Many reports have witnessed the potential of the atomic force microscope as a force sensor for probing the forces that hold molecules together. Accurate molecular design allowed us, amongst others, to study the unbinding forces between ligands and receptors (Valle et al. 2008) or to study protein unfolding (Samorì et al. 2005; Sandal et al. 2008) and to characterize on the single-molecule level the different protein conformations present in a populations. While there are many examples of force studies on proteins, the availability of data for pulling nucleic acids is more limited and it includes primarily optical tweezers studies. For example, processes such as the (over )stretching of DNA or the unfolding of RNA hairpins were characterized. We are interested in extending the possibilities for the characterization of the behaviour of nucleic-acids nanostructures under force and to employ the atomic force microscope (as it can study higher forces and apply them faster). Towards this goal, we are currently trying to upgrade our force-sensing instrumentation and to design and realize an appropriate molecular design that could be helpful in the collection of force data and in its interpretation. As previously shown by Professor Julio Fernandez, an AFM force spectroscopy apparatus can be used in a force-clamp mode, where the tensile force stretching a molecule is held constant at a pre-set value and the molecule extension is measured over time. This operational mode can yield interesting molecular information that are complementary or supplementary to those obtained with the more traditional AFM pulling mode (pulling at a constant velocity and measuring the force). In order to operate an AFM in this fashion, though, it is necessary to implement a fast force control process and to use a very fast piezoelectric actuator. In the context of a collaborative project, we built a custom-made force-clamp AFM machine based on the fastest available piezoelectric scanner. The instrument control and data acquisition is obtained through a real-time Linux application that was developed locally. The performance of this instrument has been already tested by performing protein pulling experiments on well-characterized specimens. Force-spectroscopy experiments can be significantly simplified if the molecular bridge under tension has certain known features. First, there must be a molecular spacer, so that the interesting bond or structure is put under tension when the AFM probe is far from the substrate surface, thus avoiding non-specific forces. Secondly, it is useful to design specimens to be oligomeric in their structure, as the repetition of the same force-induced event many times in a single pulling run has distinctive advantages, as shown in the pulling of multi-modular proteins or in similarly designed systems (Valle et al. 2008). According to this strategy, we are preparing a nucleic acid architecture designed for putting under tension a series of nanostructures and thus to study their breakdown under the application of forces (in the standard pulling mode and in the more novel force-clamp mode)

Probing intra- and intermolecular forces on single molecules with the AFM: towards the measurement of forces holding nucleic acids nanostructures together

ZUCCHERI, GIAMPAOLO;PASSERI, ROSITA;BRUCALE, MARCO;SAMORI', BRUNO
2008

Abstract

Many reports have witnessed the potential of the atomic force microscope as a force sensor for probing the forces that hold molecules together. Accurate molecular design allowed us, amongst others, to study the unbinding forces between ligands and receptors (Valle et al. 2008) or to study protein unfolding (Samorì et al. 2005; Sandal et al. 2008) and to characterize on the single-molecule level the different protein conformations present in a populations. While there are many examples of force studies on proteins, the availability of data for pulling nucleic acids is more limited and it includes primarily optical tweezers studies. For example, processes such as the (over )stretching of DNA or the unfolding of RNA hairpins were characterized. We are interested in extending the possibilities for the characterization of the behaviour of nucleic-acids nanostructures under force and to employ the atomic force microscope (as it can study higher forces and apply them faster). Towards this goal, we are currently trying to upgrade our force-sensing instrumentation and to design and realize an appropriate molecular design that could be helpful in the collection of force data and in its interpretation. As previously shown by Professor Julio Fernandez, an AFM force spectroscopy apparatus can be used in a force-clamp mode, where the tensile force stretching a molecule is held constant at a pre-set value and the molecule extension is measured over time. This operational mode can yield interesting molecular information that are complementary or supplementary to those obtained with the more traditional AFM pulling mode (pulling at a constant velocity and measuring the force). In order to operate an AFM in this fashion, though, it is necessary to implement a fast force control process and to use a very fast piezoelectric actuator. In the context of a collaborative project, we built a custom-made force-clamp AFM machine based on the fastest available piezoelectric scanner. The instrument control and data acquisition is obtained through a real-time Linux application that was developed locally. The performance of this instrument has been already tested by performing protein pulling experiments on well-characterized specimens. Force-spectroscopy experiments can be significantly simplified if the molecular bridge under tension has certain known features. First, there must be a molecular spacer, so that the interesting bond or structure is put under tension when the AFM probe is far from the substrate surface, thus avoiding non-specific forces. Secondly, it is useful to design specimens to be oligomeric in their structure, as the repetition of the same force-induced event many times in a single pulling run has distinctive advantages, as shown in the pulling of multi-modular proteins or in similarly designed systems (Valle et al. 2008). According to this strategy, we are preparing a nucleic acid architecture designed for putting under tension a series of nanostructures and thus to study their breakdown under the application of forces (in the standard pulling mode and in the more novel force-clamp mode)
DNA-based nanodevices
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G. Zuccheri; R. Passeri; M. Brucale; B. Samorì
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/74346
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