Amplifying the Macromolecular Crowding Effect Using Nanoparticles

Abstract

The melting temperature (T m ) of DNA is affected not only by salt but also by the presence of high molecular weight (MW) solutes, such as polyethylene glycol (PEG), acting as a crowding agent. For short DNAs in a solution of low MW PEGs, however, the change of excluded volume upon melting is very small, leading to no increase in T m . We demonstrate herein that by attaching 12-mer DNAs to gold nanoparticles, the excluded volume change was significantly increased upon melting, leading to increased T m even with PEG 200. Larger AuNPs, higher MW PEGs and higher PEG concentrations show even larger effects in stabilizing the DNA. This study reveals a unique and fundamental feature at nanoscale due to geometric effects. It also suggests that weak interactions can be stabilized by a combination of polyvalent binding and the enhanced macromolecular crowding effect using nanoparticles.Macromolecules such as proteins, RNA, and DNA occupy 20-40% of a live cell's volume. 1 Due to their mutually impenetrable nature, the excluded volume occupied by each macromolecule is larger than its geometric volume, making the addition of new polymers into this crowded system thermodynamically disfavored. This fundamental biophysical effect has a strong influence on many biochemical reactions including protein folding, oligomerization, and DNA hybridization and melting. 2 Much attention has been given to the process of DNA melting, as this reaction plays an important role in biology and biotechnology. [3][4][5][6][7][8] At the same time, DNA length and sequence can be easily varied to allow systematic studies.Upon melting, the excluded volume of a DNA is increased. Therefore, melting is usually disfavored in macromolecularly crowded environments to give rise to more stable DNA duplex and increased melting temperature (T m ). Polyethylene glycol (PEG) is the most commonly used crowding agent. PEG has been shown to both stabilize and destabilize DNA, depending on PEG molecular weight (MW), DNA length and salt concentration. For example, with 8 to 17 base pair (bp) DNAs in the presence of 1 M NaCl, only destabilization (reduced T m ) was observed even with 20% PEG 8000. 7 By reducing the salt to 100 mM, stabilization of a 20 bp DNA was observed with 15% PEG 6000. 5 On the other hand, with ~200 bp DNAs, an increase in T m was achieved when the PEG MW was greater than 400-1000. 4 This is because under otherwise identical conditions, longer DNAs produce larger excluded volume increase upon melting. The fact that decreased T m was also sometimes observed suggests that PEGs play a dual role on DNA stability. In addition to the excluded volume effect for stabilization, PEGs also reduce the water activity and solvate DNA bases leading to decreased T m . 4,5,7 In this paper, macromolecular crowding (MC) describes solely the excluded volume effect while the chemical interaction between PEG and DNA is called the destabilizing chemical effect. Efforts to dissect these two effects have been recently carried out. In general, small PEGs tend to...

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