# Nano-Polymers as Cas9 Inhibitors
## 3 Results  
### PMLA-Trileucine (LLL) Conjugate Binds to Cas9 Protein  
PMLA is a naturally occurring linear polymer with an abundance of -COOH groups that allows covalent conjugation of multi-functional modalities, such as tumor targeting mechanisms, therapeutics, and BBB-crossing moieties [23; 24; 25]. Among these conjugated functional modalities is a trileucine (LLL) peptide that we have used to facilitate endosomal escape after the internalization of nanoconjugates. The chemical structures of PMLA-polymer and PMLA-LLL are shown in Figure 1.  
To examine the potential interaction of PMLA-LLL with the Cas9 protein, we performed HPLC assays on various formulations, including free Cas9, PMLA-LLL alone, and mixtures of PMLA-LLL with Cas9 at molar ratios of 5:1, 10:1, and 20:1 (Figure 2). The chromatographic profiles reveal a clear interaction between PMLA-LLL and Cas9, with a dose-dependent pattern. Free Cas9 exhibits a distinct peak at approximately \(\
eg 9\) min, which represents its unbound state in the absence of PMLA-LLL. In contrast, PMLA-LLL alone shows a unique peak at a separate retention time, indicating its individual chromatographic behavior. Once mixed with PMLA-LLL at a 5:1 molar ratio, the Cas9 peak decreased in intensity, accompanied by the appearance of a new peak corresponding to the PMLA-LLL/Cas9 complex. Increasing the molar ratio to 10:1 and 20:1 resulted in further reduction of the free Cas9 peak and a simultaneous increase in the complex peak, demonstrating stronger interaction at higher PMLA-LLL concentrations. These findings indicate that PMLA-LLL binds Cas9 in a dose-dependent manner, with optimal binding observed at a 20:1 molar ratio. This property underscores the potential of PMLA-LLL as a nano-polymer carrier for the efficient delivery of CRISPR/Cas9 systems. Figure 1: Structure of naturally occurred PMLA polymer and PMLA-LLL conjugates. In addition, when we mixed the Cas9 protein with PMLA or PMLA-LLL, in the presence or absence of sgRNA, we found that while adding PMLA to either Cas9 protein or sgRNA had a minimal impact on the measured zeta potential [26], a measure of the electric potential at the boundary between a solid particle and its surrounding liquid environment and a low zeta potential means particles are more likely to attract and agglomerate. In contrast, adding PMLA-LLL dramatically decreased the zeta potential, suggesting a much stronger interaction between PMLA-LLL and the Cas9 protein (Table 1). Next, we performed microscale thermophoresis (MST) to measure the potential binding affinity between the nano-polymers with the Cas9 protein (Figure S1). In this experiment, we first compared the binding affinity of Cas9 to a synthetic sgRNA, a PMLA polymer backbone, and a PMLA-LLL conjugate. Our results showed that Cas9 has the strongest binding to the sgRNA with a dissociation constant (KD) of 1.24 nM, followed by \begin{table}
\begin{tabular}{c c} \hline \hline
**Product** & **Zeta Potential (mV)** \\  & **Buffer: H\({}_{2}\)O** \\ \hline PMLA & \(-5.6\) \\ PMLA + Cas9 & \(-11.5\) \\ PMLA-LLL & \(-6.16\) \\ PMLA-LLL + Cas9 & \(-22.1\) \\ PMLA-LLL + Cas9 + sgRNA & \(-24\) \\ Cas9 & \(10.8\) \\ sgRNA & \(-4.1\) \\ Cas9 + sgRNA & \(-7.1\) \\ sgRNA + Cas9 + PMLA-LLL & \(-23.5\) \\ sgRNA + PMLA-LLL & \(-9.6\) \\ sgRNA + PMLA-LLL + Cas9 & \(-19.7\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Zeta potential measurement. Figure 2: Chromatograms from HPLC analysis demonstrating binding interaction between PMLA-LLL and Cas9 protein at different molar ratios (5:1, 10:1, and 20:1). Free Cas9 and PMLA-LLL alone are represented by distinct peaks, while dose-dependent binding is evidenced by increasing intensity of PMLA-LLL/Cas9 complex peak at higher molar ratios. the interaction between Cas9 and PMLA-LLL with a KD value of 3.6 nM (Table 2). The PMLA backbone alone also exhibited some binding affinity to Cas9 with a 10-fold higher KD (36 nM).