Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening

In vitro evolution strategies have been used for >30 years to generate nucleic acid aptamers against therapeutic targets of interest, including disease-associated proteins. However, this process requires many iterative cycles of selection and amplification, which severely restricts the number of target and library design combinations that can be explored in parallel. Here, we describe a single-round screening approach to aptamer discovery that relies on function-enhancing chemotypes to increase the distribution of high-affinity sequences in a random-sequence library. We demonstrate the success of de novo discovery by affinity selection of threomers against the receptor binding domain of the S1 protein from SARS-CoV-2. Detailed biochemical characterization of the enriched population identified threomers with binding affinity values that are comparable to aptamers produced by conventional SELEX. This work establishes a highly parallelizable path for querying diverse chemical repertoires and may offer a viable route for accelerating the discovery of therapeutic aptamers.


Materials and Methods
General information.T4 DNA ligase buffer, ThermoPol buffer, Mg-free Taq polymerase buffer, and Bst 3.0 DNA polymerase were purchased from New England Biolabs.PCRBIO HiFi polymerase was purchased from PCRBiosystems.TNA polymerase and T7 DNA ligase were expressed and purified as described previously. 1 DNA triphosphates were purchased from Thermo Fisher Scientific.3][4] DNA oligonucleotides were purchased from Integrated DNA Technologies.DNA libraries were purchased from the Keck Oligonucleotide Synthesis Facility at Yale.For the aptamer selection, Ni-NTA agarose resin was purchased from Qiagen.His-tagged SARS-CoV-2 spike glycoprotein subunit 1 (S1) and SARS-CoV-2 receptor binding domain (S1-RBD) of the spike glycoprotein subunit 1 were purchased from ACRO Biosystems.Bovine serum albumin (BSA) and salmon sperm DNA were purchased from Thermo Fisher Scientific.Human a-thrombin was from Haematologic Technologies.Lyophilized powder of albumin from human serum (HSA) was bought from Sigma-Aldrich.Phenol:chloroform:isoamyl alcohol 25:24:1 (saturated with 10 mM Tris, pH 8.0, 1 mM EDTA) was purchased from Sigma-Aldrich.Amicon™ ultra centricon centrifugal filter units were purchased from EMD Millipore.DNA clean & concentrator-5 kit was from Zymo Research.For the BLI assay, streptavidin biosensors were purchased from ForteBio.For particle display, magnetic beads (Dynabeads™ M-270 Carboxylic Acid) and magnets (DynaMag-96 Side Skirted and DynaMag-2) were purchased from Thermo Fisher Scientific.Ammonium persulfate was purchased from Sigma-Aldrich.EZ-Link™ NHS-PEG4-Biotin and streptavidin-phycoerythrin (SA-PE) were purchased from Thermo Fisher Scientific.
Library synthesis.TNA libraries were designed at the DNA level to contain unbiased internal variable regions of 20, 25, 30, or 40 nucleotides (25% of A, T, G, and C) flanked on both sides by fixed-sequence primer binding sites.Each library contained a 6 nt barcode located between the forward primer and random region.Library preparation followed the same protocol as described previously 5 with a few modifications.The phosphorylated DNA hairpin primer containing a photocleavable linker was ligated to the library by combining 0.5 nmol of DNA library with 0.6 nmol of DNA hairpin in a final volume of 100 µL of 1× T4 DNA ligase buffer.The libraries were annealed to the hairpin by heating for 5 min at 95°C and cooling for 5 min at 4°C.T7 DNA ligase was then added to a final concentration of 0.5 µM and the reaction was incubated overnight at 24°C.The resulting selfpriming DNA libraries were purified by 10% denaturing polyacrylamide gel electrophoresis (PAGE), electroeluted, and quantified by A260 absorbance with the Nanodrop spectrophotometer.
Each self-priming DNA library (100 pmol) was extended with tNTPs to form a chimeric TNA/DNA heteroduplex.A reaction solution with a final concentration of 1 µM of hairpin library and 1× ThermoPol buffer was heated for 5 min at 95°C and cooled for 5 min at 4°C.TNA polymerization was initiated by adding the TNA polymerase (1 μM final concentration) and 100 μM of each tNTP (tATP/tCTP/tGTP/tTTP or C-5 modified tUTP), the solution was incubated for 2 h at 55°C.Afterwards, excess polymerase was extracted by adding an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, saturated with 10 mM Tris, pH 8.0, 1 mM EDTA) and mixing by inversion until cloudy, followed by centrifugation at 12,000 rpm for 5 min.Following extraction, the top layer was collected and the remaining tNTPs were removed by exchanging into water using a YM-30 spin filter.
