Controlling Coagulation in Blood with Red Light

Abstract Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin‐binding aptamer with a red‐light photocleavable linker derived from Cy7 by Cu‐catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood—restoring the blood's natural coagulation capability.


Chemical Synthesis
All reactions were performed under a protective argon atmosphere unless otherwise specified. Reagents and solvents were purchased from commercial sources and used without further purification. Deionised (DI) water was used for all experiments. Reaction progresses were monitored with silica gel 60-coated TLC-sheets and reaction product purifications via column chromatography were performed with silica gel 60, both by Macherey-Nagel. 1H and 13C spectra were recorded on a Bruker AV250, AV400 or AV500 MHz spectrometer. Mass spectra (MS) were obtained using a "Surveyor MSQ" for ESI measurements and high resolution mass spectra (HRMS) were obtained with a ThermoScientific "LTQ Orbitrap XL" (MALDI-HRMS).

Absorption and fluorescence measurements
The UV/vis absorption spectrum of the Cy7-linker was recorded on an Evolution 300 UV/vis spectrometer at 25 °C in a 1 cm path length cuvette in MeOH/PBS (1:2).
The extinction coefficient of the Cy7-linker in MeOH/PBS (1:2) at 660 nm is 4470 M -1 cm -1 . Figure S1: Plot of the concentration versus absorption at 660 nm to determine the extinction coefficient of the Cy7 linker at 660 nm in MeOH:PBS (1:2).
The fluorescence spectrum of the Cy7-linker was recorded on a Tecan Infinite M200 Pro plate reader in methanol (excitation wavelength 600 nm). To measure the fluorescence of the Cy7-linker in human whole blood, 16 µl of a 2 µM aqueous solution of oligonucleotide K (see also section 9. actinometry) were mixed with 64 µl human whole blood in a 96 well plate. Figure S2: Fluorescence spectrum of the Cy7-linker in human whole blood.

S3
To illustrate the decrease in fluorescence of the Cy7-linker by irradiation with red light (660 nm), 3 µM in 1xPBS of K were irradiated at 37 °C with a M660L4 LED (Thorlabs, 660 nm, operated at 1000 mA, 140 mW) using a Thorlabs LED driver DC4104 with DC4100-HUB and a ACL 2520-A lens (Thorlabs) in a cuvette (56 µl, d = 1 cm) and durations of 0-540s. This solution was transferred to a 96 well plate, made up to 100 µl and then the fluorescence was measured (excitation wavelength 605 nm).

Oligonucleotide synthesis
The used Milli-Q water was treated with DEPC (0.1%) overnight and autoclaved before usage.
The following oligonucleotides were synthesized by solid-phase synthesis: The following oligonucleotides were purchased HPLC purified from Biomers: ab m 1 m 2 m 3 Figure S4: Chemical structures of the modifier used for the oligonucleotide synthesis.
D is the equimolar solution of i-1 and i-2.

Irradiation
Irradiation of the light-inducible aptamer derivatives was performed with a M660L4 LED (Thorlabs, 660 nm, operated at 1000-1200 mA) using a Thorlabs LED driver DC2100 or a Thorlabs LED driver DC4104 with DC4100-HUB and a ACL 2520-A lens (Thorlabs). The exposure time used as well as concentration and buffer are given in the respective chapter.

CD-spectroscopy
The CD spectra were recorded on a Jasco J-715 CD spectrometer at 20 °C in a 0.01 cm path length cuvette. For measurements 1 nmol of each oligonucleotide was dissolved in 110 µl of 1xPBS buffer, heated for 5 min to 90 °C, and then allowed to cool to room temperature for at least 1 h until measurement. To measure the irradiated aptamer derivatives, the respective solution was irradiated with a M660L4 LED (Thorlabs, 660 nm, operated at 1200 mA, 170 mW) using a Thorlabs LED driver DC2100 and a ACL 2520-A lens (Thorlabs) for 60 min. Each trace is the average of 5 scans at a scanning speed of 200 nm/min, with a 1 s time constant, 1.0 nm data pitch and 1.0 nm bandwidth. All CD spectra were baseline-corrected for signal contributions of the buffer and smoothed. S17

