Literature Interpretation: How SELEX Technology Is Revolutionizing Targeted Protein Degradation-Special topic on adapter development-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Literature Interpretation: How SELEX Technology Is Revolutionizing Targeted Protein Degradation

Compared with traditional antibodies, nucleic acid aptamers offer outstanding advantages such as simple synthesis, easy modification, low immunogenicity, and good tissue penetration. Simply put, SELEX technology is a powerful platform capable of precisely screening out molecules that bind a target with high affinity and high specificity from a vast library of random sequences.

A study published in Nature in March this year, "Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation," provides strong evidence of the application value of SELEX technology: using this approach, researchers successfully screened non-inhibitory DNA aptamers targeting the novel E3 ubiquitin ligase ZYG11B and built a targeted protein degradation platform known as ZATAC. This work not only demonstrates a new strategy for targeted protein degradation, but also — more profoundly — highlights the role of SELEX technology in modern drug discovery.

To obtain E3 ligase ligands suitable for PROTAC development, the researchers optimized the classic SELEX workflow as follows:

Target Selection

They focused on ZYG11B, a substrate receptor of the Cullin 2-RING E3 ligase complex. This receptor plays a key role in protein quality control but had not yet been exploited in the PROTAC field.

Sorting Strategy

They employed magnetic bead-based selection, covalently coupling purified ZYG11B protein to magnetic beads to achieve efficient and convenient positive screening and separation.

Increasing Stringency

To eliminate sequences with low affinity or nonspecific binding, the researchers progressively increased screening stringency over seven consecutive rounds (e.g., stronger washing and the introduction of competing molecules).

Upgraded Amplification Technology

Compared with classic SELEX, they introduced emulsion PCR in the later screening rounds, partitioning PCR reactions into millions of microscopic oil droplets. This greatly eliminated PCR amplification bias, preserved sequence diversity, and improved the success rate of obtaining the optimal aptamer.

Discovery and Validation of the Apt#Z6 Aptamer

Through stringent magnetic bead screening that reached saturation in the final two rounds, the researchers analyzed the aptamers enriched in each round by next-generation sequencing and selected 10 sequences for affinity determination. In isothermal titration calorimetry (ITC), Apt#Z6 showed the strongest affinity, with a dissociation constant (Kd) of 4.69 μM against ZYG11B; microscale thermophoresis (MST) and surface plasmon resonance (SPR) further confirmed this result. More importantly, Apt#Z6 did not bind ZER1, a homologous protein of ZYG11B.

Figure 1 Discovery and validation of the Apt#Z6 aptamer

Modular Application of SELEX Outputs

After identifying Apt#Z6 as the E3 ligase-recruiting ligand, the research team further linked it to aptamers targeting different proteins of interest via chemical conjugation or self-assembly through complementary base pairing, and found that a 20 bp flexible double-stranded DNA linker delivered the best performance, thereby constructing a series of bispecific degraders — ZATACs. As shown in Figure 2a, they linked Apt#Z6 to an aptamer targeting nucleolin (NCL) to construct NCL-ZATAC#20, and systematically demonstrated through binding affinity assays, protein–protein interaction experiments, and live-cell imaging that high-quality aptamers obtained via SELEX can serve as standardized, plug-and-play functional modules for rapidly building therapeutic platforms against different disease targets.

Figure 2 SELEX-enabled targeted delivery and intelligent therapeutics

To further maximize the power of aptamers, the researchers connected an aptamer targeting a protein highly expressed on the surface of cancer cells, the E3-recruiting aptamer Apt#Z6, and an aptamer targeting an intracellular pathogenic protein, thereby constructing a trispecific ZATAC (as shown in Figure 3a). In vitro and in vivo experiments demonstrated that the trispecific ZATAC not only achieves precise drug delivery but also substantially improves the therapeutic index.

Figure 3 3WJ-ZATAC achieves targeted delivery and dual-target degradation

TekBiotech (Tianjin) Co., Ltd. has established a comprehensive SELEX platform, offering customers multiple screening modalities — including MB-SELEX, Cell-SELEX, and CE-SELEX — for targeted aptamer discovery against different types of targets. In addition, we provide high-quality downstream development and validation services, including aptamer modification, bispecific aptamer development, aptamer–drug conjugate (ApDC) development, in vitro affinity validation (including EC50 determination and BLI/SPR affinity assays), cytotoxicity and targeting validation, in vivo animal imaging validation, and efficacy evaluation in animal models, providing new ideas and technical support for customers' research projects and small-molecule drug development.

References

[1] Yang, Z., Chen, M., Ge, R. et al. Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation. Nat Commun 16, 2494 (2025).

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