Application Notes

4 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Throughout the assays, the Cy5-labeled RNA was used at concentrations of 100 pM, and the highest Cas9 concentration in the serial dilutions was 10 nM. The affinity measurements of the Cy5-labeled RNA constructs yielded excellent MST traces and subsequent signal-to-noise ratio (Figure 4). Overall, 10 dgRNA constructs displayed affinities in the lower picomolar range (between 1.3 and 12.9 pM, Figure 4 and 5, Table 2); sgRNA/Cas9 binding affinity was 7.3 pM. As shown in Figure 5, all MST experiments are characterized by an excellent signal-to-noise ratio that enables reliable determination of picomolar affinities for RNA-Cas9 interactions (Table 1). Although cysteine labeling might be a valid approach for monitoring sgRNA/Cas9 affinity, this study suggests that the unintended labeling of the binding- pocket-proximal cysteine could affect the binding of hybridized dgRNA. We therefore recommend using a Cy5-labeled probe for hybridization with an array of different RNA constructs. By using such probes, MST allows for a rapid and straightforward determination of affinities between RNA and Cas9 protein. Importantly, we noticed an abrupt loss of Cas9 binding for the probe containing crRNA hybridized with the corresponding tracrRNA (Table 1). sgRNA ID (> 90 nt) crRNA/ tracrRNA ID (42 nt, 68 nt) K d (pM) sgRNA1 — 1.1 ± 1.3 sgRNA1 ivt — 10.7 ± 3.9 sgRNA2 — 2.4 ± 2.3 sgRNA3 — 6.7 ± 4.0 sgRNA4 — 2.8 ± 2.4 sgRNA5 — 1.0 ± 1.4 sgRNA6 — 2.4 ± 2.5 — dgRNA1 No binding We recognized that the proximity of Cys808 to the binding pocket might influence the binding of RNA to Cas9 (Figure 3, Table 1). To determine if the loss of RNA binding is the result of our labeling strategy, we hybridized a Cy5-labeled probe to crRNA and measured its affinity for Cas9. The same Cy5-crRNA was then hybridized with various tracrRNAs. In this manner, we were able to label different dgRNAs with a single fluorescent probe. Additionally, we hybridized the same Cy5 probe to a sgRNA. Table 1: Affinities of various sgRNAs and a crRNA/tracrRNA hybrid as determined by MST measurements using the Monolith™ NT.115Pico instrument. MST experiments were performed with 50 pM Cas9-NT647 at 85 % LED and 40 % MST power.

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