SPM Applications in Biology

     Nucleic acids especially RNA and DNA are being extensively investigated since the very beginning of AFM. To show the variety of studies in this field we summarized the results and details of samples preparation in the table below. The material from sources in this table was taken from extended review of prof. A. Ikai [256] with permission of the author and publisher.

ID	What was studied	Details of sample preparation
1506	Nucleosomal DNA with 146 nucleotide base pairs. Both aggregates and individual molecules with lengths corresponding to theoretical values of 52 nm were discerned	Electrochemical deposition
1507 1508	15 mer DNA of solely pyrimidine bases
1510	Circular plasmid DNA and 623 base pair fSK14 restriction fragments.	First receipt) Deposition of DNA on an untreated mica surface followed by first blotting, then washing in an ammonium acetate solution, and finally air drying before scanning in AFM Second receipt) Heating the washed sample to 80?°C for 30-60 min in a vacuum oven before imaging since DNA molecules after heating is much more stable against scanning compared with air dried ones.
1511	Nanodissection of supercoiled plasmid DNA. A part of 100-150 nm in length consisting of 300-400 base pairs was excised (it was stated that it is a much shorter fragment of DNA than that of cut using a glass needle and a micromanipulator).
1529	The effects of adhesion and scanning force were examined on DNA and a linear fd phage. The width and height of DNAs in the images taken under various environmental conditions were carefully measured. Authors concluded that the AFM image resolution depended more on the adhesion than the force exerted by the tip on samples.
1532	Double stranded renovirus genome DNA of 10 nm or more in width
1534	Two DNA complexes: 1) Cytochrome c:DNA 2) DNA:polymerase	1) Cytochrome c was added to DNA in solution 2) The Klenow fragment of DNA polymerase I was added together with dNTP to start a random priming reaction
1536	Circular plasmid DNA strings of 8 nm in width	Incubating in aqueous DNA solutions at 35?°C.
1542	Binding of RNA polymrase on DNA as a transcription complex was studied. 681 bp fragments of DNA containing lPL promotor and E.coli RNA polymerase were used. DNA appeared bent (about 54?°) in an open promotor complex containing RNA polymerase and more severely bent in an elongation complex (average 92?°) where the enzyme completed RNA synthesis up to 15 nucleotides.
1543	DNA was imaged wider and higher (about twice) in aqueous environment than in propanol presumably because the AFM tip was imaging hydration layers around DNA, according to the authors.	DNA was extensively dehydrated by baking in vacuum before being transferred to an aqueous environment containing MgCl2.
1544	DNA was imaged with an expected length for B-DNA, its width being thinner at the two ends where it was anchored to the substrate and wider in the middle, which, the authors believed, allowed the freedom for DNA molecules to react with other molecules such as enzymes. The result in propanol was better in the sense that DNA was imaged thinner than in water and the substrate surface was clean. Inclusion of dithiothreitol (DTT) in DNA solution was necessary to disaggregate DNA.	DNA of a specific length was PCR amplified (see the paper for details). The amplified double helical DNA fragments were thiolated at both ends. An aqueous solution of thiolated DNA was applied on a gold-coated mica surface for 40-60 min and dried without blotting so that the number density of chemisorbed DNA could be increased. The dried sample was washed to remove free DNA and submerged in either an aqueous salt solution containing dithiothreitol (DTT) or in propanol for AFM imaging.
1545	High quality images of DNA with excellent resolution revealing a periodic corrugation along a DNA strand with repeats corresponding to the double helical structure of DNA are presented.
1546	Binding dynamics of the E.coli RNA polymerase complex to a short fragment of DNA of 1258 base pairs that contained lPR promotor was studied. A binding-unbinding-rebinding process of RNA polymerase complex on a single piece of DNA imaged at roughly every 70 s.	DNA in a buffer containing 1 mM MgCl2 was deposited on freshly cleaved ruby mica for 10 s and washed thoroughly with clean water. The sample was then dried under nitrogen and put in a desiccator. It was then placed in a liquid cell and scanned with a contact mode AFM
1547	A plasmid DNA was imaged with good reproducibility and its width was as small as 3 nm. Occasionally some reproducible bumps were observed along the double helical strand of DNA and they were tentatively interpreted as representing a feature associated with the double helical nature of DNA.	DNA was deposited onto mica surface from a solution containing MgCl2 and, after drying, immersed under either n-propanol, n-butanol or isopropanol.
1548	Contact mode imaging of DNA labeled with gold particle and attached to a mica surface were performed. The height of nominally 5 nm gold spheres was estimated as 5.8?±0.12 nm from the image and that of DNA was 0.54?±0.12 nm in air of relative humidity less than 10%.	DNA labeled with a spherical gold particle at one end and deposited on the mica surface was dried with nitrogen. It allowed a more stable imaging of DNA than with an unlabeled DNA since gold sphere at the end of DNA stuck to the mica substrate rather firmly.
1549	The location of the binding protein (Ferric uptake repression protein, Fur repressor) on DNA was studied by Electron Microscopy and AFM. No bend in the DNA structure were observed after Fur binding. The repressor protein bound on DNA had a shape like a peanut shell with two globular domains. The domain structure was clearer in the AFM image than in the dark field TEM image of a metal coated sample.
1550	A class of compounds with a capability of binding to DNA and straightening it were studied. The AFM images of kinetoplast DNA with and without distamycin or MGT-6b showed a distinct difference in the shape of the samples.
1551	A dynamic process of degradation of DNA in the presence of Dnase I, an enzyme that hydrolyzes phosphodiester bonds of DNA without requirement for specific base sequences was studied. After an AFM scan was started in a tapping mode for imaging of DNA, Dnase I was added to the DNA solution and progressive splitting and degradation of DNA were continuously monitored with repeated scans.	A double stranded DNA was adsorbed on a mica surface that was treated with NiCl2 prior to application of a solution containing DNA and the loosely adsorbed DNAs were washed off with high pressure water.
1552 1553	Z-DNA and G-wire DNA have been treated. Z-DNA was biochemically identified by using a specific antibody. The antibody was imaged as a globular protrusion at a fixed position in a linear DNA molecule. G-wire DNA is a member of the quadruple helical nucleic acid family and its structure was imaged clearly with AFM.	Z-DNA was inserted as a d(CG)11 fragment into pAN022 DNA plasmid.

