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. 2014 Feb 26;136(8):3264-70.
doi: 10.1021/ja412934t. Epub 2014 Feb 17.

Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates

Lynn M McGregor  1 , Tara Jain, David R Liu
Affiliations

Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates

Lynn M McGregor et al. J Am Chem Soc. .

Abstract

We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand-target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding affinity from Kd = 0.2 nM to 3.2 μM out of 67,858 possible combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.

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Figures

Figure 1
Figure 1
(A) Antibody-mediated interaction determination using unpurified proteins (IDUP) uses DNA-linked antibodies to recognize a target protein or epitope tag. (B) Alternatively, a covalent bond can be formed between the target and identifying DNA strand in IDUP by fusing the target to a self-labeling protein tag such as SNAP-tag, CLIP-tag, or HaloTag. After primer extension and PCR, the resulting DNA encodes all ligand+target combinations.
Figure 2
Figure 2
(A) The ability of IDUP to enrich the sequence corresponding to a particular interaction is evaluated by incubating mixtures of binding and nonbinding DNA-linked ligands with a target protein and a DNA-linked antibody. After primer extension and PCR, a restriction digest is used to determine the fraction of the amplified sequences corresponding to the target+ligand interaction. (B) IDUP with DNA-αSA and 0.01% SA in HeLa lysate shows selective amplification of a sequence corresponding to SA+desthiobiotin in qPCR (ΔCT = 4.7). (C) IDUP on a mock library containing mixtures of DNA-desthiobiotin and DNA-GLCBS shows ∼1000-fold enrichment of a sequence corresponding to SA+desthiobiotin. (D) When analyzed by qPCR, IDUP with DNA-αCAII and 0.01% CAII in HeLa lysate shows rapid amplification of sequences corresponding to CA+GLCBS and CA+CBS, but not CA+desthiobiotin or CA+amine (ΔCT = 4–5). (E) IDUP on a mock library containing mixtures of DNA-GLCBS and DNA-desthiobiotin shows ∼10-fold enrichment of a sequence corresponding to CAII+GLCBS.
Figure 3
Figure 3
(A) IDUP using DNA-αHis and genetically encoded His6-tagged target proteins. (B) IDUP using DNA-αHis with 293T cell lysate expressing CAII-His6 shows rapid amplification of sequences corresponding to CA+GLCBS and CA+CBS but not CA+desthiobiotin (ΔCT = 5–6). (D) IDUP using a mock library shows ∼10-fold enrichment of the sequence corresponding to CAII+GLCBS and (F) ∼100-fold enrichment of a sequence corresponding to CAII+CBS. (E) When the transfected lysate was diluted 1:10 into untransfected lysate, the enrichment of the CAII+GLCBS sequence increased to ∼100-fold. (C) IDUP using DNA-αHis with 293T cell lysate expressing His6-BclxL shows rapid amplification of DNA-Bad but not DNA-GLCBS or DNA-biotin (ΔCT = 8) and ∼100-fold enrichment of a sequence corresponding to Bcl-xL+Bad (G).
Figure 4
Figure 4
(A) IDUP in cell lysates expressing a SNAP-tagged target protein. DNA was rapidly amplified in samples corresponding to known interactions: (B) SNAP/CAII+(GL)CBS (ΔCT = 5–6), (C) SNAP/Bcl-xL+Bad or Bak (ΔCT = 8–9), (F) SNAP-FRB+rapamycin (ΔCT = 6), and (G) FKBP-SNAP+rapamycin (ΔCT = 6). (D) A sequence corresponding to CAII+CBS was enriched ∼100-fold in a sample expressing either SNAP-CAII (lane 7) or CAII-SNAP (lane 8). (E) In samples expressing SNAP-Bcl-xL, a sequence corresponding to the interaction between Bcl-xL+Bak was enriched ∼100-fold (lane 11), but no enrichment was observed for a sequence corresponding to BclxL+BakL78A, a weakly binding mutant of the Bak peptide (lane 9). (H) Overexpression of FKBP with SNAP-FRB increased the enrichment of a sequence encoding FRB+rapamycin by 10-fold compared to a sample transfected with SNAP-FRB alone. (I) Overexpression of FRB with FKBP-SNAP also increased the enrichment of a sequence corresponding to FKBP+rapamycin by 10-fold compared to a sample transfected with FKBP-SNAP alone.
Figure 5
Figure 5
(A) Cell lysates expressing SNAP-CA, SNAP-Bcl-xL, and SNAP-FKBP were individually labeled with one of three DNA sequences and combined with a cell lysate expressing SNAP and labeled with 256 DNA sequences. The pooled lysates were combined with a library of 262 DNA-linked small molecules, including DNA-linked GLCBS, CBS, Bad, Bak, BakL78A, and rapamycin, for a model library×library IDUP “selection”. (B) IDUP using a library of cell lysates expressing SNAP-target fusions identified all five known target+ligand pairs, including A - FKBP+rapamycin, B - Bcl-xL+Bad, C - Bcl-xL+Bak, D - CAII+GLCBS, and E - CAII+CBS, despite having affinities from 0.2 nM to 3.2 μM. (C) For interactions with Kd = 40 nM–26 μM, we observed a strong relationship between the log of target–ligand Kd and the number of sequence counts after selection.
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