The technical process of DNA-encoded libraries (DELs) technology involves DELs design, library production, affinity selection against targeted proteins, next-generation sequencing of binders' DNA tags, data analysis, re-synthesis of on-DNA & off-DNA compounds, and hit validation. Among these steps, hit validation after selection is a crucial step, which aims at classifying DELs selection results, eliminating false positive results, and confirming the binding properties and activities of featured molecules against targets.
Alfa Chemistry has been using DELs technology as a drug discovery method for many years, we have a lot of experience in this area, and our researchers have worked on several related projects. After DELs selection, we can use a variety of strategies to complete validation services for the hit compounds.
On-DNA Hit Validation Services
Library decoding after the affinity selection step often results in the identification of preferentially binding molecules, which need to be resynthesized and tested in appropriate assays in order to confirm the specificity of binding and provide information on the affinity constants. Alfa Chemistry has the ability to provide On-DNA approaches for validation of enrichment compounds to accelerate the hit validation process.
Advantages of on-DNA validation approaches
Compared to synthesizing a given number of Off-DNA compounds and performing biochemical or biophysical assays, On-DNA approaches have unique advantages in terms of low reagent consumption, purification, and ease of characterization by mass spectrometry, as listed below [1,2].
- The synthesis protocols of On-DNA follow the experimental steps of library construction, which may reduce the tediousness of synthesis and the consumption of reagents.
- On-DNA compounds are easy to purify and can be easily purified by selective precipitation.
- Mass Spectrometry (MS) used in the On-DNA validation approach can more easily characterize the structural features of On-DNA compounds.
Our validation strategy
Importance of AS-MS (affinity selection-mass spectrometry)
It is often assumed that there is a "one-to-one" relationship between a DNA tag and the chemical structure of the attached small molecule it encodes. However, during library synthesis, chemical reactions are usually not complete, which generate by-products and intermediates in addition to the main product. The presence of the product mixture may cause the target compound to share a DNA coding strand with other compounds, thus leading to the generation of false positive signals. Therefore, effectively identifying the structure of the small-molecule compound that each DNA tag in the DELs selection result actually corresponds to is an important challenge [3].
(Schematic Diagram of AS-MS [4])
Our ability
AS-MS is a binding assay technique for analyzing interactions between targets and ligands to discover small molecules that engage a specific target.
Alfa Chemistry can use the AS-MS validation strategy to analyze on-DNA compounds to further identify which ligands (products or by-products) actually bind to target proteins in DELs selection, and ultimately characterize its structural information.
Off-DNA Hit Validation Services
Alfa Chemistry offers Off-DNA hit validation services to further confirm preferentially binding molecules identified by sequencing and determine whether or not they are hits. This involves synthesizing Off-DNA compounds and analyzing their binding affinity to the target of interest using biochemical or biophysical methods.
Our validation strategies
Fluorescence Polarization (FP)
MicroScale Thermophoresis (MST)
Thermal Shift Assay (TSA)
Surface Plasmon Resonance (SPR)
Isothermal Titration Calorimetry (ITC)
Insolution diffusional sizing
Fluorescence anisotropy technique
Choose Us
Alfa Chemistry has extensive experience in the field of DELs technology and we offer a one-stop service for DELs technology, including on-DNA and off-DNA hit validation services. If you need help, please feel free to contact us.
References
- Luca, Prati.; et al. On-DNA hit validation methodologies for ligands identified from DNA-encoded chemical libraries. Biochemical and Biophysical Research Communications. 2020, 533(2): 235-240.
- Gunther, Z.; et al. Hit-Validation Methodologies for ligands isolated from DNA-encoded chemical libraries. ChemBioChem. 2017, 18(9): 853-857.
- Bing, X.; et al. DNA-encoded library hit confirmation: Bridging the gap between on-DNA and off-DNA chemistry. ACS Med Chem Lett. 2021, 12(7): 1166–1172.
- Motoyaji, T. Revolution of small molecule drug discovery by affinity selection-mass spectrometry technology. Chemical and Pharmaceutical Bulletin. 2020, 68(3): 191–193.