Site-directed conjugation of single-stranded DNA to affinity proteins: quantifying the importance of conjugation strategy

Affinity protein–oligonucleotide conjugates are increasingly being explored as diagnostic and therapeutic tools. Despite growing interest, these probes are typically constructed using outdated, non-selective chemistries, and little has been done to investigate how conjugation to oligonucleotides influences the function of affinity proteins. Herein, we report a novel site-selective conjugation method for furnishing affinity protein–oligonucleotide conjugates in a 93% yield within fifteen minutes. Using SPR, we explore how the choice of affinity protein, conjugation strategy, and DNA length impact target binding and reveal the deleterious effects of non-specific conjugation methods. Furthermore, we show that these adverse effects can be minimised by employing our site-selective conjugation strategy, leading to improved performance in an immuno-PCR assay. Finally, we investigate the interactions between affinity protein–oligonucleotide conjugates and live cells, demonstrating the benefits of site-selective conjugation. This work provides critical insight into the importance of conjugation strategy when constructing affinity protein–oligonucleotide conjugates.


Protein-DNA conjugation
Below are general protocols for protein-DNA conjugation.Incubation times and ssDNA lengths and equivalences were changed according to the experiments described above.Ultrafiltration was performed using VivaSpin devices (Sartorius, Germany).UV-Vis spectroscopy or microBCA assay were used to determine protein concentrations, with extinction coefficients; ε280 = 215,000 M -1 cm -1 for ONT, ε280 = 68750 M -1 cm -1 for ONT-Fab, ε280 = 7450 M -1 cm -1 for ADAPT6, and ε335 = 9,100 M -1 cm -1 for pyridazinedione scaffolds.A correction factor at 280 nm of 0.25 (of ε335) was employed to correct for the absorbance of the pyridazinedione scaffold. [3]Appropriate buffers were used as blanks for baseline correction.Affinity protein-ssDNA conjugates were purified using anionic exchange spin chromatography (ThermoFisher Scientific, USA), and eluted into an ammonium acetate buffer (pH = 3.75, 500 mM acetate).

Surface plasmon resonance analysis
Surface plasmon resonance experiments were run on Biacore 3000 and T200 instruments (GE Healthcare) at 25 °C with PBST as a running buffer.HER2 (SinoBioligical, China) was diluted to 10 μg ml -1 in 10 mM NaOAc pH 4.5 and immobilised on CM5 chips by amine coupling.Alternatively, biotinylated HER2 (SinoBiological, China) was diluted to 3 µg/mL and immobilised onto a streptavidin-coated CM5 chip.Immobilisation levels are detailed in Tables S4 and S7.The analytes were diluted into PBST and injected at 30 μl min -1 .Surfaces were regenerated using 10 mM HCl. Sensorgrams were double-referenced using a blank flow cell and a buffer injection.Data were fitted to a Langmuir 1:1 interaction using BiaEval 4.1 software, and dissociation equilibrium constants were calculated from the association and dissociation rate constants.As a negative control to rule out non-specific binding, TCO-ssDNA29 was diluted to 400 nM in PBST and injected (no response observed).

Immuno plate-based assay
A sandwich-style immuno-assay was performed on 384-well NUNC Maxisorp flat-bottomed black plates (Thermo-Fisher Scientific) in triplicate.Capture antibody (Pertuzumab Biosimilar) (Proteogenix) was added to each well (50 µL, 2 µg / mL in carbonate buffer), a coverslip was placed over the wells, and the plate was incubated at 4°C overnight.The wells were washed four times (100 µL, PBST).HER2 was added as a dilution series to each well (40 µL, 0.0128-200 nM in PBST 1% BSA) and incubated at 21°C for 1 hour.As a control, one well was filled with PBST 1% BSA.The wells were then washed four times (100 µL, PBST).Affinity protein-ssDNA conjugate was added to all wells (40 µL, 0.2-5 nM in 0.5X PBST 0.1% BSA) and incubated at 21°C for 30 minutes.The wells were finally washed six times (100 µL, PBST).Collection solution was added to all wells (50 µL, 0.5X PBST 0.1% BSA) and incubated at 95°C for 20 minutes.Samples were collected and stored for PCR analysis on collection tubes blocked with 0.5X PBST 0.1% BSA (100 µL, 30minute incubation).qPCR qPCR was performed on the samples from the sandwich assay using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific, USA).A PCR MasterMix was prepared using DreamTaq Hot Start PCR Master Mix (Thermo Fisher Scientific, USA), containing 1X EvaGreen (Biotium, USA), 50 nM ROX reference dye (Biotium, USA), and 1 nM forward/reverse primers (Microsynth, Switzerland).The samples obtained from the plate-based sandwich assay were diluted 1 in 2 with UltraPure DNA/RNAse-free distilled water (Thermo-Fisher Scientific).The diluted samples were added to the prepared PCR mix (10 µL sample, 15 µL MasterMix), and qPCR was performed under the following cycling conditions: 95°C for 30 seconds; 40 cycles of 95°C for 7 seconds, 67°C for 30 seconds, 72°C for 10 seconds; final extension step for 10 seconds.The fluorescence data were processed using a Python script developed using Pycharm Professional Edition software (JetBrains, Czech Republic) employing Matplolib, Numpy, Pandas, Seaborn and Scipy packages.

