Molecular Insights into the Binding of Linear Polyethylenimines and Single-Stranded DNA Using Raman Spectroscopy: A Quantitative Approach

Abstract

Establishing how polymeric vectors such as polyethylenimine (PEI) bind and package their nucleic acid cargo is vital toward developing more efficacious and cost-effective gene therapies. To develop a molecular-level picture of DNA binding, we examined how the Raman spectra of PEIs report on their local chemical environment. We find that the intense Raman bands located in the 1400-1500 cm^-1 region derive from vibrations with significant CH₂ scissoring and NH bending character. The Raman bands that derive from these vibrations show profound intensity changes that depend on both the local dielectric environment and hydrogen bonding interactions with the secondary amine groups on the polymer. We use these bands as spectroscopic markers to assess the binding between low molecular weight PEIs and single-stranded DNA (ssDNA). Analysis of the Raman spectra suggest that PEI primarily binds via electrostatic interactions to the phosphate backbone, which induces the condensation of the ssDNA. We additionally confirm this finding by conducting molecular dynamics simulations. We expect that the spectral correlations determined here will enable future studies to investigate important gene delivery activities, including how PEI interacts with cellular membranes to facilitate cargo internalization into cells.

Figure 1:

Schematic of model compounds used in this study.

Figure 2:

Raman spectrum of high molecular weight PEI (2500 Da) prepared at 30 mM in methanol. The spectroscopic contribution of methanol has been subtracted out. The asterisk (*) symbol shows an artifact of subtracting out the spectral contribution of methanol.

Figure 3:

Raman spectra of low molecular weight (103 − 232 Da) PEIs prepared at equimolar (200 mM) concentrations in water. (a) PEHA (black trace); (b) TEPA (black trace); (c) DETA; (d) PEHA - TEPA difference spectrum. The difference spectrum closely approximates the spectrum of internal monomers in PEIs, while the DETA spectrum approximates the spectrum of terminal monomers. This is demonstrated by the fact that the PEHA and TEPA spectra can be modeled as a linear combination of the spectra shown in (c) and (d). PEHA can satisfactorily modeled as 3×(PEHA - TEPA) + DETA (red trace in panel a), while TEPA can be modeled as 2×(PEHA - TEPA) + DETA (red trace in panel b). All spectra were normalized to the 2250 cm⁻¹ nitrile stretching band of acetonitrile, which was used as an internal intensity standard. The spectral contribution of solvent was subtracted out. The asterisk (*) symbol shows an artifact of subtracting out the spectral contribution of the nitrile stretching band of acetonitrile.

Figure 4:

Raman spectra of NEPA in: (a) H₂O; (b) D₂O; (c) CDCl₃; and (CD₃)₂SO. All spectra were measured using a high-resolution (2400 gr/mm) grating. The spectral contributions of solvents were subtracted from all spectra. For (c) and (d), the Raman bands that derive from CH₂ scissoring modes are highlighted to show the intensity changes that occur between low and high dielectric environments.

Figure 5:

Raman spectra of: (a) PEHA (100 mM)-ssDNA (3 mM) in solution (black trace); (b) PEHA (100 mM); and (c) ssDNA (3 mM). The red trace in (a) is the sum of the spectra shown in (c) and (d). The difference spectrum shown in (d) was calculated by subtracting the red trace from the black trace in (a). The fact that the experimentally measured spectrum in (a) shown in the black trace cannot be modeled as a linear combination of the ssDNA (c) and PEHA (b) spectra indicates binding is occurring between the two species. All spectra were normalized to the 2250 cm⁻¹ nitrile stretching band of acetonitrile, which was used as an internal intensity standard. The asterisk (*) symbol shows an artifact of subtracting out the spectral contribution of this nitrile stretching band.

Figure 6:

MD simulation results of PEHA binding ssDNA in water. (a) Solvent accessible surface area (SASA) time evolution plot. Structures are inlayed corresponding to before and after the confirmational change. Disordered secondary structure is established with polymer bound to the DNA surface. (b) Ratio per residue of backbone contacts vs. base contacts (4 Å cutoff, averaged over total frames). (c) Pie chart of overall backbone/base interactions. The chart shows PEHA exhibits a higher affinity for the DNA backbone (electrostatics) than for the DNA bases (hydrogen bonding). Polymer binding sites correspond to areas of high electrostatic potential shown in Figure S8a.