Detection of chain backfolding in simulation of DNA in nanofluidic channels Peter Cifra * and Tom a  s Bleha Received 16th May 2012, Accepted 5th July 2012 DOI: 10.1039/c2sm26128f The DNA extension in cylindrical nanochannels under moderately strong confinements in the ‘‘transition’’ region, where experiments are conducted, is examined by Monte Carlo simulations. The focal point is estimation of the amount of backfolding structures such as hairpins and loops in extended (linearized) DNA molecules. At the chain-ends of DNA an extensive folding (mainly as the J-type hairpins) is detected under all confinements, covering the Odijk and de Gennes regimes and the transition region between them. In contrast, in the DNA chain interior, the backfolding into Z-type hairpins is abundant in the de Gennes regime, significantly reduced in the transition region, and practically absent in the Odijk regime. The linear relationship between the DNA extension and its contour length is validated also in the transition region where explicit theories are absent. I. Introduction With recent nanotechnology advances single DNA molecules can be manipulated and analyzed in new ways. Stretching (linear- izing) of double-stranded (ds)DNA in nanofabricated channels has emerged as an innovative technique for fragment length analysis and genome mapping. 1–8 Confinement-induced stretch- ing of dsDNA is accomplished in fluidic channels of the dimen- sions much smaller than the radius of gyration R g of the DNA molecule in free solution. Combination of nanofluidic devices fabricated by chip lithography with fluorescence microscopy allows direct experimental visualization of single DNA molecules. It can be expected that in nanochannels the chain dimensions of DNA molecules such as the mean span R sp or the end-to-end distance R will depart significantly from their values in bulk. Confining and stretching of DNA molecules inside nanochannels have provided evidence that DNA molecules can be extended to a substantial fraction of their contour length L. 9–13 In experi- ments, especially DNA extension as a function of the channel width and ionic strength has been explored. Interpretation of experimental data on confined DNA molecules is assisted by the polymer physics theories and coarse-grained computer simula- tions. The extension of nanoconfined DNA molecules is described by two current theories of polymer confinement: by the Odijk theory 14,15 at strong confinement and by the scaling (blob) theory at moderate confinement. 16–18 The Odijk deflection theory 14,15 is based on the interplay of confinement and chain elasticity in narrow channels. The undulated chain extended along the channel axis can be viewed as a sequence of deflection segments of a characteristic length l (Fig. 1). In the scaling theory of de Gennes a moderately confined polymer is represented by a one-dimensional sequence of spherical blobs of the size of the channel diameter 16,17 (Fig. 1). It is assumed that within each blob the chain segments have the same structure as in the bulk solu- tion and the monomer density along the channel is uniform. The blob theory was generalized 17,18 for semiflexible polymers such as DNA on condition that the channel width D is much greater than the persistence length P of the polymer. Computer simulations using the coarse-grained mesoscopic models offer a powerful option to experimental and theoretical studies of confined DNA. Monte Carlo (MC) simulations based on a non-charged worm-like chain model have been employed to compute the chain extension R of DNA as a function of channel dimension D at high salt concentrations. 19,20 The chain extension profiles R(D) computed for a rectangular channel, tube and slit displayed three sectors, in a qualitative agreement with nano- fluidic measurements of DNA. Another simulation 21 using a slightly different model confirmed the existence of transitional regimes in between the Odijk and de Gennes (blob) limits pre- dicted earlier 22 by the scaling analysis. For the wormlike chain model the details of polymer behavior in the Odijk regime were determined 23 by a special simulation algorithm. Recently, simu- lations of nanoconfined DNA involving explicit electrostatic interactions between charged segments were reported. 8 The ionic strength dependence of DNA extension from simulations 8 showed a reasonable agreement with experimental findings and Odijk theory. Nanochannel-induced DNA elongation is now being devel- oped along the route to robust analytical devices for biological bioassays. In the ideal case of narrow nanochannels, of the diameter slightly larger than the diameter of DNA molecule, macromolecules would stretch up to their full contour length L. However, the very difficult loading of DNA into such narrow channels presents a major handicap for application in high- Polymer Institute, Slovak Academy of Sciences, 845 41 Bratislava, Slovakia. E-mail: cifra@savba.sk 9022 | Soft Matter , 2012, 8, 9022–9028 This journal is ª The Royal Society of Chemistry 2012 Dynamic Article Links C < Soft Matter Cite this: Soft Matter , 2012, 8, 9022 www.rsc.org/softmatter PAPER