# Nano-Polymers as Cas9 Inhibitors
## 3 Results
### PMLA-Trileucine (LLL) Conjugate Binds to Cas9 Protein
Interestingly, when we pre-incubated the Cas9 protein with PMLA-LLL or PMLA, we found that the Cas9-sgRNA binding was dramatically decreased, especially with PMLA-LLL preincubation that led to a 425-fold decrease in the Cas9-sgRNA binding affinity (Table 2). Upon confirmation of the binding affinity between the PMLA-LLL polymer and Cas9 protein, we asked whether this interaction may have any impact on Cas9 nuclease function. To answer this question, we used a pGEM vector and designed an sgRNA (TCGTAGT-TATCTACAGACG) targeting the ampicillin resistance gene in this plasmid. The pGEM plasmid was first linearized by the Pei-I restriction enzyme, resulting in 3.2 kb linear DNA. After cleavage by Cas9/sgRNA ribonucleoprotein (RNP), this vector DNA would produce two fragments of 2.3 kb and \(\sim\)90 bp in length. When the Cas9/sgRNA was pre-incubated with the PMLA-LLL polymer, we found that the DNA cleavage was completely blocked, while PMLA incubation did not affect the DNA cleavage by Cas9/sgRNA RNP (Figure 3A). Next, we asked whether we could achieve the same Cas9 inhibition by incubating the Cas9 protein with the pre-mixed PMLA polymer and free LLL peptide, since we found that the PMLA backbone failed to inhibit Cas9 function (Figure 3A). Our DNA digestion results clearly demonstrated that PMLA plus free LLL peptide had no effect on Cas9 activity (Figure 3B). Furthermore, when we incubated Cas9 with a concentration series of PMLA-LLL with a polymer/Cas9-sgRNA molar ratio of 5:1, 3:1, 2: 1, and 1:1 (Figure 3C), we observed a dose-dependent pattern of Cas9 inhibition by PMLA-LLL, with a minimal 3:1 polymer/Cas9 molar ratio to completely block Cas9 nuclease activity. Lastly, although PMLA showed a moderate binding affinity to Cas9 via the MST assay (Table 2), we did not detect any functional inhibition of Cas9 nuclease activity by PMLA even at an extremely high polymer concentration (PMLA/Cas9 = 600:1) (Figure 3D), suggesting that the naked PMLA backbone lacks a structural basis to block Cas9 function. Tumor-targeting mechanisms are essential to allow nano-polymers to selectively bind to the tumor cell surface. This tumor-specific binding can be achieved by conjugating an antibody or a peptide with a selective binding affinity to cancer cells, such as an AP-2 (angiopep-2) peptide [7; 27]. The next question we asked was whether the addition of a tumor-specific targeting moiety could affect Cas9 inhibition by PMLA-LLL. To answer this question, we modified the PMLA-LLL polymer by further conjugating an AP-2 peptide to generate PMLA-LLL-AP2. AP-2 is a 19-amino-acid synthetic peptide (TFFYGGSRGKRN-NFKTEY) that can specifically interact with the LRP-1 receptor to facilitate blood-brain barrier crossing [28]. In the DNA digestion assays, our data showed that further conjugation of AP-2 to the PMLA-LLL does not affect Cas9 inhibition. As shown in Figure 3E, compared to PMLA-LLL, the PMLA-LLL-AP2 exhibited a similar dose-dependent inhibition of Cas9 with a minimal polymer/RNP molar ratio of 5:1 to completely block Cas9 function. \begin{table}
\begin{tabular}{p{56.9 pt} p{56.9 pt} p{56.9 pt} p{56.9 pt}} \hline \hline
**No.** & **Target** & **Ligand** & **K\({}_{\text{D}}\) (nM)** \\ \hline
1. & Cas9 & sgRNA & 1.24 \\
2. & Cas9 & PMLA-LLL & 3.6 \\
3. & Cas9 & PMLA & 36 \\
4. & Cas9 + PMLA-LLL & sgRNA & 528 \\
5. & Cas9 + PMLA & sgRNA & 23 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Polymer–Cas9 binding affinity determined by MST assays. * PMLA-LLL can effectively block Cas9-mediated DNA cleavage. A linearized pGEM DNA vector (\(\sim\)3.2 kb) was used as the DNA substrate and a sgRNA targeting the ampicillin resistance gene sequence in the vector was used to direct the Cas9-mediated DNA cut. Only when the Cas9/sgRNA RNP was present in the digestion reaction was the DNA vector cut, resulting in two fragments (\(\sim\)2.3 kb and 900 bp). However, when pre-incubated with PMLA-LLL (with a molar ratio of polymer/RNP = 50:1), the DNA digestion was completely blocked.