The strand displacement step was performed by extending a DNA primer annealed to the hairpin loop with dNTPs.Accordingly, the library (1 µM as the final concentration) was resuspended in 1× ThermoPol buffer containing 500 µM dNTPs and 2 µM strand displacement primer.After heating for 5 min at 95°C and slowly cooling to room temperature, the displacement reaction was initiated by adding Bst 3.0 DNA polymerase to a final concentration of 80 U/mL.The reaction was incubated for 1 h at 50°C.Bst polymerase was extracted with phenol/chloroform/isoamyl alcohol as described above.The TNA library displayed on dsDNA was separated from the residual phenol and reaction components in a YM-30 spin filter.Any remaining phenol was removed by adding 900 µL of ethanol to the solution and drying completely with the SpeedVac vacuum concentrators.The libraries were resuspended in 400 µL of the binding buffer (10 mM HEPES pH 7, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, 0.5 mg/mL of BSA and 0.05 mg/mL of salmon sperm DNA).
Optimization of the binding and wash conditions.Aptamer selections were performed by incubating library L24.1 prepared with the Phe chemotype (10 pmol, ~10 12 unique sequences) with Ni-NTA agarose resin for 30 min at 24°C with rotation in one of two different selection buffers: buffer 1 contained 25 mM Tris pH 8.0, 150 mM NaCl; and buffer 2 contained 10 mM HEPES pH 7, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, 0.5 mg/mL BSA, 0.05 mg/mL salmon sperm DNA.The material collected from the flow-through of the negative selection (Ni-NTA agarose resin only) was incubated with the His-tagged S1 poised at a 100:1, 10:1 or 1:1 (library:target) ratio for 15 min at 24°C with rotation.The aptamer/S1 solution was then added to a column with Ni-NTA agarose resin and incubated for 15 min at 24°C with rotation.The column was drained and washed 3 times with 400 µL of selection buffer to remove unbound and weakly bound molecules.A stringent wash step was performed using buffer supplemented with a) 1 M NaCl, b) 0.5 M urea, c) 1 M urea, d) 2 M urea, e) no additive.The column was then washed twice with Mg-free Taq polymerase buffer [10 mM Tris-HCl, 50 mM KCl pH (8.3)].The column was exposed to UV radiation for 1 hour (UVP crosslinker CL-1000L, 365 nm) to release the encoding dsDNA from the S1-RBD bound Ni-NTA agarose resin.Conditions that included an aptamer refolding step involved heating the solution to 65°C for 5 min and cooling on ice for 15 min prior to the initial negative selection step.qPCR analysis.The starting DNA library and eluted material recovered after photocleavage were evaluated by qPCR to assess the stringency of each binding and wash condition.For each measurement, a linear calibration plot was obtained by performing a qPCR analysis on dilution samples of the starting ssDNA library (L24.1,Table S1) that spanned a concentration with 5-fold dilutions from 10 nM to 3.2 pM.The analyzed samples correspond to the DNA display library before selection and after elution via photocleavage (see above).The DNA display library and eluted material were diluted such that their final concentration for qPCR occurred within the linear range of the calibration plot.Reported values were the average of 3 experimental replicates.An example of the qPCR data and analysis can be found on Supplementary Figure S1.
Aptamer selection.Aptamer selections were performed by first refolding the library (100 pmol, ~10 12 unique sequences) as described above in binding buffer 2 (10 mM HEPES pH 7, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, 0.5 mg/mL BSA, 0.05 mg/mL salmon sperm DNA) and incubating with Ni-NTA agarose resin for 30 min at 24°C with rotation.The material collected from the flow-through of the negative selection (Ni-NTA agarose resin only) was incubated with the His-tagged S1-RBD poised at a 10:1 (library:target) ratio for 15 min at 24°C with rotation.The aptamer/S1-RBD solution was then added to a column with Ni-NTA agarose resin and incubated for 15 min at 24°C with rotation.The column was drained and washed 3 times with 400 µL of selection buffer to remove the unbound and weakly bound molecules.A stringent wash step was performed with 2 M urea to reduce the occurrence of nonspecific binding followed by 2 washes with Mg-free Taq polymerase buffer (10 mM Tris-HCl, 50 mM KCl pH 8.3).The column was exposed to UV radiation for 1 hour (UVP crosslinker CL-1000L, 365 nm) to release the encoding dsDNA from the S1-RBD bound Ni-NTA agarose resin.The recovered DNA was amplified first by droplet PCR to minimize sequence bias, followed by a regular bulk PCR with library-specific PCR primers and purified using DNA Clean and Concentrator columns from Zymo Research.

