The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics. DOI: 10.1103/PhysRevLett.94.196101 PACS numbers: 81.16.Nd, 82.35.Lr, 82.39.Pj Top-down approaches to nanotechnology have the potential to revolutionize biology by making possible the construction of chip-based devices that can not only detect and separate single DNA molecules by size [1-4] but also-it is hoped in the future-actually sequence at the single molecule level [5]. While a number of top-down approaches have been proposed, all these approaches have in common the confinement of DNA to nanometer scales, typically 5-200 nm. Confinement alters the statistical mechanical properties of DNA. A DNA molecule in a nanochannel will extend along the channel axis to a substantial fraction of its full contour length [1,6]. Moreover, confinement is expected to alter the Brownian dynamics of the confined molecule [1]. While the study of confined DNA is interesting from a physics perspective, it is also critical for device design, potentially leading to new applications of nanoconfinement (for example, the use of nanochannels to prestretch and stabilize DNA before threading through a nanopore [5]). Moreover, available models [7][8][9][10][11] and simulations [12,13] are unable to account for the effect of varying confinement over the entire range of scales used in nanodevices. The theory gives asymptotic results valid only in limits that are not necessarily compatible with device requirements [1].Consider a DNA molecule of contour length L, width w, and persistence length P confined to a nanochannel of width D with D less than the radius of gyration of the molecule. When D P, the molecule is free to coil in the nanochannel and the elongation is due entirely to excluded volume interactions between segments of the polymer greatly separated in position along the backbone (see Fig. 1). de Gennes developed a scaling argument for the average extension of a confined self-avoiding polymer [8,12] which was later generalized by Schaefer and Pincus to the case of a persistent self-avoiding polymer [14]. The de Gennes theory predicts an extension r that scales with D in the following way:If the aspect ratio of the channel is not unity, i.e., the width D D 1 does not equal the depth D 2 , then Eq. (1) is still valid provided that D is replaced by the geometric average of the dimensions. As the channel width drops below the persistence length, the physics is dominated not by excluded volume but by the interplay of confinement and intrinsic DNA elasticity. In the strong confinement limit D P, backfolding is energetically unfavorable and contour length is stored exclusively in deflections made by the polymer with the walls. These deflections occur on average over th...