Actinometry
The quantum yield of the Cy7-linker was determined using a fulgide actinometer, according to our previous published protocol. [2] For this purpose, i-2 was conjugated to both sides of the Cy7-linker 3 using the click protocol described previously, thus obtaining K.
Sequence of K = 5'-T GGT TGG m 3 -Cy7-linkerm 3 GGT TGG T-5' K was irradiated at 37 °C with a M660L4 LED (Thorlabs, 660 nm, operated at 1000 mA, 140 mW) using a Thorlabs LED driver DC4104 with DC4100-HUB and a ACL 2520-A lens (Thorlabs) in a cuvette (48 µl, d = 3 mm) and durations of 0-1200s. The sample volume consisted of 8 µM K and 0.83 µl internal standard (uridine) in PBS buffer. After irradiation, the samples were analyzed by RP-HPLC on a Agilent 1200 equipped with a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 µm, 4.6 mm x 250 mm) using gradient 8 (400 mM HFIP/16.3 mM TEA buffer pH 7.9, methanol, gradient 8: 5% MeOH for 5 min, 5% to 40% MeOH in 3 min, 40% to 55% MeOH in 15 min, 55% to 100% MeOH in 3 min, flow rate 0.7 ml/min, 60 °C). Via fulgide actinometry a photon number of 40.47 pmol/s could be measured for the used M660L4 LED. The amount of uncaged K was determined by peak integration of the HPLC chromatogram ( Figure S15). The initial slope at t=0 was determined by differentiation of the experimental fit of the uncaging kinetics. The quantum yield was then calculated as the ratio between initial slope and the absorbed photon flux. Each experiment was performed in triplicates The calculated quantum yield is Φp = 0.12% and the quantum product is Φp·Ɛ660 = 5.3. Figure S14: Typical example of a RP-HPLC. Figure S15: Decrease of starting material K as a function of exposure time. S18 10. Clotting assay to measure the thrombin time in human pooled plasma The anticoagulation activity of the aptamer derivatives was measured in a plasma-based coagulation assay measuring the thrombin time (TT) using an KC 10 A Amelung-Coagulometer. For this, human α-thrombin (CellSystems) was diluted in the assay buffer (1xPBS with 1 µg/µl BSA, pH 7.4) to reach a final concentration of 3 µg/ml. The lyophilized aptamer derivatives were dissolved in Milli-Q water and a dilution series was prepared for each. 50 µl of the thrombin solution was spiked with 50 µl aptamer solution of the respective concentration. After an incubation period of 4 min at 37 °C, 100 µl of pooled human plasma (citrate pooled plasma, in-house preparation, prepared from whole blood taken from healthy blood donors after informed consent, approval of ethics board of the medical faculty of the University Hospital Bonn #070/05) were added and the thrombin time was measured. To measure the anticoagulation activity of the pre-irradiated samples, the light-inducible aptamer derivatives were dissolved 3 µM in HFIP/TEA buffer (400 mM HFIP/16.3 mM TEA buffer pH 7.9) and irradiated with a M660L4 LED (660 nm, Thorlabs, operated at 1200 mA, 170mW) using a Thorlabs LED driver DC2100 and a ACL 2520-A lens (Thorlabs) for 60 min at 37 °C to guarantee the complete deprotection of the photocage and then lyophilized. Each concentration point was measured in triplicates.
11. Irradiation of the aptamer-thrombin-complex and fibrinogen coagulation assay 85 µl of a aqueous TBA, I or J solution (0.6 µM) was mixed with 85 µl thrombin solution (0.125 µg/ml in 1x PBS containing 1 µg/µl BSA, pH 7.4) and incubated for 4 min at 37 °C. To measure the irradiated samples, this solution was placed in a cuvette and was irradiated for 30 min at 37 °C with a M660L4 LED (660 nm, Thorlabs, operated at 1000 mA, 140 mW) using a Thorlabs LED driver DC2100 and a ACL 2520-A lens (Thorlabs). Then, the solution was left at 37 °C for another hour. 50 µl of the irradiated or not irradiated aptamerthrombin-mixture were mixed with 50 µl fibrinogen solution (4 µg/ml, diluted in 1x PBS) respectively in a clear 96 well plate. Absorption at 360 nm was measured on a Tecan Infinite M200 Pro plate reader over 1 h. To measure the pre-exposed samples TBA, I or J was dissolved 3 µM in HFIP/TEA buffer (400 mM HFIP/16.3 mM TEA, pH 7.9) and irradiated for 30 min at 37 °C with a M660L4 LED (660 nm, Thorlabs, operated at 1000 mA, 140 mW) using a Thorlabs LED driver DC2100 and a ACL 2520-A lens (Thorlabs). After another hour at 37 °C, the samples were lyophilized. To measure the activity, the respective sample was dissolved in Milli-Q water and treated in the same way as described above. Each sample was measured in triplicates.
12. Irradiation in human whole blood and coagulation assay 80 µl human citrate-blood (from healthy blood donors after informed consent, approval of ethics board of the medical faculty of the University Hospital Bonn #070/05) was spiked with 20 µl of TBA or I solution (2 µM in Milli-Q water). For irradiation, the solution was placed between two petri dishes (d=0.5 mm), with the upper one filled with water to form a thin layer of water. The irradiation was performed with a M660L4 LED (660 nm, Thorlabs, operated at 1200 mA, 350 mW) using a Thorlabs LED driver DC2100 and a ACL 2520-A lens (Thorlabs) over different time periods.
To measure the activity, 60 µl of the respective irradiated or not irradiated blood-aptamer-mixture (or as a control blood-water-mixture) were placed in a PCR tube and heated to 37 °C over 2 min, then 10 µl thrombin solution (1 µg/ml in 1x PBS containing 1µg/µl BSA, pH 7.4) were added and incubated over 2 min at 37 °C. After 2 min at room temperature, the PCR tubes were turned upside down, tapped and then the photo was taken. Temperature changes due to irradiation were < 4°C. A) B) Figure S16: Coagulation tests with human whole blood, where no aptamer was added (A) or with TBA (B). Irradiation time = 20 min. S19 13. Plasma Stability 10 µl oligonucleotide solution (25 µM in Milli-Q water) were mixed with 40 µl human pool plasma. The resulting assay mixture was incubated at 37 °C for a total of 24 hours. Samples were taken at different time points as indicated in the figures. The degradation process was stopped by mixing the sample (4 µl of the assay mixture) with 6 µl gel-lading buffer (80% 5 M urea in formamide, 8% 500 mM EDTA in water, 12% formamide), heated to 95 °C for 3 min, and then stored at -20 °C. The subsequent analysis was carried out by gel-electrophoresis on 20 % denaturing polyacrylamide gels using 7 M urea in 1xTBE (Tis-borate-EDTA) as running buffer. The gels were run at room temperature for 40 min at 200 V, stained with a SYBRGold solution and visualized with a gel imager (BioRad, GelDoc XR+).

a) b)
Figure S17: 20 % denaturing polyacrylamide gels for analysis of stability of I (a) and TBA (b)in human pool plasma. Incubation in 1x PBS buffer served as control.