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New Progress in the Investigation of Macromolecular Interactions

The application of Atomic Force Microscopy (AFM)-based Single-molecule Force Spectroscopy (SMFS) in the investigation of polymer structure and macromolecular interactions is one of the most important subjects in polymer science. Great progress has been made on the study of nanomechanical properties of single macromolecules in solutions (Wenke Zhang, Xi Zhang Prog. Polym. Sci., 2003, 1271-1295).

More recently, supported by the National Natural Science Foundation of China (NSFC), Prof. Wenke Zhang from Jilin University and his collaborators have further extended AFM-based SMFS to the investigation of inter-macromolecular interactions in more complicated systems, namely, polymer single crystal and native plant virus, respectively.

A good understanding of the mechanism of nucleic acid-protein interactions will help us gain the control in many important biological processes, such as virus infection and cancer cell growth. Furthermore, the investigation of nucleic acid-protein interactions in real biological system will provide more accurate information and direct guidance in practice. Due to the limitation of detection method, such study was quite difficult to carry out. As a proof-of-concept study, Wenke Zhang together with his collaborators from Changchun Institute of Applied Chemistry, CAS (Prof. Zhaohui Su) and Department of Chemistry, University of South Carolina, US (Prof. Qian Wang) have chosen tobacco mosaic virus (TMV) as a model system to study the interactions between genetic RNA and its coat proteins. By subtle experimental design/control, TMV particles were immobilized perpendicularly on a solid gold substrate leaving their 5’ openings (especially the 5’ end of RNA) exposed for picking up by the AFM tip. The genetic RNA was pulled step-by-step out of the TMV particle by the AFM tip via physical adsorption, and unbinding forces between RNA and protein coat have been measured directly at the single molecule level (see Figure 1).

Figure 1. Single-molecule force spectroscopy study of RNA-protein interactions in TMV

It has been found that the unbinding forces  increases with the increase of stretching speed, and decreases with the increase of pH. In addition, the detached RNA can find its way back to the protein coat with the help of intact RNA-protein complexes during relaxation. In doing this, they have extended AFM-based SMFS to study protein-nucleic acid interactions in more complicated biological system, and the established method may be a gateway toward investigations of the mechanism of virus infection. The relevant results have been published in J. Am. Chem. Soc. 2010, 132, 11036-11038.

The mechanical properties of polymer materials are governed by their chain composition/structure as well as the polymer chain interactions. However, due to the complexity of the system, the single-molecule investigation of inter-macromolecular interactions in their condensed states was difficult to realize. Zhang and his collaborator Prof. Xi Zhang from Tsinghua University, China, have made the first successful attempt in the investigation of macromolecular interactions in a polymer single crystal by a good combination of AFM imaging and SMFS. They used a polyethylene oxide (PEO) with a sulfur-containing end group as a model polymer system. PEO single crystals were prepared from their dilute solution by using self-seeding method. Gold nanoparticles (GNPs) were then used to label the thoil-terminus of the PEO chain by gold-thiol chemistry. AFM imaging was firstly used to locate the specific GNP, then thiol-group functionalized AFM tip was brought to interact with the GNP forming the bridge structure. During the separation of AFM tip with the sample, the specific PEO chain was extracted from its single crystal and the interaction strength between folded polymer fragments was measured quantitatively, as shown in Figure 2. This study extends the usefulness of AFM-based SMFS to the investigations of polymer interactions in their condensed states (e.g., in polymer single crystal). The method established here can be used to study crystallization of various polymers at the single-molecule level. The above result has been published in J. Am. Chem. Soc. 2011, 133, 3226-3229. This work has been highlighted by Nature Materials titled “Getting a grip” (http://www.nature.com/nmat/journal/v10/n5/full/nmat3022.html) after being published. In addition, NPG Asia Materials also highlighted this research with a title of “Polymer science: Investigating interactions” (http://www.natureasia.com/asia-materials/highlight.php?id=889).

Figure 2. AFM-based SMFS study of inter-macromolecular interactions in a polymer single crystal

The above mentioned work has been funded by the NSFC (Grant No. 20844003, 20834003, 20974039), NSFC-DFG joint grant (TRR61) and NSFC International Cooperation and Exchange Program (20640420622).

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