# Nano-Polymers as Cas9 Inhibitors
## 3 Results
### Computational Prediction of Cas9-Polymer-LLL Interaction  
The significant difference in Cas9 inhibition by PMLA and PMLA-LLL suggested that the polymer-conjugated LLL moiety could have unique interactions with Cas9 or the Cas9-DNA complex. The resolved structure of the SpCas9 protein in a complex with the protein inhibitor AcrIIA4 revealed that the dsDNA binding pocket is rich in positive amino acids [29]. These amino acids could form hydrogen bonds with AcrIIA4, or a 'positive ring' around the pocket to connect with dsDNA substrates (Figure 6A). Because the backbone of the PMLA polymer is rich in electronegative groups like ester and carboxyl, it is possible that PMLA may have a certain affinity with this pocket.  
To explore the potential mechanism of Cas9 inhibition by PMLA-LLL, we performed molecular docking simulations to investigate the binding mode of PMLA-related monomers to Cas9, including a mono unit of PMLA (mPMLA), a capped mPMLA (closed), mPMLA conjugated with a LLL (mPMLA-LLL), or a double LLL (mPMLA-2 LLL) (Figure 6B). After induced-fit docking and scoring, the top-scored pose of mPMLA-LLL revealed that the LLL moiety was close to a hydrophobic surface formed by Leu1318, Gly1319, Ala1320, Pro1321,  
Figure 6: Molecular docking results of different monomers on Cas9.  
Ala1322, Ala1323, and Phe1324 within the PAM-interacting domain (Figure S3A-C). Also, the connected PMLA backbone was close to a nearby hydrophilic site, including Arg1335 and Arg1333. Moreover, Lys1325 is another hydrophilic site next to the hydrophobic surface, which could form hydrogen bonds with the terminal carboxy group of LLL or other carboxy groups in the PMLA backbone (Figure S3A-C). Meanwhile, the docking results of the naked PMLA backbone predicted various interactions with the hydrophilic pocket, but only one interaction with the hydrophobic surface, suggesting the hydrophobic pocket may not be suitable to bind to the negatively charged PMLA backbone (Figure S3D-F).  
Consistently, monomers with a cap moiety showed fewer effects on the polarity matching between the PAM-interacting domain and PMLA-LLL (Figure 6C,D). Furthermore, mPMLA-2 xLLL showed that the adjacent LLL tends to be close to another hydrophobic site and did not disturb the interactions between Cas9 and mPMLA-LLL (Figure 6E-H). These results suggested that the hydrophobic surface of PAM could be a preferable site for the binding of the LLL moiety, and the inconsecutive hydrophilic sites within the pocket formed by spaced positive and neutral residues would be suitable for the interaction with PMLA-LLL but not naked PMLA, which could explain their difference in Cas9 inhibition.  
A. Vacuum electrostatic analysis of SpCas9 protein: Blue, white, and red represents the positively, neutrally, and negatively charged surface. The white circle represents the 'positive ring' in the pocket.  
B. The structures of monomers involved in the docking assay.  
C, E, and G: The binding poses of the (C) mPMLA(closed) structure. (E) mPMLA-LLL and (G) mPMLA-2 LLL. The monomers are represented in magenta, the hydrophobic residues are represented in green, and the residues with a positive charge are represented in blue.  
D, F, and H: The Cas9 binding surfaces with the abovementioned polymers. The monomers are represented in green; blue, white, and red represent the positively, neutrally, and negatively charged surfaces.