Characterizing miniprotein – DNA interactions
Three key questions and where current methods fall short
In the biophysical characterization of DNA-targeted miniprotein therapeutics, three key questions consistently arise:
1
What is the binding affinity (KD) of each variant for the target DNA?
2
Can each variant distinguish the correct sequence from non-specific ones?
3
How do these variants behave in physiological matrices, such as 100% plasma?
In practice, answering all three questions is not straightforward – no single biophysical platform can address them comprehensively. SPR and ELISA can measure affinity but rely on immobilization. Circular dichroism and EMSA provide qualitative confirmation, but not quantitative KD. Meanwhile, behavior in plasma typically remains uncharacterized, as none of these methods are compatible with complex backgrounds such as undiluted biological matrices.
The result is fragmented workflows, inefficient sample use, and a missing translational data point which is critical for clinical and regulatory decision-making.
How can we help?
Microfluidic Diffusional Sizing (MDS), the proprietary technology behind the Fluidity One-M, addresses all three questions in a single, simple, in-solution workflow.
Quantitative KD
MDS quantifies binding affinity directly in solution by measuring changes in hydrodynamic radius (Rh) as interactions occur. By titrating miniprotein variants against a constant concentration of DNA, concentration-dependent shifts in Rh generate binding curves from which precise KD values are derived. This enables accurate comparison of affinities across variant panels, even for tight binders in the nanomolar range – without the need for immobilization or detection reagents.
Specificity assessment
MDS determines sequence specificity by comparing binding interactions across different DNA probes under identical conditions. By measuring KD and complex formation for both canonical target sequences (e.g. E-box) and non-specific DNA, it reveals whether binding is truly sequence-driven. Differences in Rh shifts and affinity directly reflect selective recognition, enabling clear and quantitative discrimination between on-target and off-target interactions.
Protein behavior in 100% plasma or serum
MDS operates directly in solution and is compatible with complex biological matrices, including up to 100% plasma or serum. This enables measurement of miniprotein behavior without dilution, purification, or assay reformatting. By tracking Rh in these environments, MDS provides direct insights into non-specific binding and stability under physiological conditions – bridging the gap between in vitro characterization and in vivo relevance.
APPLICATION NOTE
From Nanomolar Binding to Neat Plasma: Surface-free Mini-Protein Characterization
Explore how Peptomyc’s team used the Fluidity One-M to characterize the Omomyc miniprotein variants behind their OMO-103 clinical program (Phase 1 trial published in Nature Medicine). They resolved DNA-binding affinities from 69.8 nM to 7,490 nM across three variants, confirmed E-box sequence discrimination, and profiled non-specific binding from buffer through 100% mouse plasma. Each measurement required 4 uL of sample and completed in under 25 minutes, with no immobilization, no purification, and no buffer restrictions.
Make better, faster selection of candidates with the right balance of affinity, specificity, and in-matrix behavior.
True Size
Using MDS, the hydrodynamic radius (Rh) of fluorescently labelled DNA probes – both canonical E-box and non-standard sequences – were measured as Omomyc variants (OLS, V110, V113) were titrated in solution. Binding-induced changes in Rh enabled direct quantification of KD while simultaneously resolving DNA–protein complex sizes, revealing clear differences in affinity and sequence-dependent binding across variants.
True Environment
As MDS operates entirely in solution, no immobilization to surfaces or chips is required. The Rh of labelled Omomyc variants (OLS, V110, V113) were measured directly in PBS-T and across increasing concentrations of plasma, including 100%, without assay reformatting – enabling characterization in native physiological conditions.
True Insights
By tracking Rh changes from buffer through to 100% plasma, MDS distinguishes variants that retain specificity from those prone to non-specific interactions. Assuming plasma contains negligible concentrations of the target DNA, any increase in Rh reflects off-target binding to plasma components. The magnitude of this shift provides a direct, quantitative measure of non-specific interactions – revealing developability risks early and enabling more informed candidate selection.
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