title: AFM volumetric methods for the characterization of proteins and nucleic acids.
abstract: The atomic force microscope overestimates lateral dimensions and underestimates heights of nanometer size objects such as proteins and nucleic acids. This has made researchers cautious of AFM measurements, even though there is no other technique capable of measuring topography with sub-nanometer precision. Nevertheless, several approaches for determining the stoichiometry of protein and protein-DNA complexes have been developed which show that, although the absolute values may be incorrect, the AFM volume is essentially proportional to the mass. This has allowed the determination of the mass of protein complexes with the help of a calibration curve. Here we review the main techniques for AFM volume measurements and detail a methodology that significantly reduces the associated errors. This method uses a fragment of DNA as a fiducial marker by which the volume of a protein is normalized. The use of fiducial markers co-adsorbed together with the protein of interest minimizes the contribution of tip-induced artifacts as they affect both the object of interest and the marker. Finally, we apply this method to the measurement of the length of single-stranded DNA. A linear relationship between length and volume was obtained, opening the door to studies of ssDNA intermediates formed during complex DNA transactions such as replication, recombination and repair.
authors: Centro Nacional de Biotecnología, CSIC, Campus UAM, Darwin 3, 28049 Cantoblanco, Madrid, Spain.
authors: Maria Eugenia
authors: ME
authors: Fuentes-Perez
authors: Mark S
authors: MS
authors: Dillingham
authors: Fernando
authors: F
authors: Moreno-Herrero
mesh_terms: D000465:Algorithms; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000648:ultrastructure*; D003627:Data Interpretation, Statistical; D057918:Fiducial Markers; D018625:Microscopy, Atomic Force / Q000592:standards*; D009690:Nucleic Acid Conformation; D010316:Particle Size; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000648:ultrastructure*
chemical_list: D004277:DNA, Single-Stranded; D011506:Proteins