Cell culture
Breast cancer cell lines were provided by François M. Cuenot (Aceto lab, ETHZ), and originally purchased from ATCC.Cell lines tested negative for mycoplasma contamination before commencement of the cell studies (March 2023).The SK-BR-3 cell line (ATCC HTB-30, 43-year old white human female) was cultured in RPMI 1640 (Roswell Park Memorial Institute, Thermo Fischer Scientific, USA), while the BT-20 (ATCC HTB-19, 74-year old white human female) cell line was cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F12 (Thermo Fischer Scientific, USA).Both culture media received a 10% supplementation of fetal bovine serum (FBS) and 5% penicillin-streptomycin (Invitrogen, USA).They were maintained during culture at 37°C in an incubator (Galaxy 170 S, New Brunswick Scientific, USA) with an atmosphere of 95% air and 5% CO2.Cells were cultured until they reached approximately 90% confluency, as determined by visual observation, after which they were harvested using a 0.25% trypsin-EDTA mix (Thermo Fischer Scientific, USA).Once harvested, the cells were washed with their medium at 160 r.c.f for 5 minutes.The supernatant was removed, and the cells were resuspended in medium to continue the culture or in FACS buffer for further experiment.
The supernatant was removed, and the cells were resuspended with the affinity protein-ssDNA29-TEX conjugate or control ssDNA29-TEX solutions (100 µL, 0.0046-10 nM, in FACS buffer) and incubated in the dark at 21°C for 30 minutes.As a control, the cells were also incubated with pure FACS buffer.The cells well were washed with FACS buffer to remove unbound protein.The cells were subsequently analysed using a CytoFLEX S (Beckman Coulter, USA).Each sample was measured at a 30 µL/min flow rate and detected based on their forward scattering height (FSH) (factory default values).The data were subsequently analysed with FlowJo Software v10 (BD Life Sciences, USA) and a Python script developed using Pycharm Professional Edition with Matplotlib, Numpy, Pandas and Seaborn packages.

Preparation of cells for Imaging
Cultured cells were seeded into a glass-bottom 18 well µ-Slide (ibidi GmbH, Germany) at a concentration of 10 4 cells per well, with each well containing their respective growth media (100 µL).Following a 24-hour incubation period, the cells were gently washed (PBS, 2 × 100 µL) to remove any non-adherent or excess media.Subsequently, the cells were blocked (1% BSA 0.1% Sperm Salmon DNA in PBS, 100 µL) and incubated at room temperature for 60 minutes under gentle shaking.The cells were washed (PBS, 2 ×100 µL), and each cell group was stained with the protein-conjugate solution (50 nM in PBS) and incubated at room temperature for 80 minutes under gentle shaking.As a control, a group of cells underwent a similar incubation with PBS alone.After incubation, the cells were washed (PBS, 3 × 100 µL).Paraformaldehyde (100 µL, 4% in PBS, Bio-Rad) was added to each well and incubated for 5 minutes.The cells were then washed (PBS, 1 × 100 µL, 2 min incubation).Subsequently, the cell membrane was permeated with Triton-X (100 µL, 0.1% in PBS) and incubated for 5 minutes before the cells were washed (PBS, 3 × 100µL, 2 min incubation).Following that, the nuclei were stained with DAPI (100 µL, 1 µg/mL in PBS) and incubated for 5 minutes before being washed (PBS, 2 × 100 µL).As a control, a group of cells underwent a similar incubation with PBS alone.Finally, to prevent cell desiccation, PBS was added to each well (100 µL).

