Clearsynth stands at the forefront of NMR technology and our BIO NMR Services are designed to cater to diverse research needs across the life sciences. Here's why you should entrust us with your biomolecular analysis
BioNMR is a powerful and versatile technique that can provide detailed structures of macromolecules and information on how they interact with ligands. BioNMR can be used as a detection method for screening. It is especially well-suited for fragment screening, where weak binding is expected. BioNMR is complementary to X-ray crystallography since many molecules do not crystallize and BioNMR is performed in solution. Further, X-ray crystallography produces full structures, and is an all-or-nothing technique. BioNMR is more flexible; while it can produce full structures, it can also deliver high-value information with lower resolution, but at much greater speed.
Clearsynth has an established reputation for its expertise and capabilities in NMR research and analysis. With the aid of advanced NMR equipment, we can provide timely and precise NMR data to accelerate our clients’ product development programs.
BioNMR is the gold standard method for sorting out binding behaviors. A 15N-labeled version of the protein is used, so only the protein is observed in the NMR spectrum. Solution conditions can be changed in real time and additions can be made. The status of each residue in the protein is monitored. Upon addition of a potential binder, the response of the protein reveals if the added molecule binds to the protein or not, and if the binding is clean or causes the protein to unfold or aggregate. Based on which residues respond, the binding location can be determined (sometimes called “footprinting”).
After any screen, hit validation is essential, especially for difficult targets (e.g., protein-protein interactions) where false hits may bind more strongly than authentic hits.
For true binders, the binding footprint can immediately allow modeling of the docked ligand, which is essential for guiding a chemistry optimization strategy. Also, absence of a BioNMR response reliably indicates non-binding; no other technique can do this.
The amount of protein required for this type of experiment would be approximately 1-3 mg and the amount of compound needed would be about 0.2 mg. Labeling of the protein with 15N can be done easily if the protein is expressed in E. coli, but labeling can also be accomplished in yeast or mammalian cells, if necessary.
Beyond examining molecules that are already identified as hits by other methods, BioNMR can be used as the primary detection method in a screen. Potential binders can be added as mixtures to increase throughput. When there is a positive response, the mixtures are broken out to find the actual hit. Because BioNMR is so sensitive, it can reliably detect weak binding and therefore is the method of choice for a fragment screen.
At first glance, BioNMR would appear to be a low-throughput method. However, it is exceptionally efficient since detection and validation are obtained simultaneously. The overall throughput from screen to authentic leads is therefore comparable to other approaches.
Because chemical shift patterns are unique to every protein, it is possible to perform a BioNMR screen with multiple proteins present. The response can clearly identify the protein to which the hit binds. This methodology can dramatically increase throughput for a screening library or can be used to assess selectivity. BioNMR is the only technique that can do this.
BioNMR can provide 3D structures of proteins, protein-ligand complexes, oligonucleotides, peptides, macrocycles, and TPDs. While many proteins and oligonucleotides are amenable to X-ray crystallography and cryogenic electron microscopy, some are not. Further, because it is performed in solution, BioNMR can resolve concerns about the effects of crystal packing. BioNMR can also usually provide medium-resolution information faster than those other methods.
Typically, medium-sized macromolecules (e.g., peptides, macrocycles, TPDs) do not crystallize, but can be readily studied by BioNMR. BioNMR also allows the selection of any solvent condition. Detailed structures of these molecules can guide the design of analogs that are more potent, more stable, and/or more bioavailable.
The sophisticated NMR techniques needed to deal with macromolecules can be applied to smaller molecules, to answer complex questions of structure or stereochemistry. NMR can provide a rigorous verification of primary structure.