     It is very important to find out the best substrate for imaging of biological objects since both tight and almost loose bonding to the substrate does not allow for the best scanning conditions. To a great extent it concerns DNA and RNA imaging. Apart from freshly cleaved mica a number of substrates or methods to anchor DNA or RNA to this substrates were proposed: mica surface pretreated with cadmium arachidate and coated with LB film [1507, 1508], mica surface modified with 3-aminophopyltriethoxy silane (APTES) [1532], mica surface treated with NiCl₂ [1551] and so on. It was shown, for example, that supercoiled structure of DNA can be imaged directly only using mica substrate treated with spermine but not the freshly cleaved mica or APTES-mica [384]. Mou at al. [523] found that cationic lipid bilayer is an excellent support for DNA anchoring due to relatively strong electrostatic interaction between DNA phosphate groups and positively charged lipid headgroups. Using AFM they received reproducible images of double stranded DNA helix and measured its period with high accuracy of 3,4?±0,4 nm which is in an excellent agreement with known pitch of the dsDNA. Refer to paper of P. Wagner [1024] for summary on immobilization techniques for SPM.

     One of the most interesting DNA related phenomenon is its supercoiling mentioned above. Supercoiling is the matter of investigation in many studies [384, 560, 1000, 1119, 1124]. Atomic force microscopy has always contributed significantly to the understanding of this phenomenon and has proved to be preferable among other powerful direct imaging methods such as electron microscopy mainly due to natural physiological conditions of DNA imaging.

     Stevenson et al. [1622] reported fabrication of DNA biosensor on the AFM microcantilever platform. In their study, gold-coated rectangular cantilevers (MikroMasch) were functionalized with double-stranded thiolated DNA and exposed to reaction buffer without and then with the small reducing agent DTT (dithiothreitol). Significant cantilever deflection occurred upon exposure to buffer containing DTT indicating either reaction of the DTT on the gold side (at interstitial locations) or exchange of immobilized DNA for DTT.

     Radiation is considered one of the main causes of cell inactivation due to damage of DNA. Till the last years the consequences of such damaging was studied with various methods but AFM. Recently AFM become a powerful tool in radiation biological research. Murakami at al [321] report on nanometer-level-structure analysis of DNA damage. Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single strand break) and linear form (DNA with a double strand break) were visualized after g-irradiation. Authors stressed that torsional feature of the plasmid DNA was imaged better with AFM than with a transmission electron microscope (TEM) and structural changes of DNA were discernible by AFM at nanoscale resolution. See also the latest paper on DNA and chromosome irradiation effect [708].

     The prospects seemed fantastic a decade ago now little by little are coming true. For example, researchers from IBM reported on the attempts to make AFM based sensor for identifying gene mutations [1589]. DNA strands immobilized on cantilever surface react in specific way dependent upon match or mismatch of the complementary strands of DNA in solution. The heat evolved upon match force cantilever to bend.

     Finally, let us simply encounter tasks have been successfully solved with AFM: high resolution imaging of DNA [523, 652, 1588] and synthesized nucleic acids [1557], investigation of DNA stiffness or elasticity [790, 996, 1586, 1587], segmental dynamics of DNA [1119], [1306], processes of gene transfection [354], recovering DNA from biological samples [684].

     It is hardly to attend to every paper in the huge amount of excellent works related to studying of nucleic acids with atomic force microscopy [1554-1575]. We believe such a remarkable interest will grow instantly in the future.