Cell Imaging Procedure
The cells were imaged on a Ti Eclipse fluorescence microscope (Nikon, Japan) equipped with a SpectraX-6-LCR light source (Lumencor, USA) and a Nikon S Plan Fluor ELWD 40x/0.60Objective (Nikon, Japan).To visualise Texas Red fluorescence, a filter cube setup with a 550/49 excitation filter, a 630/69 emission filter and a FF593-Di03 dichroic mirror was used.To visualise DAPI fluorescence, a filter cube setup with a 377/50 excitation filter, a 442 long pass emission filter and a FF409-Di03 dichroic mirror was used.To visualise FAM fluorescence, a filter cube setup with a 478/28 emission filter, a 525/45 excitation filter and a HC500 dichroic mirror was used.Images were captured by a C11440 ORCA-Flash 4.0 fluorescence camera (2048x2048 pixels, Hamamatsu Photonics, Japan) with a 100 millisecond exposure time and light power at 50%.Micro-manager software (v1.4.22,Arthur Edelstein et al. [4] ) was used to control the camera and light source simultaneously.The images were processed using the Fiji software (v2.14.0,Schindelin et al. [5] ).

General
Unless stated otherwise, all reagents and starting materials were obtained from chemical suppliers and were used as received.Reactions were monitored by thin layer chromatography using pre-coated SIL G/UV 254 plates purchased from VWR. Flash chromatography was carried out manually using Kieselgel 60 M 0.04/0.063mm silica gel or automatically using a BioTage Isolera with KP-Snap or KP-Sil columns.NMR spectra were recorded using a Bruker AC300, AC500, or AC600 spectrometer (300 MHz, 500 MHz, and 600 MHz, respectively).Chemical shifts (δ) are given in ppm units relative to the solvent reference and coupling constants (J) are measured in Hertz.Proton (1H) NMR multiplicities are shown as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet), dt (double triplet), etc. HMBC, HSQC, and DEPT were employed to aid with accurate assignments.

Sequences
Table S2.Sequences, theoretical molecular masses, and predicted extinction coefficients for the ssDNA oligonucleotides used in this study.Theoretical molecular masses were calculated from the molecular formulas.Extinction coefficients were calculated from the sequences.  .UV-Vis spectrum for ONT-dis.The peak at 335 nm is characteristic of the pyridazinedione and suggests a pyridazinedione:antibody ratio of 3.3 (see equation 2, main text). [3]creasing the lysine labelling of ONT-Fab  SPR data summary tables Table S3.Summarised values for kon, koff, KD, and RmaxO:RmaxT obtained from the SPR plots in Fig. 2 and equation 1.
Table S6.Summarised values for kon, koff, KD, and RmaxO:RmaxT obtained from the SPR plots in Fig. 3 S7.Theoretical Rmax and RmaxO:RmaxT calculations for ONT-F(ab)-dis and ONT-F(ab)-dis-ssDNA6-50.The molecular weight of each ligand was calculated using the respective molecular weights of the native ligands, the linkers, and TCO-ssDNA29.Protein:ssDNA ratios, as obtained from densitometry or UV-Vis spectrometry analysis, were taken into account.
Table S8.Transport rate constants (ktr), transport coefficients (Lm), and Onsager coefficients (Lr) for the affinity protein-ssDNA conjugates studied in Fig, 3.These values were calculated from the molecular weight, RmaxO, and association constants (kon) as described above.
Fig. S3.TCO groups can be easily installed onto azide-containing oligonucleotides.LCMS ion traces for the native (a) and modified (b) oligonucleotides used in this study.The predicted molecular mass for each TCO functionalised oligonucleotide was calculated as the sum of the theoretical molecular mass of the starting material + the mass of the TCO linker.The observed molecular mass for each TCO functionalised oligonucleotide was calculated from the m/z values, accounting for the charge states.

Figure S6
Figure S6.UV-Vis spectrum for ONT-dis.The peak at 335 nm is characteristic of the pyridazinedione and suggests a pyridazinedione:antibody ratio of 3.3 (see equation 2, main text).[3]

Figure S7 .
Figure S7.SDS-PAGE analysis of the conjugation between ONT-Fab-lys and TCO-ssDNA29, with an average ssDNA:protein ratio of 1.35 : 1. ONT-Fab-lys was incubated with 4. equiv of ssDNA29-TCO.A visible band just below 50 Kda indicates the presence of leftover ONT-Fab-lys.The ladder was run on a different gel, as indicated by the white gap.

Figure S11 .
Figure S11.The optimised conjugation protocol is transferable to larger ssDNA oligonucleotides.SDS-PAGE analysis of the modified ONT and ONT-Fab conjugates employed for the immuno-PCR assay (Fig.4).Once again, more homogenous products are obtained using site-selective approaches (lanes 3 and 8). qPCR

Figure S12 .Figure S14 .Figure S15 .
Figure S12.qPCR curves obtained for the immuno-PCR assays of HER2 titrated against ONT-ssDNApcr and ONT-Fab-ssDNApcr.The probes were evaluated at 5, 1, and 0.2 nM.The solid curves and shaded areas represent the mean and standard deviation values of three measurements.