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DNA Folding In ‘Crowded’ Conditions

August 8, 2011 | A version of this story appeared in Volume 89, Issue 32

We read with great interest, but then with consternation, the article “DNA Folding in Cell-like Setting,” by Stu Borman (C&EN, June 13, page 10). The story misconstrues the phenomenon of “crowding” and greatly exaggerates the actual physiological relevance of the structure obtained by Anh Tuân Phan’s laboratory.

“Macromolecular crowding” has a precise definition and underlying physical chemical basis. “Crowding” results from the addition of large, inert solute molecules that exclude accessible volume from reactants and products and therefore must favor the most compact state (or least hydrodynamic volume). Because the parallel “propeller” quadruplex structure determined by Phan is hydrodynamically bigger than other competing quadruplex forms (“basket,” “hybrid 3+1”), it cannot be favored by “crowding.”

What Phan and coworkers in fact showed is that formation of the “propeller” structure is driven by dehydration; that is, by the addition of small-molecule osmolytes that diminish water activity. We showed the same thing in a recent paper that was not cited (J. Am. Chem. Soc., DOI: 10.1021/ja105259m). Notably, we discussed the conditions for crowding and also showed that a true inert “crowding” agent (serum albumin) did not promote the hybrid to propeller quadruplex transition. A more recent publication from Robert Hänsel and coworkers (Nucleic Acids Res., DOI: 10.1093/nar/gkr174) confirmed that observation and, importantly, showed that crowding by a cell extract or by Ficoll did not favor formation of propeller structures.

What Phan and coworkers used is more properly called “osmotic stress,” in which diminished water activity pushes the reaction equilibrium to the least hydrated form, as actually demonstrated in the paper in question. The danger lies in extrapolating the results in solution to the “cell-like setting.” Cells are indeed “crowded” but are not very dehydrated in a thermodynamic sense. So the syllogism: “Crowding favors the propeller form. Cells are crowded. Therefore, the propeller form is the relevant G-quadruplex structure in a solution like that in cells” is logically unfounded. Phan and coworkers have not demonstrated “crowding” but rather have studied the effects of dehydration and/or differential binding of cosolute (such as PEG 200). The interior of cells may be “crowded,” but it does not resemble a 50% solution of alcohols or ethylene glycols.

By Jonathan B. Chaires, M. Clarke Miller, Robert Buscaglia, Robert D. Gray, Andrew N. Lane, John O. Trent
University of Louisville

We read Borman’s News of the Week article with mixed feelings. The article extrapolates the results in 40% solutions of polyethylene glycol and ethanol to the “cell-like setting,” whereas the recent structural evidence clearly demonstrates that the parallel “propeller” quadruplex structure, as observed by Phan in polyethylene solution and Stephen Neidle in crystalline state, is not the preferred conformation under physiological conditions either in vivo (J. Am. Chem. Soc., DOI: 10.1021/ja9052027) or in cellular extracts (Nucleic Acids Res., DOI: 10.1093/nar/gkr174).

Formation of the propeller structure under conditions used by Brahim Heddi and Phan is connected with low water activity diminished by the application of molar concentrations of small osmolytes. As recently shown by M. Clarke Miller and coworkers and Hänsel and coworkers, the conditions used by Heddi and Phan have little relation to true molecular crowding inside cells, as mostly larger biomolecules such as proteins and nucleic acids are known to be the relevant crowders (Gary J. Pielak and Andrew C. Miklos, Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1013095107).

The real danger lies in calling low-water-activity conditions such as solutions of PEG or the crystalline state to be “cell-like,” because so far all available structural data on non-B-DNA motifs, including G-DNA, inside cells show that DNA adopts different conformations in the cell or in cellular extract compared with structures observed in the crystalline state and in polyethylene solutions.

By Volker Dötsch, Lukáš Trantírek
University of Louisville

Heddi and Phan respond:

We have shown (J. Am. Chem. Soc., DOI: 10.1021/ja200786q) that four different G-quadruplex conformations of human telomeric DNA can be converted to a propeller-type parallel-stranded G-quadruplex in K+-containing crowded solution owing to water depletion.

We emphasize that our results agree with those of other groups, including Daisuke Miyoshi (J. Am. Chem. Soc., DOI: 10.1021/ja061267m), Michaela Vorlíčková and coworkers (Biopolymers, DOI: 10.1002/bip.20672), Miller et al., and Hänsel et al. in the notion that water content is the main cause of the observed structural transition.

In their letters, Chaires et al. and Dötsch et al. question the extrapolation of our results to a “cell-like setting.” They argue against the use of small cosolutes such as PEG 200 and ethanol but totally neglect that our data have also shown the structural transition induced by cosolutes of various types and sizes, including the polysaccharide Ficoll 400. Furthermore, additive effects have been observed for mixtures of different cosolutes. These indicate the predominant role of water depletion rather than specific cosolute-DNA interactions on this conformational transition, a possibility raised by Chaires et al. in their letter.

In our opinion, the crowding condition in cells may have two main effects on DNA: (1) steric repulsion between DNA and other macromolecules and (2) partial volume exclusion of water accessibility to DNA surface. The crowding conditions induced by various cosolutes can simulate at least the latter effect, and this drives the equilibrium of telomeric G-quadruplexes toward the parallel form. In cells, the water volume content next to DNA is clearly reduced due to molecular crowding, although the exact number might depend on the cell type. The population of the parallel G-quadruplex and other forms at the telomeres (if any) should depend on the local water content next to DNA, as well as other factors such as their folding kinetics.

By Anh Tuân PhanBrahim Heddina
Nanyang Technological U, Singapore



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