Bio-AFM for Single Molecular Level Analysis The nanotechnology for life sciences, nano-biotechnology, is the convergence of nanoscience/technology and biological science, leading to new “eyes and hands” for understanding and manipulating biological systems. In this field, atomic force microscopy (AFM) has attracted keen attentions, because it is compatible with non-conductive materials, does not require labeling, and can be operated under the physiological condition, while allowing single molecular level analysis. Therefore, AFM has become a central tool for nano-biotechnology. Also, imaging surfaces with the nanoscale resolution, probing local mechanical properties, and measuring a variety of interaction forces from nN to pN are possible with the instrument. In particular, the microscopy measuring the force of a picoNewton range has been utilized for a variety of single molecule measurements, and the examples include protein unfolding process, DNA-DNA, protein-protein, and ligand-receptor interaction. At the same time, a significant growth of the genome and proteome studies for drug discovery, as well as for disease diagnosis and prevention, has placed a strong demand for advanced biomolecular recognition probes with high sensitivity and enhanced specificity. It is therefore clear that this type of approach finds important applications not only within basic life science research, but also within emerging areas for the analysis of larger biomolecular libraries.
When the force measurement between individual biomolecules is carried out with AFM, the accuracy and the resolution of the force value is a critical factor for the reliability. In order to enhance the reliability, it is necessary to control density and orientation of the immobilized biomolecules on surface. It used to be hard to control small scale variations due to spatial differences in surface topography and chemistry, and such factor is some of the remaining bottle-necks to the future progress of such force-based approaches. To resolve such issues, NB POSTECH offers a new surface modification of AFM tips and substrates for measuring specific biomolecular interaction forces reliably (J. Am. Chem. Soc. 2007, 9349; Adv. Mater. doi:10.1002/adma.200801323). The dendron-modification of the surfaces can optimize the density of immobilized biomolecules, remove steric hindrance between interacting biomolecules, and avoid unwanted nonspecific binding and/or the formation of multiple biomolecular complexes. Therefore, dendron modified-AFM tip of NB POSTECH provides a superb option to the existing biomolecular surface immobilization methods. We believe that thus-modified AFM tips are ideal for studying the interactions between DNA and DNA/RNA, ligand and protein, protein and protein, as well as receptor on cell surface and ligand.
Figure 1. A schematic drawing of the experimental setup employing the dendron-modified AFM tip and substrate.
1) DNA-DNA Interaction at Single Molecule Level (J. Am. Chem. Soc. 2007, 9349) The dendron modified-AFM tip simplifies the force-distance curves for the specific biomolecular interaction, and enhances the reliability of the analysis. We confirmed effect of the spacing provided by the dendron layer on the force analysis by using dendrons of different generations. In case of DNA force measurement, the use of the 9-acid gave a narrow histogram in comparison with the cases of the lower generation dendrons (3-acid). It is clear that the control of the spacing between the biomolecules is essential for the fine analysis.
Figure 2. Effect of spacing on the adhesion forces. Distributions of adhesion forces (recorded with a retract velocity of 0.10 μm/s) obtained for 30 mer oligonucleotide functionalized with (a) the first generation dendron (3-acid), (b) the second generation dendron (9-acid). The sequence of the DNA is 5′-NH2-GCT GCT ATG GAG ACA CGC CCT GGA ACG AAG-3′, and its complementary DNA sequence is 5′-NH2-CTT CGT TCC AGG GCG TGT CTC CAT AGC AGC-3′.
The adhesive events were observed by a chance of 50 – 80 %, and the retraction traces with a single clean pull-off event were recorded by using a substrate and an AFM tip with the optimal lateral spacing. The adhesive force histograms displayed the narrow distribution in comparison with the case other immobilization methods provided.
Figure 3. Distribution of adhesive forces recorded at 0.10 μms-1 for all of the fully complementary sequences. The y-axis shows the probability of observing the specific force for each case.
In particular, the attractive force associated with the specific molecular binding event is observed reproducibly during the approach mode.
Figure 4. A typical force-distance curve for the complementary 30-mer DNAs. Approach and retract traces are in red and blue, respectively.
2) Imaging of mRNA on a Tissue Surface (Nucleic Acids Research doi:10.1093/nar/gkn965 (2008)). We demonstrated successfully the feasibility of mapping mRNA distribution in a biological tissue sample by measuring DNA-RNA interaction forces. For this end, we utilized a DNA probe attached to a dendron-modified AFM tip to measure the specific adhesive force to the complementary RNA and Pax6 mRNA of 802 bases, and to map the Pax6 mRNA distribution on the surface of sectioned mouse embryonic tissues.
The dendron modified-AFM tip of NB POSTECH is readily adaptable to other biomolecular species, such as proteins, oligopeptides, RNAs, aptamers, PNAs, etc. Therefore, dendron modified-AFM tip of NB POSTECH should not only be of interest to researchers using AFM for studies of label-free DNA hybridization, but also those utilizing a range of force measurement approaches for basic life sciences research, as well as researchers who are interested in developing new methods for biomolecular screening.
NB POSTECH recommends AFM tips of a small spring constant for studying DNA-DNA, DNA-RNA, RNA-RNA, and protein-protein interaction. When we modified an AFM tip from Veeco (MSCT-AUNM), the spring constant changed from 10 pN/nm to a value between 10 pN/nm to 16 pN/nm. NB POSTECH is more than happy to coat other AFM tips with the dendron upon customer’s request.
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