Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening

Abstract

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.

Figure 1

Aptamer screening. (a) Cartoon representation of the distribution of functional aptamers within a library. Libraries comprising function-enhancing chemotypes increase the abundance of active sequences relative to that of conventional standard base libraries. Color: binders (pink) and nonbinders (gray). Green plane denotes the threshold for the detection of binding affinity by an analytical technique. (b) Chemical structures of TNA triphosphates (tNTPs) used to generate (3′,2′)-α-l-TNA libraries using a laboratory-evolved TNA polymerase to transcribe DNA into TNA. Libraries are prepared with diverse chemotypes ranging from standard bases only to uracil residues equipped with functionally diverse side chains. (c) Library preparation. DNA display establishes a genotype–phenotype relationship by covalently linking each TNA molecule to its encoded dsDNA template. (d) Library screening. The TNA library is incubated with the target protein and bound molecules are separated from the unbound pool by affinity capture on Ni-NTA beads. Information carrying DNA molecules is recovered by photocleavage, uniformly amplified, and subjected to high-throughput sequencing (HTS).

Figure 2

Selection conditions. (a) Parallelized single-round aptamer screening demands stringent binding conditions to partition functional and nonfunctional sequences. Cartoon representation of an evaluation of the binding and wash conditions for a starting library tested against S1. (b) Summary of the selection conditions evaluated to increase the separation of active and inactive sequences. Buffer 1: 25 mM Tris (pH 8.0) and 150 mM NaCl. Buffer 2: 10 mM HEPES (pH 7), 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, 0.5 mg/mL BSA, and 0.05 mg/mL ssDNA. Washing conditions refer to selection buffer supplemented with (a) 1 M NaCl, (b) 0.5 M urea, (c) 1 M urea, (d) 2 M urea, and (e) no additive. Library refolding involves heating to 65 °C and cooling on ice. T/L refers to target/library molar ratio and [L]/[E] denotes the molar ratio of the starting library and elution sequences.

Figure 3

Secondary screening. (a) Cartoon representation of a flow cytometry assay performed using TNA aptamer hydrogel particles prepared by primer extension and template stripping in a hydrogel particle format. Sequences with affinity to a biotinylated S1-RBD fluoresce when incubated with PE-labeled SA. The binding activity level of sequences isolated from diverse chemotype pools is assessed in 96-well format by flow cytometry. (b) Flow cytometry analysis of TNA aptamers isolated from diverse chemotype libraries. (c) Flow cytometry analysis of TNA aptamers isolated from Trp libraries with random regions of varying lengths. All assays were performed in 96-well format in binding buffer (10 mM HEPES at pH 7, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20). Fold change represents the average fluorescence observed from sampling 100,000 particles per sequence. Sample size (n = 96 sequences) per chemotype and/or library length. Abbreviations: standard (std), leucine (Leu), cyclopropyl (cP), phenylalanine (Phe), dioxol (Dx), and Trp.

Figure 4

Kinetic binding analysis of Trp-modified threomers to S1. Background subtracted BLI sensorgrams comparing threomers Apt-132 (left), Apt-133 (middle), and Apt-126 (right). Curve fitting was performed with a 1:1 binding model. Binding assays were measured in binding buffer [10 mM HEPES (pH 7), 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20]. Kinetic values provided in Table S3. Trp-modified residues in the sequence are indicated in red. Threomers were enzymatically prepared using a 5′ DNA primer (not shown).

Figure 5

Aptamer specificity. (a) Cartoon representation of a target specificity assay performed using TNA aptamer hydrogel particles. Sequences with affinity to their cognate biotin-modified protein fluoresce when incubated with PE-labeled SA. Sequences with low off-target binding yield low fluorescence. (b) Flow cytometry analysis of Trp-modified TNA aptamers selected to bind the receptor binding domain of the S1 viral coat glycoprotein (S1-RBD) of SARS-CoV-2. Each aptamer sequence was evaluated for on-target binding to S1-RBD and S1 and for off-target binding to HSA and SA. Error bars denote ±standard deviation of the mean for 2 independent replicates. Two-tailed Student’s t-test reveals a statistically significant difference (p ≤ 0.001) between on-target and off-target values for all samples tested. All reactions were performed in binding buffer (10 mM HEPES pH 7, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20).