Application Notes

3 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. free in solution, and a chemical equilibrium is reached. Unlike other approaches, a Kd value can be measured in less than 10 min using NanoTemper Technologies Monolith NT.115 Pico , or less than 2 min using the Monolith NT.Automated. Importantly, binding affinities in picomolar range of either modified sgRNA or hybridized crRNA/tracrRNA (so-called dual guide RNA (dgRNA)) are easily and precisely determined using MST. Results and Discussion In order to measure the binding affinities of various RNAs with Cas9, we followed two different approaches. The first approach was the direct fluorescent labeling of Cas9. Cas9 has 150 lysine residues, some of which are involved in RNA/DNA binding (Figure 2A). 8 Hence, lysine-directed NHS labeling was not amenable to our study. Instead, two cysteine residues, one residing on the outer rim of the binding crevice 8 (Figure 2B), were chosen as suitable targets for fluorescent labeling using maleimide chemistry. We fluorescently labeled Cas9 using the cysteine- directed maleimide-NT647 dye. Since we anticipated very high affinities of the RNA for Cas9 protein, we performed MST measurements on the Monolith NT.115 Pico instrument, which enables determination of K d values in the picomolar range. We combined 50 pM labeled protein with RNA in a serial dilution series, with 10 nM as the highest RNA concentration. Using this approach, we were able to successfully measure the affinities of various sgRNA for Cas9 (Figure 3, Table 1). A B Figure 2: Crystal structure of catalytically-active S. pyrogene Cas9 in complex with single-guide RNA 8 . A) Lysines (red spheres) in the contact with the sgRNA; other lysines are not shown for clarity. B) Position of cysteines (orange spheres). Figure 3: The binding of sgRNAs to fluorescently labeled Cas9. MST traces (A) and dose-response curve (B) for the sgRNA1 interaction with Cas9. C) Dose-response curves for the interaction of various sgRNAs and a dgRNA with Cas9. The resulting dose-response curves were fitted to a one-site binding model to extract K d values. MST experiments were performed with 50 pM of Cas9-NT647 at 80 % LED and 40 % MST power. F norm = normalized fluorescence A B C

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