Okada Group

SPM and Optical Analysis for DNA and Organic Molecular structures
Takao Okada

We develop a new type of multi-sensing/functional scanning probe microscopy (MS/F-SPM) based on scanning probe microscopy and a novel Laser-induced fluorescence spectroscopy (LIFS). We use these techniques for observation and manipulation of organic molecular structures at atomic or molecular level. And our final goal is to apply them to highly efficient analysis of DNA sequence.

■Results in Fiscal Year

1. Observation and Manipulation of Organic Molecular Structures by Multi-sensing/Functional SPM.

The subjects of our research are investigation for conservative AFM of biological applications in high resolution and development of novel SPM to approach organic molecules from the analytic point of view. Meanwhile we are trying to investigate the technique of sample preparation and to analyze the interaction between sample and substrates.

(a) Conservative AFM imaging of DNA.

It was confirmed that the circular DNA molecules would also be fixed on substrates in the same orientation with liquid flow as same as linear DNA observed before. We tried to fix DNA on mica substrates with magnesium, which revealed that Mg-treated kept the surface flatness than silane-treated. As shown in Fig.1, AFM image of double-stranded DNA was obtained with this treatment. Meanwhile the preparation method on a flat substrate was investigated. And we could successful imaged fiber structure of nucleosome with AFM (see Fig. 2).

(b) Improvement of Multi-sensing/Functional SPM

AFM/SNFOM images of chromosomes were taken simultaneously. In order to discriminate between a topological image and the features of specimen, we tried to obtain fluorescence images with SNFOM and succeeded in taking fluorescent signals from fluorescent latex microspheres.

(c) Simulation for mononucleotide structure

The energy of each mononucleotide on the structurally fixed graphite was minimized. As a result, the base planes of adenine, guanine and cytosine were arranged in parallel with the plane of the graphite.

2. Single-molecule detection and identification based on laser-induced fluorescence measurement and its application to ultrafast DNA sequencing.

(a) Visualization and manipulation of individual DNA double strands and evaluation of the activity of exonuclease III.

The activity of exonuclease III inhibited by YOYO-1, which is used for the selective visualization of DNA chain, is retrieved by washing the stained DNA with a 1 M NaCl solution. We also observed decrease in the area tracked by the Brownian motion of single DNA chains modified with SSB proteins; we evaluated the relation between DNA lengths and the tracked area.

(b) Arrangement of the set-up for single-molecule detection based on fluorescence measurement.

We found a procedure making possible to control the number of scattered single dye molecules on silicon wafers for fluorescence single-molecule detection.

Fig. 1 AFM image of DNA

Fig. 2 AFM image of fiber structure of nucleosome