High Throughput Sequencing (HTS).
The recovered amplicons were prepared as barcoded libraries and sequenced on a NovaSeq6000 with the S4 cell with the paired end setting for 100 cycles for an estimated 200 M reads per library.Sequencing data was then filtered, trimmed, and aligned.Among the filtering conditions were full-length reads on both directions and £4 consecutive bases.The sequences were organized based on their percent T content for the std library, and C-5 modified U for the functionally-enhanced libraries.
Hydrogel bead preparation.Hydrogel particles were prepared as previously described. 6In brief, 8×10 8 carboxylic magnetic beads were transferred to a 5 mL Eppendorf tube.After removal of the supernatant by placing the tube in the magnet, 1056 uL of 6% degassed acrylamide:bisacrylamide (19:1), 120 µL of 1 mM acrydite-modified primer PBS8 and 24 µL of freshly made 30% (w/v) ammonium persulfate (APS) were added and the beads were carefully mixed with the pipette without introducing any air to the mixture.Then 3.6 mL of previously degassed oil mixture (4%/20%/76%; w/w/w; KF6036 / mineral oil / DMF H6CS) were added to the top of the acrylamide solution without mixing and the top oil layer was degassed again for 10 min.Then 4 µL of TEMED were added to the oil mixture and the headspace of the tube was purged for another 60 seconds.The tube was placed on a Benchmark Bead bug-6 for 65 s at 2500 rpm and then placed on ice to polymerize for 4 hours.The beads were then washed with breaking buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% (v/v) SDS, 1% (v/v) Triton x100) until no oil was observed.The beads were resuspended in 4 mL of breaking buffer and counted on a cell counter hemocytometer.
Transcription in hydrogel beads.Aptamer hydrogel particles were prepared as previously described 6 .In brief, 3 million of 5' acrydite-modified PBS8 polymerized hydrogel-coated beads were transferred to each well of a 96-well plate and washed twice with 100 µL of 1x Thermopol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton® X-100, pH 8.8@25°).Then a reaction mixture with a final volume of 25 µL was prepared in each well with 1×Thermopol buffer, 2 µM of template, 0.01% of KF6012, and water.The plate was heated at 90°C for 3 minutes and then cooled down to 4°C for 5 minutes to anneal.TNA polymerization was initiated by adding 100 µM of each tNTP and 2 µM of TNA polymerase and the reaction was incubated at 55°C for 4 hours.Once the reaction was completed, the beads were washed twice with breaking buffer and resuspended for a final concentration of 25,000 beads per µL.
Screening of hydrogel beads.Approximately 500,000 hydrogel beads with transcribed TNA products were transferred to a new plate.The same amount of primer-polymerized only hydrogel beads was set as the negative control.The template strand was removed by adding 100 mM NaOH containing 0.5% Tween 20 and incubated for 3 minutes, then adding an equal volume of breaking buffer and removing all the supernatant.The stripping step was done twice.Then the beads were washed with 100 µL of breaking buffer twice and BLI binding buffer (10 mM HEPES pH 7, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) twice more.The plate was heated at 90°C for 3 minutes and then cooled to 4°C for 5 minutes to fold the aptamers into their active structure.RBD was biotinylated using 20 molar fold excess of EZ-Link™ NHS-PEG4-Biotin at room temperature for 30 minutes and followed by three PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4) washes using the YM-10 Centricon spin column.Biotinylated RBD was then added to each well to a final concentration of 100 nM in 50 µL and incubated for 30 minutes at room temperature.After 30 minutes, the supernatant was removed, and beads were resuspended in 50 µL of BLI binding buffer and 0.5 µL of SA-PE was added to the solution.After 15 minutes of incubation, the supernatant was removed, the beads were resuspended in 50 µL of BLI binding buffer, and samples were run in the flow cytometer in sets of 24.Flow cytometer conditions: 600 rpm mix every 8 samples for 5 seconds, rinse after every sample, 20 µL of sample is taken, gating: SSC-H (10 6 ) x FSC-H (10 6 ) (0.4 x 0.4 for 2.8 µm Dyna beads).
