INTRODUCTION
Biopolymers are macromolecules of biological origin, which include nucleic acids (DNA and RNA), proteins, peptides, and polysaccharides. Although these macro-molecules influence biological processes in different ways, most biological processes are associated to some extent with the physical properties of biopolymers (chain structure, flexibility, and excluded volume interactions). For example, the conformation of bacterial surface bio-polymers affects their adhesion to host tissue in the establishment of infection. In other biological processes such as protein synthesis, the specific structural units of the biopolymers (nucleic acids and proteins) control the biological function.[1]
Interest in analyzing the physical properties and structural features of biopolymers stems from the wide variety of functions they can perform in living systems of humans,[2] animals,[2,3] plants,[4] bacteria,[5] and fungi[6,7] or the important roles they play in industrial operations. The ultimate aim behind the characterization of bio-polymer properties is to provide a better understanding and control of their behavior in biological, medical, and/ or industrial processes. Examples of applications affected by biopolymer properties are environmental biore-mediation,’8-10-1 biomedical applications such as wound healing,’11-13-1 gene therapy,[14] growth mechanisms of macromolecular crystals,[15] food technology,[16,17] and bacterial adhesion.[18] OVERVIEW
A wide range of techniques and instruments have been used to characterize biopolymer properties. Examples include the use of vibrational circular dichroism (VCD) to investigate DNA condensation,1-19-1 fluorescence correlation spectroscopy (FCS) to study the diffusion properties, size, and conformation of native and denatured schizophyllan in dilute solutions,1-20-1 size exclusion chromatography (SEC) to characterize Pseudomonas putida KT2442 surface biopolymers,1-21-1 X-ray diffraction (XRD) to study the ordered conformation of gel-forming polysaccharides,1-22-1 transmission electron microscopy (TEM) to image well-characterized algal cellulose microfibrils,1-23-1 scanning electron microscopy (SEM) to study the extracellular matrix of nutrient-limited adherent bacterial populations,[24- Fourier transform infrared (FTIR) spectroscopy to study the effect of protein immobilization on birnessite,[25- and laser light scattering (LLS) to investigate the effect of pH on gelatin self-association in dilute solutions.1-26-1 Other techniques such as optical tweezers1-27-1 and the surface-force apparatus1-28-1 have also been used to measure the interactions between biopolymers and surfaces.
Because of the accelerating developments made in atomic force microscopy (AFM) as a surface characterization technique, AFM is now a preferred instrument in the study of biological macromolecules. Atomic force microscopy is characterized by high lateral (6 A) and vertical (1 A) resolutions'-29,30-1 and a high signal-to-noise ratio.1-31-1 These features give rise to the AFM's ability to image detailed structures of individual or groups of delicate biopolymers.'31- In contrast to conventional biological imaging methods such as SEM and TEM, AFM can be used to image biopolymers in their native state without the need for staining,'3- labeling,'31- or coating with a conducting gold layer.'8- In addition, AFM can be used to probe biopolymers in ambient air or liquid without the need to operate under vacuum.'3- In particular, the ability to image biopolymers in liquid with AFM allows for investigation of macromolecules under native conditions for several hours or even days without damage.'31,32- Although AFM can be used to image biopolymers in air, precautionary measures have to be taken to ensure that the sample is not damaged and that artifacts are not created. When samples are allowed to air-dry, there is the possibility of coagulation or rearrangement of the molecules on the substrate during the drying '33- process.' -
Interaction forces between biopolymers and surfaces should be measured in a liquid environment to minimize the presence of large capillary forces that are present in air 30 nN).[34] The high adhesive forces caused by capillary forces are destructive to many biological samples and mask other interaction forces of lesser magnitudes, such as van der Waals forces and electrostatic interactions.
Atomic force microscopy has evolved from an imaging technique to a versatile tool that also allows for investigation of molecular forces at interfaces with great detail. Atomic force microscopy can also be used to probe the chemical nature,[35] elasticity,1-36-1 roughness,[13] and surface charge[37] of biopolymers. We review the use of AFM as a state-of-the-art tool to characterize biopolymers. Examples will be provided on the use of AFM to characterize DNA, proteins, and polysaccharides.
