Surface Plasmon Fluorescence Spectroscopy (SPFS) in Studies of Interfacial DNA Hybridization

W. Knoll

Surface Plasmon Resonances(SPR) are electromagnetic surface modes. They only exist on metal surfaces. The propagation along the surface and decay evanescently into both the dielectric medium and the metal. The surface plasmon is usually detected via a dip in the angle-dependent reflectivity. The angle of minimum reflectivity is the coupling angle. The coupling angle is the central observable of SPR spectroscopy. It shifts upon adsorption of an analyte to the metal surface. Using the SPFS, the hybridization event is detected from the study of the emitted light from a labelled oligonucleotide excited by the evanescent electromagnetic field of the surface plasmons.

Major decay channels for excited chromophores near metallic surface:

1. While at close distances quenching to the metal layer is dominant
2. The excitation of plasmons by the red-shifted fluorescence light is found for distances up to about 20 nm
3. At large distance to the metal the free emission of photons can be found

The current DNA Track EU Project is based on detection and analysis of the DNA content in the GMO (Genetically Modified Food) and develop PNA and DNA chip technology for fast measurements. For this purpose Surface Plasmon Fluorescence Microscopy (SPFM) and Spectroscopy (SPFS) techniques are being used in the group. The real time observation of DNA binding process is being carried out by SPFM technique.

Most of the detection formats include the interaction of immobilized DNA probes and targets with surfaces. The analysis of the kinetic behavior of oligonucleotides at the sensor surface is hence of major importance for the improvement of known detection schemes. Furthermore, the characterization of DNA structure on surfaces can lead to the development of novel detection formats.

PCR (Polymerase Chain Reaction) is one of the most important biochemical techniques in use today. It is basically an invitro primer extension reaction, which allows the exponential amplification of a specific deoxyribonucleic acid (DNA) region that lies between two regions of known DNA sequence. Our work aims at detecting single nucleotide mutation in PCR DNA, and clarifying the hybridization behavior of PCR DNA on surfaces with the aid of SPFS.

Peptide Nucleic Acids (PNA) are polypeptide-derived synthetic compounds that mimic the natural properties DNA. PNA can be chemically synthesized to any specific sequence and used for hybridization measurements with DNA. Its uncharged pseudo-peptide backbone is responsible for its enhanced binding behavior to DNA. This study further investigates the role of charge effects on hybridization processes by introducing positive charges onto the otherwise neutral backbone of PNA probes. Experiments are performed in biological pH and ionic strength conditions, with three different DNA targets carrying 0, 1, and 2 base mismatches respectively. Experimental data sets are then simulated using Langmuir Model. Finally, such theoretical modeling translates data quantification into kinetic constants that allow for numerical modeling.

The Klenow Fragment (KF) is a prokaryotic DNA Polymerase that catalyzes template-directed DNA synthesis. The enzyme requires all four desoxynucleoside 5' phosphates (dNTP), Mg2+ a DNA template hybridized to a primer with a 3'OH. The synthesized DNA chain grows in 5' to 3' direction. The project employs SPFS as tool for studies on polymerase/DNA interactions. Using this method one may address several steps in the kinetic scheme of the KF in their temporal order, whereas most conventional biochemical methods provide only snapshots on single steps. Being involved in the faithful replication of genetic information and DNA repair processes, DNA polymerases are enzymes of high biological relevance. Introducing mismatched bases into the DNA primer region will elucidate how these enzymes cope with DNA alterations to ensure high DNA replication fidelity.

One of the detection method of the DNA binding process in our group is Surface Plasmon Flourescence Microscopy (SPFM). The system is mainly based on SPR set-up, the only difference in SPFM is that the photomultiplier tube is being replaced with a sensitive CCD camera. SPFM technique allows to observe binding process in the real time. When it combined with array technology, it also allows to detect more than one sample on the same chip and so it becomes cost and material efficient method.

An ink-jet spotter is being used for preparing the microchips.

Flourescence image of Biotinylated PNA probes spotted onto Streptavidin coated Au after hybridization with 50 nMCy5 Labeled DNA target.