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Single Molecule Analytical Raman Tools based on DNA nanostructures

Periodic Reporting for period 1 - SMART-DNA (Single Molecule Analytical Raman Tools based on DNA nanostructures)

Reporting period: 2018-04-01 to 2019-09-30

The monitoring of single molecule reactions promises unrivalled insight into chemical reaction mechanisms, but represents one of the most challenging tasks in chemistry. Surface-enhanced Raman scattering (SERS) is a particularly attractive single molecule (SM) technique due to its high chemical specificity, which allows to directly detect relevant intermediates and molecular subpopulations. However, SM-SERS is still at a premature state due to the highly challenging task to place single molecules precisely in nanoscale gaps of plasmonic nanostructures. These are required to provide sufficiently high electromagnetic field enhancement to reach SM sensitivity. The aim of SMART-DNA is to exploit artificial DNA nanostructures to provide sufficient structural control to assemble both, nanoparticles and target molecules, with nanometer precision. By means of novel DNA origami nanostructures the distance between two nanoparticles will be controlled, and at the same time target molecules will be placed at the positions of highest Raman enhancement through DNA aptamers.
Apart from Raman enhancement the excitation of the localized surface plasmon resonance of the metallic nanostructures results in other plasmonic effects such as heating and possibly the transfer of hot electrons. This can lead to diffusion, conformational changes or even dissociation of the target molecules. These issues do not only concern SM-SERS, but also make quantitative SERS and the SERS analysis of complex (bio)molecules very challenging. By the improved structural control achieved by SMART-DNA, nanoscale heating and hot electron transfer and their effect on SERS spectra will be studied on an ensemble and a SM level. Finally, reactions induced by plasmonically generated electrons in DNA and DNA modified with electrophilic molecules will be studied by SERS with the aim to develop novel strategies to improve cancer radiation therapies such as the photothermal therapy.

Objectives of SMART-DNA:
I. Provide control over multiple structural parameters using bionanotechnology. SMART-DNA will create and apply advanced DNA origami based SERS substrates with aptamer binding sites. The SERS substrates should have the following properties: i) Tunable SERS enhancement, (ii) Control of analyte number and orientation in the SERS hot spot using aptamers, (iii) Possibility of analyte diffusion into hot spots, (iv) SERS measurements should be possible with SERS substrates dispersed in solution and immobilized on surfaces.
II. Establish reliable, continuous SM-SERS detection for a broad range of molecules. Field enhancement and structural control will be further optimized to establish SM-SERS measurements for small and complex molecules and for resonant and non-resonant molecules.
III. Explore nanoscale interactions between NPs, molecules and excitation light. Especially changes of temperature, geometry and molecular integrity upon LSPR excitation will be explored for small and complex molecular systems.
IV. Explore electron-transfer induced reactions in DNA and modified DNA. A specific focus will be put on hot-electron induced reactions in DNA derivatives, since these are rather unexplored in the context of SERS but have great potential to become relevant for cancer radiation therapy.
The action started successfully and the major achievements of the first reporting period are:
WP1: Optimization of DNA origami fork design has been carried out along with an in-depth characterization by gel electrophoresis, AFM and TEM. An optimization of the fork bridge using oxDNA simulations in cooperation with Dr. Antonio Suma was done resulting in an improvement of single-molecule measurements using dyes. Milestones 1.1 and 1.3 are basically completed.
WP2: Hemin was bound to the DNA origami fork via aptamers and detected by SERS using both gold and silver nanoparticle dimers. The SERS spectra are very promising, i.e. we can evaluate the charge and spin state of hemin on a single-molecule level. Furthermore, 6-helix bundles were covalently attached to DNA origami forks to align them by molecular combing. Milestones 2.1 and 2.2 are therefore close to being completed.
WP3: Proteins (CytC and horseradish peroxidase (HRP)) are bound by different coupling strategies to the DNA origami fork and detected by single-molecule SERS. HRP was chosen as an extra protein to be studied due to its high stability. MS 3.1 will be completed soon.
WP4: The kinetics of hot electron induced reactions on gold nanoparticles have been studied using nitrothiopenol, halogenthiophenols and brominated nucleobases. A detailed kinetic model is currently developed. Furthermore, we have studied electron transfer from nanoparticles to DNA modified with brominated adenine and determined the distance dependence of the reaction. Milestones 4.1a and 4.2 can be completed soon. Two papers arising from this WP have been already published.
The SMART-DNA project represents a fundamentally different approach to SM- and single nanostructure SERS spectroscopy compared to previous studies and enables the control of a large number of parameters that are critical for significant advancement of this field. The unprecedented degree of structural control on the nanometer scale enabled by the DNA origami technology will be exploited to establish a versatile tool for spectroscopic investigations with significantly improved control over the target molecules. SMART-DNA represents frontier research of high risk, but the interdisciplinary approach (DNA-nanofabrication combined with SERS spectroscopy) promises high gain by advancement of novel fields of application for SERS that showed only slow progress during the last years or even decades due to the general complexity of SERS substrates. The highest risk concerns the Raman enhancement that is required for SM analysis especially of molecules that are not in resonance with the excitation laser. Several innovative strategies will be explored to reduce the interparticle gaps of sufficiently large NPs and to control the target molecule positioning to provide sufficient Raman enhancement. SMART-DNA addresses a broad range of fundamental scientific challenges such as the origin of SERS blinking in SM and few-molecule SERS and its role for ensemble measurements, the SERS signal distribution and a fundamental mechanistic understanding of electron transfer induced reactions in modified DNA. The latter mechanisms will contribute to develop a complete picture of physical and chemical radiosensitization in cancer radiation therapy, which will enable the improvement of currently applied therapeutics and will enable the development of novel treatment strategies. Furthermore, the tools developed within SMART-DNA are not only restricted to SERS, but can also be exploited by other surface-enhanced spectroscopy methods such as surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced fluorescence (SEF).