PROBING DNA WITH ATOMIC FORCE MICROSCOPY
Earlier Efforts to Establish High-Resolution DNA Imaging
Since the invention of AFM, continuous improvements have been made in the ability to image and characterize deoxyribonucleic acid (DNA), which carries the genetic code of all living organisms. Most early attempts to image DNA were performed in air[38] because early attempts to image DNA in liquid (especially water) showed that if the molecules were not fixed properly, they could move during the imaging process. Their movement limited the ability to obtain reproducible results.[39] This challenge was overcome by stable binding of double-stranded DNA molecules to flat mica using chemical modification of the mica with 3-aminopropyltriethoxysilane. DNA molecules were bound to mica by this technique and molecules with contour lengths of 20-80 nm were stably imaged under repetitive scanning.[40] With techniques available at that time, the highest resolution achievable in DNA imaging was 2-3 nm. Because this was an order of magnitude less than the resolution required for DNA sequencing,[41] better substrate and sample preparation methods were required. Continuous and accelerated efforts were focused on reaching high-resolution AFM imaging of DNA. For example, Bensimon et al.[42] invented a technique for alignment of DNA on a silanated mica substrate, known as ”molecular combing.” With this preparation technique, DNA could be elongated and even minute quantities (103 molecules) could be imaged, which opened the way for faster physical mapping of the genome and increased detection abilities. Many variations of DNA immobilization based on coating of a surface with an aminosilane compound or a self-assembled monolayer can be found in the literature.[43-45]
Imaging of DNA Structure
Although there are numerous successful examples of high-resolution DNA imaging, we will focus on selected examples. Atomic force microscopy imaging has been widely used to discern the structure of DNA. Single- and double-stranded DNA were each imaged in propanol, butanol, and air. Measured molecular lengths were ~ 1 mm.[46] From imaging DNA on mica, single- and double-stranded DNA could be differentiated.1-47-1 The images of the double-stranded DNA showed an open circular shape without drastic contortions and a contour length within 7% of the calculated length. Single-stranded DNA was present as compact open circles with nodes or lumps almost uniformly distributed or as highly elongated circles with a few nodes, the latter being more common.[47]
Because of much effort, rapid characterization of the structure of DNA by tapping mode AFM imaging in ambient conditions has become a relatively routine technique.’48- Examples on the use of tapping mode AFM imaging to discern differences between linear double-stranded l-DNA, circular double-stranded plasmid DNA, and supercoiled double-stranded DNA plasmids with twists and writhes are shown in Fig. 1. DNA supercoiling has also been observed. The intramolecular triplex H-DNA formed by mirror-repeated purine-pyrim-idine repeats and stabilized by negative DNA supercoiling was imaged. These images showed that the H-DNA is a protrusion with a different thickness than the DNA duplex (Fig. 2).’49- The conformation of DNA can be also studied with AFM imaging. For example, pH was shown to affect DNA conformation because l-DNA could be denatured by HCl addition and renatured upon NaOH addition.’50-
Fig. 1 Top-view AFM images of different double-stranded (ds) DNA topologies on amino-terminated mica (vertical color scale = 3 nm) taken in AFM tapping mode under ambient conditions. (a) Linear l-DNA (with 48.5 kbp), (b) nontwisted circular DNA plasmids (vector with 3.2 kbp) and (c) circular supercoiled DNA with twists and writhes due to internal supercoiling (supercoiled DNA ladder 216 kbp). The measured width of dsDNA of all geometrical topologies was ~ 3-7 nm and affected by the tip geometry. The molecular height was ~ 1 nm.
Fig. 2 Atomic force microscopy images of DNA deposited at pH = 5.0. These images represent high-resolution images of H-DNA. The two images are schematics of the height and width.
Characterization of DNA Molecular Properties
Analyzing AFM images provides a wealth of information on the size and conformation of DNA. For example, DNA imaged under ambient conditions using near-contact mode showed width values up to four times smaller than values measured in noncontact mode, even with the same tip.’51-The discrepancy was attributed to the greater resolution achievable through near contact compared with noncon-tact mode imaging. Estimation of the length of double-stranded DNA molecules as short as 100-200 base pairs from AFM images in air was considered an advance in DNA characterization. The measurements gave lengths consistent with the known dimensions of A-DNA.’52- It was not possible to image shorter DNA molecules (25-50 base pairs) because intermolecular cross-bridging and base pairing in the molecules caused only globular forms to be viewed.’52-
In a revolutionary development, carbon nanotube AFM tips are starting to provide a new dimension in AFM imaging. These tips are ideal for AFM work because of their small diameter, high aspect ratio, large Young’s modulus, mechanical robustness, well-defined structure, and unique chemical properties.’53- With the use of carbon nanotube tips, high-resolution images of RecA-double stranded DNA complexes were obtained (Fig. 3). The images revealed the 10-nm pitch of RecA-double stranded DNA complexes and RecA filaments as three-dimensional surface topographical features, without reconstruction analysis. The depth of the notch between two pitches was <1 nm.[54]
Fig. 3 Topographic AFM images of RecA-dsDNA filaments observed with A) a CNT tip and B) with a standard tip (TESP-type tip).
