Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase

Abstract

Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3'-ends of DNA. If a target protein is expressed as a TdT fusion and incubated with a DNA-encoded library (DEL) in the presence of dATP, the binders of the target induce proximity between TdT and the DNA, promoting the synthesis of a poly-adenine (polyA) tail. The polyA tail length is proportional to the binding affinity, effectively serving as a stable molecular record of binding events. The polyA tail is also a convenient handle to enrich binders with magnetic poly(dT)₂₅ beads before sequencing. In a benchmarking system, we show that ligands spanning nanomolar to double-digit micromolar binding can be cleanly identified by TdT extension, whereas only the tightest binding ligands are identified by classical affinity selection. The method is simple to implement and can function on any DEL that bears a free 3'-end.

Figure 1

Conceptual design for a molecular recorder of encoded library affinity data. Binding events are recorded by TdT (A), providing a handle to separate binders from non-binders (B). After NGS, the counts can be compared to a selection without the POI, revealing binders (C).

Figure 2

Induced proximity can control the activity and selectivity of TdT (A). TdT fused with a SNAP-tag (SNAP–TdT) is coupled to a short DNA with a 5′-benzylguanine and a 3′-deoxy end (see Supporting Information for complete structure of DNA) to create SNAP_DNA–TdT; the proficiency of this chimeric fusion in creating 3′-extensions is then tested with fluorescently labeled oligonucleotides which are either matched (FAM) or mismatched (A590); lane 3 is the test condition and shows that the matched DNA is extended by TdT more efficiently than the unmatched. (B) Comparison of background activity of SNAP–TdT and SNAP_DNA–TdT: lanes 4–6 in (A) show that SNAP–TdT has a substantial background extension; this is further confirmed by lane 3 in (B). A version of SNAP_DNA–TdT, which is mismatched to the FAM oligonucleotide (SNAP_mmDNA–TdT) shows a dramatically reduced background (lane 4). (C) The binding affinity of the FAM oligonucleotide is systematically varied through base-pairing rules and mismatches (calculated by the nearest neighbor method) to test whether affinity correlates to 3′-extension activity; comparing lanes 2–6 shows that as binding strength increases, 3′-extension also increases.

Figure 3

(A) PolyA tailing with TdT is selective and can be purified with Poly(dT)₂₅ beads. (B) The polyA tailing occurs on both ssDNA and dsDNA but is more efficient on ssDNA (compare lanes 2 and 5); qPCR measurement of DNA after enrichment in the templated (red bars) versus untemplated (blue bars) reactions show similar enrichment values for ssDNA and dsDNA. The error bars represent the standard error of mean across triplicates.

Figure 4

Enrichments in a model library and the effect of library size. (A) Structures of CAII binders as DNA conjugates and the binding affinities of their parent molecules. (B) PolyA tailing using a CAII–TdT fusion gives a positive correlation between binding strength and tail length, while the non-binding control shows no tailing. Smear in all lanes comes from the background elongation of sssDNA. (C) qPCR data show strong enrichment of binders relative to non-binding control. The error bars represent the standard error of mean across triplicates. (D) NGS and qPCR data for a simulated library size of 196 compounds with known binders AAZ, CTZ, and SAA compared with AE or DELSTAR. Uncertainties are given as the 90% confidence interval from triplicates. (E) Same as (D), except the simulated library size is 19,204 compounds. (F) Same as (D), except simulated library size is 1,920,004 compounds.

Figure 5

Construction of a chemically identical ss- and dsDEL. (a) S_NAr of 5′-amine DNA with TCT followed by a second S_NAr to install diversity element 1. (b) Diversification of dp2 by Suzuki–Miyaura cross-coupling. (c) 1. S_NAr of dp2 with DABCO and NaN₃, 2. CuAAC. (d) Diversification of dp2 by S_NAr; (b–d) diversity elements were encoded by splint ligation. Before pooling of the three sublibraries, it was encoded for the type of reaction performed in the dp2 diversification either by splint ligation (e) ssDEL or by Klenow extension (f) dsDEL.

Figure 6

Selection results of ssDEL and dsDEL. (A) In the ssDEL (top panels), DELSTAR reveals a more complete binding picture than that of AE. DELSTAR performs similar to AE in the dsDEL (bottom), although acetazolamide is less prominent. The shaded area differentiates the three sublibraries. (B) Expected binders in the DEL (blue and red) and putative binders identified by DELSTAR (black). (C) Log-fold distributions in affinity enrichment for ssDEL (top) and dsDEL (bottom) of all retrieved library members [phenylsulfonamides (red), acetazolamides (purple), or all others (gray)]. The shaded area contains binders below 3 times the standard deviation and were not used for the scatter plot in panel (A). (D) DELSTAR performs better with ssDEL than with dsDEL at an equivalent incubation time (30 min). (E) Lowering the amount of protein (top panel) has little effect on DELSTAR results, while increasing incubation time to 60 min with dsDEL (bottom panel) improves the enrichment values of known binders [cf. with bottom panel in (D)]. (F) Counting the unique molecular identifier (UMI) shows that the DELSTAR signal is derived from 10-fold more individual binders than AE (top panel); this is also seen when counting the number of individual molecules with >10 copies (bottom panel). The shaded area in the top panel and the error bars in the bottom panel indicate the standard error of mean across three selections.