorescence, Concentration Assay, Microscopy

Journal: bioRxiv

Article Title: Small amphiphilic DNA for programmable transmembrane signaling and amplification

doi: 10.1101/2025.10.29.685379

Figure Lengend Snippet: (a-c) NUPACK simulated structure of extended target (T ′ -Cy3, 60-nt) bound to HALOS 31 (a), linear and SA-Tr dual-reporter (Cy5 and AF488) labeling to the opened hairpins (b-c), confirming thermodynamic stability and no cross reactivity between reporter.S (b) Native PAGE of dual reporter labeling to open HALOS conformation in solution. 10% Native PAGE was run using 1x TBE buffer + 12.5 mM MgCl 2 at 100 V for 45 min at room temperature. The gel was post-stained using EtBr (0.5 µg/mL) for 10 min and imaged using UV-transillumination. The fluorescence images were scanned using Image Quant 800 gel imager. Addition of both R-Cy5 and R-AF488 resulted in dual labeling of T ′ -Cy3 (60 nt) treated HALOS 31 (lane 2). Absence of either extension from extend domain from target (T-Cy3, 41 nt) or HALOS (lacking open hairpin domain) showed no labeling of R-AF488 and R-Cy5 respectively(lane 3-4), confirming no cross reactivity between R-Cy5 and AF488 reporter.SThe single color reporter (either R-Cy5 or R-AF488) labeling to T ′ -Cy3 (60 nt) treated HALOS 31 resulted in specific reporter labeling as depicted by fluorescence image (lane 7-8). In the absence of target (T ′ -Cy3), HALOS 31 remained uncreative to any reporters (lane 9).

Article Snippet: Cy3 and Cy5 fluor conjugated DNA were synthesized using DNA synthesizer while AF488 fluor coupling with DNA was carried out using amine modified DNA and AF488 NHS ester (Lumiprobe, 11820).

Techniques: Labeling, Clear Native PAGE, Staining, Fluorescence

Journal: bioRxiv

Article Title: Small amphiphilic DNA for programmable transmembrane signaling and amplification

doi: 10.1101/2025.10.29.685379

Figure Lengend Snippet: (a) Extended-view confocal images for dual reporter labeling in GUV.SWhen treated with R-Cy5 (100 nM) or R-AF488 (100 nM) individually, T ′ -Cy3 detected GUVs showed specific fluorescence on the respective Cy3 or AF488 channel; both were observed when both reporters were applied simultaneously. (b) Dual reporter labeling with control GUVs (Coumarin, Cyan). Cy3 target GUVs display dual reporter labeling while no reporter labeling on coumarin GUVs, suggesting no-non-specific labeling (c) Live-cell compatibility of dual reporter probe.SLive HEK293T cells were incubated with HALOS 31 (250 nM) in Opti-MEM containing 50 µM ATA and 12.5 mM MgCl 2 for 15 min under live-cell culture environment. After washing, cells were sequentially treated to T-Cy3 (250 nM) and R-Cy5 + R-AF488 (250 nM each) with washing steps (3x) between each addition. Confocal imaging showed Cy3, Cy5 and AF488 fluorescence at the cell membrane, demonstrating compatibility of HALOS-mediated signal transduction using dual reporter labeling on live-cell membrane. Scale bar: 20 µm (a-c).

Article Snippet: Cy3 and Cy5 fluor conjugated DNA were synthesized using DNA synthesizer while AF488 fluor coupling with DNA was carried out using amine modified DNA and AF488 NHS ester (Lumiprobe, 11820).

Techniques: Labeling, Fluorescence, Control, Incubation, Cell Culture, Imaging, Membrane, Transduction

Journal: bioRxiv

Article Title: Design of solubly expressed miniaturized SMART MHCs

doi: 10.1101/2025.03.14.643101

Figure Lengend Snippet: A) Size exclusion chromatogram trace of empty CSM8 A*02:01 purified from the soluble fraction of E. coli and thereafter from the monomeric fraction by SEC. B) FP data (filled circles) and fitted binding curve (line) for n=3 technical replicates of CSM8 A*02:01 binding to the AF488-NY-ESO-1 peptide. C) Equilibrium binding data (filled circles) and fitted binding curves (lines) from SPR experiments for CSM8 A*02:01 presenting variants of the NY-ESO-1 peptide to immobilized 1G4 TCR. D-E) yeast display data for hit6 (blue) or full-length SCT (gray) A*02:01 stained with an anti-HA antibody and either an anti-A*02 antibody (dark) or streptavidin A6c134 TCR tetramers (light). D) Surface expression levels (measured as anti-HA MFI) for the HA+ population of each sample. E) Fraction of HA+ yeast cells that are also stain+ for the anti-A*02 or TCR tetramer stain in each sample.

Article Snippet: AF488 carboxylic acid was purchased from Lumiprobe.

Techniques: Purification, Binding Assay, Staining, Expressing