Fig. 4 Magnetic alternating contact (MAC) mode AFM topographical images in air of the DNA biosensor surface prepared by 3 min of free adsorption onto HOPG from A) 10 mg/ml and B) 5 mg/ml dsDNA in phosphate buffer (pH 7.0, 0.1 M).
New imaging modes also helped improve DNA characterization. In a recent study, magnetic-mode AFM was used to characterize the process of DNA adsorption on a highly oriented pyrolytic graphite (HOPG) electrode surface. The images of single- and double-stranded DNA molecules showed that both types have the tendency to self-assemble from solution onto the HOPG surface (Figs. 4 and 5). The adsorbed film heights were dependent on the DNA concentration and were held to the surface with noncovalent interactions such as hydrogen bonding, base stacking, and electrostatic, van der Waals, and hydrophobic interactions.[55]
Stretching DNA to Study Biomechanical Properties
The biomechanical properties of DNA can be obtained through stretching these molecules in a technique known as single molecule force spectroscopy (SMFS). DNA molecules are tethered to a surface at one end and stretched through application of an external force, which may be magnetic,[56] caused by hydrodynamic flow,[56] or result from the AFM cantilever stiffness[57] or an electrical field.[58] Typically, one DNA molecule is picked up from an adsorbed layer of DNA molecules by the AFM tip because of an applied contact force of several nanoNew-tons. Upon retraction of the tip from the layer, the DNA strand is stretched. The resulting force from this stretching is measured as cantilever deflection, which can be converted to force by accounting for the spring constant of the cantilever. Several methods exist for determining these spring constants, as reviewed in Ref. [59]. Stretching experiments provide important information about the mechanical properties of DNA. We will discuss three examples: probing DNA elasticity, quantifying interactions between complementary strands of DNA, and DNA sequencing.
Elasticity of DNA
The elasticity of single molecules can be estimated directly from force-extension measurements by applying random-walk statistical mechanical-based models. The most frequently used models are the freely jointed chain (FJC), extensible freely jointed chain (FJC+), and wormlike chain (WLC) models.[36] The elasticity of DNA molecules was first estimated by applying the FJC model to force-extension data between single DNA molecules (chemically attached by one end to a glass surface and by the other end to a magnetic bead) and an AFM tip under three different salt concentrations.[56] The FJC model failed to explain the force-extension data because of the fact that it does not account for the extensibility of the molecules. It appeared that the DNA molecules could deform when exposed to stress. The authors discounted the WLC model because they speculated that the latter model would also fail due to the inability to account for the extensibility of DNA.
However, in some cases, the WLC model was appropriate for explaining DNA’s mechanical properties. For example, the elasticity of l-phage DNA was explained well with the WLC model, although the FJC model showed a large deviation from the experimental data.[58] The WLC model provided a contour length of 32.8±0.1 mm and a persistence length of 53.4±2.3 nm.[58] An additional difference between the FJC and WLC models is that the FJC model accounts for entropic effects only, while the WLC model also accounts for enthalpic interactions.
Fig. 5 Magnetic alternating contact mode AFM topographical images in air of the DNA biosensor surface prepared by 3 min of free adsorption onto HOPG from a 5 mg/ml single-stranded DNA in phosphate buffer (pH 7.0, 0.1 M).
Fig. 6 The mechanical compliance of DNA strongly depends on the specific base paring in the double helix. a) For double-stranded poly(dG-dC) DNA, there was a transition from B-DNA to a new overstretched form (S-DNA) that occurred at 65 pN (see arrows), similar to the transition observed in l-DNA. The melting transition occurred at 300 pN for this DNA, compared to 150 pN in l-DNA. b) In duplex poly(dA-dT) DNA, the force of the B to S transition is reduced to 35 pN and the strands melt during this transition, so that no separate melting transition can be observed.