Aptamer preparation for kinetic analysis.Aptamers identified by sequencing were prepared as biotinlabeled TNA molecules using the 5' biotinylated PBS8 20 nt DNA primer and the corresponding DNA template, which contained an additional 20 nucleotides (AAAC)5 at the 3′ end to facilitate the band separation by PAGE.A 750 µL reaction volume containing 1 µM of both primer and template as well as 1× ThermoPol Buffer was heated for 5 min at 95°C and then cooled down to 4°C to anneal.TNA polymerization was initiated by adding 100 µM of each tNTP and 2 µM of 10-92 TNA polymerase and then incubated for 2 h at 55 °C.Full-length biotinylated aptamers were purified by 10% denaturing polyacrylamide gel electrophoresis for 1.5 h at a constant 16 W. TNA was recovered from the gel by electroelution at 60 V overnight and then buffer-exchanged into water and concentrated using a YM-10 Centricon centrifugal filter device.TNA concentration was quantified by measuring its absorbance at 260 nm with a NanoDrop spectrophotometer.BLI assays.Biotinylated aptamers at a concentration of 50 nM were folded into their active conformation in BLI binding buffer (10 mM HEPES pH 7, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) by heating to 95°C for 5 minutes and then cooling to 4°C for 5 minutes.Streptavidin-coated optical biosensors were previously equilibrated in BLI binding buffer for 30 min.For measuring full kinetics, the BLI run was performed with the following steps: a buffer only baseline for 60 s to equilibrate sensors, loading the aptamer for 200 s, a second buffer only baseline for 200 s, an association phase with the target protein for 600 s, and a dissociation phase for 600 s.Serial diluted target concentration ranged from 50 nM to 3.125 nM.The experiments were conducted at 30ºC.Data were analyzed using the Octet Data Analysis HT software.The buffer only baseline measurement was used to subtract the background from all samples before applying Savitzky-Golay filtering and fitting the association and dissociation curves and applying a global fit to determine KD.

Aptamer specificity test.
From the initial hydrogel bead screening with 4 lengths of the tryptophan-modified sequences, 17 TNA aptamer sequences were selected, which displayed the highest fold-change values with respect to the beads that contained only the primer.These 17 aptamers were synthesized on the PBS8polymerized hydrogel beads using the method described above.Each specific synthesis was split into 4 different wells (with approximately 500,000 beads per well).The 96-well plate was heated at 90°C for 3 minutes and then cooled to 4°C for 5 minutes to promote the folding of the aptamers.Then, 100 µM of biotinylated S1, S1-RBD, HSA, or streptavidin was added to each well and incubated for 30 min respectively.After removal of the supernatant, samples were incubated in 50 uL of BLI binding buffer with 0.5 µL of SA-PE for 15 min.The supernatant was removed, and the beads were resuspended in 50 µL of BLI binding buffer, and samples were run in the flow cytometer.The experiment was performed twice independently.
Chemotype swap.Hydrogel aptamer particles were separately prepared for the top 17 S1-RBD binding threomers with the std, Phe, and Trp chemotypes.Each of the three aptamer sets were prepared using the appropriate set of TNA triphosphates.Following synthesis and template stripping, the sequences were refolded by heating at 90 °C for 3 minutes and then cooled to 4°C for 5 minutes.The samples were then incubated with 100 µM of biotinylated S1 for 30 min, followed by a 15 min incubation with 0.5 µL of SA-PE.The supernatant was removed, and the beads were resuspended in 50 µL of BLI binding buffer.Lastly, the samples were run in the flow cytometer.The experiment was performed twice independently to verify its reproducibility.

Thermal stability measurements
Melts were performed with 1:1 oligonucleotide stoichiometry (1 µM of each strand, Table S2) in 500 mM NaCl, 10 mM sodium phosphate, 0.1 mM EDTA at pH 7 with and without 2 M urea.Prior to melting, the strands are annealed in an Eppendorf tube by heating to 95ºC for 5 minutes and cooling on ice.The heating curves were obtained in a quartz cuvette with a temperature gradient of 20º to 90ºC and a ramping rate of 1°C per min by monitoring the change in UV absorbance at 260 nm at each temperature.Tm values were either undeterminable (due to the value being outside the measured range of 20-90˚C) or determined by the maximum calculated from the first derivative of the heating curve.

Table S3 .
Summary of BLI analysis for threomers binding to the S1 protein.