4P050
Properties of Water at the Lipid-DNA Interfaces Revealed byHeterodyne-Detected Vibrational Sum Frequency Generation(HD-VSFG) Spectroscopy(Molecular Spectroscopy Laboratory, RIKEN) Prashant Chandra Singh, Satoshi
Nihonyanagi, Shoichi Yamaguchi, Tahei Tahara[Introduction]
Cationic lipids are widely used as in vitro DNA transfection vectors and show promise
as non-viral carriers for in vivo gene therapy. In solution, DNA and cationic lipids form
complexes that can enter the cell through endocytosis. Understanding and ultimatelycontrolling the electrostatic interactions between these lipids and DNA is therefore
essential to optimize these genetic carriers. Although numerous in vivo and in vitro
studies have been performed to explore the structural properties of the lipid-DNAinteraction, yet the molecular level understanding of this interaction is very limited.1
Vibrational spectroscopy is a useful tool to get the information about the molecularlevel interactions in lipoplexes. Several groups have conducted infrared absorption
measurements of various DNA/lipid systems. However, due to the lack of inherent
surface sensitivity/specificity of infrared absorption, we still do not get clear
understanding of the complexation of DNA with lipids. In this study, we overcome thesurface specificity/sensitivity challenge by applying heterodyne-detected vibrationalsum frequency generation (HD-VSFG)2 spectroscopy to gain a molecular level pictureof the interaction between DNA, lipids, and interfacial water.
[Experimental]
A narrow band visible (1) and broadband IR (2) beams are used for the HD-VSFG.
The SF, visible and IR beams were s-, s-, and p-polarized, respectively, (ssppolarization). All the spectra were normalized to a quartz spectrum.
1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP) was purchased as lyophilized
powders from Avanti Polar lipids. λ-phage DNA (each molecule specified as 48502
base pairs in length by the manufacturer) was purchased from Sigma Aldritch. The
surface pressure was monitored continuously during the HDVSFG measurement. All
the measurements were done in liquid condensed (LC) phase of DPTAP with surfacepressure of 31 ± 3 mN/m.

[Results and Discussion]
Figure 1 shows complex (2)spectra in the OH stretchingregion of the DPTAPmonolayer/ aqueous solution
interfaces containing DNA atvariousconcentrations
ranging from 0 to 220 pM.
Two broad negative band
1

1around 3200 cm and 3450 cm
in the Im(2) spectra at 0 pM
DNA have been assigned to thesymmetric OH stretching ofhydrogen bonded water, split

Figure 1. Im(2) spectra of air / DPTAP monolayer /H2O interfaces in the OH stretching frequency region
containing DNA of the concentrations at 0, 10, 30, 70,
130, 220 pM. Black dotted line shows the Im(2)spectrum of air/water interface.

by Fermi resonance with the bending overtone. The sign of the Im(2) spectra in theOH stretching region is negative, indicating that the water molecules at these interfaces
are oriented with their hydrogen pointing downward to bulk water. When no DNA isadded, the OH band intensity is about five times larger than that at the neat water
surface because the static electric field created by the DPTAP monolayer inducesorientation of more water molecules along the field. Addition of DNA to the DPTAP
subphase results in a reduction of water signals even at concentrations as low as 10 pM.
Around 100 pM, the signal reaches to the level of the neat air/ water interface. Thisimplies that already at these low concentrations, the static electric field is fully
screened by the polyanionic DNA. This result indicates that DNA reaches the interface
and has specific interaction with the lipid. As the DNA concentration increases further(>200 pM), the sign of Im(2) in the OH stretch region becomes completely positive
which shows that the orientation of the water molecules change from hydrogen down
to hydrogen up at the DPTAP interface. This flipping of water orientation at theinterface depicts that DNA makes a contact adsorption to the lipid head group and net
surface charge of the interface turn to be negative. This study clearly exhibits theapplicability and novelty of the HD-VSFG technique to get the inherent molecularlevel understanding of biological interfaces.
[References]
1. Radler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810-814.
2. Nihonyanagi, S.; Yamaguchi, S.; Tahara, T.; J. Chem. Phys. 2009, 130, 204704-204710.