In another example, the elasticity of a single super-coiled DNA molecule was probed via SMFS. Sharp transitions were observed in the elasticity of the molecules at ~ 0.45 and ~ 3 pN for underwound and overwound molecules, respectively. These transitions were attributed to the possibility of the formation of alternative DNA superstructures or because of DNA transcription and replication.’60-
Interaction Forces Between Complementary Strands of DNA
Understanding the intermolecular forces within the DNA double helix is important to control the behavior of DNA in various applications, such as DNA sequencing. In the first effort to measure the forces between single DNA strands, DNA oligonucleotides were covalently attached to a spherical probe and to a silica surface.’57- Force measurements between these strands showed three distinct force regimes, centered at 1.52, 1.11, and 0.83 nN. The forces were directly associated with the rupture of the interaction between a single pair of molecules involving 20, 16, and 12 base pairs, respectively. This study demonstrated the importance of AFM in detecting the presence of, and relative positions of, specific base sequences with angstrom resolution.’57-
In a study on double-stranded l-phage DNA, the split of the molecule into single strands was observed via force microscopy.’61- Stretching experiments revealed a transition in the force-extension measurement at 65 pN attributed to the conversion of B-DNA to a new overstretched conformation called S-DNA. This transition was followed by a nonequilibrium melting transition at 150 pN (Fig. 6). The melting transition is the part of the curve at which the double-stranded DNA split into single strands that fully recombined upon relaxation.’61-
DNA Sequencing
Because of the continuous increase in the resolution of AFM to the angstrom level, sequencing of DNA became possible. The principle behind this application is that the force-extension curve that arises when DNA is stretched is sequence-dependent. In one of the first studies to address DNA sequencing, a comparison was made between the force measurements on poly(dG-dC) and poly(dA-dT), where ”d” represents the deoxynucleotide.’61- With knowledge of the melting transition for l-phage DNA,’62-single strands of poly(dG-dC) and poly(dA-dT) were prepared. Upon relaxation, these strands reannealed into hairpin structures as a result of their self-complementary sequences.’61- Studying the unzipping of these hairpins with AFM directly revealed the base pair unbinding forces for G-C and for A-T, which were 20±3 and 9±3 pN, respectively. In another study of DNA unzipping, the force needed to open the double strands upon tip retraction was measured as a function of extension of the DNA molecule (Fig. 7). The required force was between 10 and 20 pN on a length scale of 10 bases. The force profiles were characteristic for each specific DNA sequence.’63-
Fig. 7 Complementary DNA oligonucleotides are chemically attached to an AFM tip and a glass surface. As the tip is brought in contact with the surface, a double strand forms, which can subsequently be unfolded upon retraction of the tip.
PROBING PROTEINS WITH ATOMIC FORCE MICROSCOPY
High-Resolution Imaging of Proteins
High-resolution protein imaging with AFM is a well-established technique. Proteins are often bound to mica for imaging because their tight binding facilitates imaging.’64-Alternately, proteins are covalently bound to chemically modified surfaces.’65- Examples of AFM imaging studies on proteins are too numerous and diverse to be contained in this review. We focus on two illustrative examples: 1) imaging of ligand-receptor interactions between cholera toxin B-oligomers bound to bilayers of biologically relevant lipids’66- and 2) imaging microtubules, protein structures of eukaryotic cells.’67-
In the first example, the interactions between cholera toxin B-oligomers (CTX-B) and a dipalmitoylphosphati-dylcholine (DPPC) bilayer containing 10 mol% GM1 (the membrane receptor for CTX-B) were characterized. Images taken before and after the CTX-B oligomers were added to the lipid bilayer confirmed the high binding affinity of CTX-B to the receptor GM1 (Fig. 8). The high quality of the images revealed the ability of AFM to image membrane proteins without the need for cross-linking.
As a second example, a comparison was made between AFM, scanning tunneling microscopy (STM), and trans-mission electron microscopy (TEM) for their abilities to image microtubules isolated from pig brains. Atomic force microscopy was the easiest to use and most reproducible imaging technique.’67- The AFM images revealed the linear structure of the microtubules (Fig. 9) and the possibility of crossing of various tubules.
Fig. 8 A) A typical AFM image of a DPPC bilayer with 10 mol% GM1 before CTX-B was added. B) The CTX-B bound to the ganglioside in the bilayer is clearly seen. The coverage is complete indicating that the distribution of the ganglioside is uniform.
Fig. 9 Atomic force microscopy top-view image of chemically immobilized microtubules (MTs) on silicon imaged under buffer solution (MES buffer with 0.7 M glycerol). Protein concentration was 2.0 mg/ml at the start of polymerization. Microtubules fixed with 5% glutaraldehyde before immobilization and imaged without damage under liquid, demonstrating an improved resistance to tip pressure.
The capabilities of AFM imaging of proteins were extended to many other interesting applications such as capturing the conformational changes of proteins under physiological conditions’68- and visualization of DNA-protein complexes.’69-
