Rotation-Induced Macromolecular Spooling of DNA

Genetic information is stored in a linear sequence of base pairs; however, thermal fluctuations and complex DNA conformations such as folds and loops make it challenging to order genomic material for in vitro analysis. In this work, we discover that rotation-induced macromolecular spooling of DNA around a rotating microwire can monotonically order genomic bases, overcoming this challenge. We use single-molecule fluorescence microscopy to directly visualize long DNA strands deforming and elongating in shear flow near a rotating microwire, in agreement with numerical simulations. While untethered DNA is observed to elongate substantially, in agreement with our theory and numerical simulations, strong extension of DNA becomes possible by introducing tethering. For the case of tethered polymers, we show that increasing the rotation rate can deterministically spool a substantial portion of the chain into a fully stretched, single-file conformation. When applied to DNA, the fraction of genetic information sequentially ordered on the microwire surface will increase with the contour length, despite the increased entropy. This ability to handle long strands of DNA is in contrast to modern DNA sample preparation technologies for sequencing and mapping, which are typically restricted to comparatively short strands, resulting in challenges in reconstructing the genome. Thus, in addition to discovering new rotation-induced macromolecular dynamics, this work inspires new approaches to handling genomic-length DNA strands.

Popular Summary

Genetic information is encoded in a linear sequence of bases in threadlike DNA, but that thread can become tangled like a long strand of yarn. Previous attempts to organize DNA have amounted to threading it through the eye of a needle, which is not how one would unravel yarn. Instead, one would wrap it around a spool. While relatively short strands of DNA may be fully stretched in linear geometries using previous approaches, fully ordering genomic-length DNA has been impossible at micrometer scales. Here, we discover the nanoscale equivalent to spooling yarn, which can be used to deterministically unravel a significant portion of DNA around a rotating micrometer-sized cylinder. We refer to this effect as “rotation-induced macromolecular spooling.”

With a $50\text{−}\mu \mathrm{m}$-diameter tungsten wire, this single-molecule manipulation technique explicitly exploits disordering thermal fluctuations through hydrodynamic drag; it is independent of the chemistry of the DNA. We show that long segments of DNA can be stretched over the curved surface of the rotating microwire. Because rotation-induced macromolecular spooling relies on thermal fluctuations, the fraction of genetic information sequentially ordered on the surface of the rotating microwire increases with DNA length despite the increased entropy. We show that untethered DNA elongates substantially, but strong extension of DNA only becomes possible by tethering one end of it. With tethered DNA, we observe previously unseen conformational states of deformed DNA. In one of these states, a substantial portion of the chain can be deterministically spooled into a fully stretched, single-file, sequentially ordered, curvilinear progression of base pairs.

We expect that our findings will enable better inventories of genomic material.

Figure 1

(a) The untethered DNA system: The rotating cylinder of radius $a$ generates a flow profile $v_{\varphi }(r)$, which shears the DNA and deforms it. A model for this is a procession of identical blobs of size $\xi$. (b) Simulation snapshot portraying a typical steady-state conformation of an untethered DNA chain. (c) The tethered system with the French-horn DNA conformation: The blob size $\xi$ is a function of angle $\varphi$ from the untethered end. For sufficiently large Weissenberg numbers Wi, a portion of the DNA is fully stretched into a stem section, while the rest is in a horn conformation. For smaller Wi, there is no stem and the polymer is in the shofar conformation. (d) Simulation snapshots portraying typical steady-state tethered conformations. Increasing Wi changes the state from relaxed, to shofar, to French-horn conformations. Sufficiently long, rapidly rotated chains wrap around the circumference forming a rim of strongly stretched DNA.

Figure 2

The apparatus for untethered strands. (a) Schematic representation of the apparatus, described in detail in the text. (b) Sample image of a $50\text{−}\mu \mathrm{m}$-diameter tungsten wire (dark horizontal line) rotating in a dilute solution of T4-phage DNA (166 kbp). The contour length of YOYO-1 stained T4-DNA is $(65\pm 2)\mu \mathrm{m}$. Strands of DNA are drawn towards the wire and elongated by the shear flow. (c) Image of the microwire 1s after it has stopped rotating: DNA strands have begun to relax to more entropic states. (d) Conformational relaxation of a single DNA strand in the period of time immediately after the microwire has stopped rotating (see Movie 1 in Ref. [16]). (e) Quantitative measurements of the extension of a single DNA strand during relaxation, along with the fit and associated studentized residuals, used to determine the fully extended length of the polymer ($t=0$).

Figure 3

The apparatus used to collect data on the rotation-induced macromolecular spooling of DNA. The microwire is situated directly above the cover slip using the $z$ stages of the two motor mounts, and the tension stage is adjusted so that the microwire is taut. Solution containing DNA is dropped onto the cover slip such that a portion of the wire is submerged. The motors are driven simultaneously by an Arduino microcontroller.

Figure 4

Curvilinear end-to-end extension of untethered T4-DNA (166 kbp) and DPD polymers for varying rotation rates. The model-dependent shift between the high Weissenberg-number simulations and experiments is removed by setting the $\mathrm{Wi}=10$ value to the fitted strong extension value. Dotted lines show the predicted scaling for weak extensions at low Wi [Eq. (2); $L\sim \mathrm{Wi}$]. Moderate extensions at intermediate Wi are shown as dash-dotted lines [Eq. (4); $L\sim {\mathrm{Wi}}^{(1-\nu )/3\nu }\sim {\mathrm{Wi}}^{0.26}$], while dashed lines show strong extensions at the highest Wi [Eq. (5)]. Inset: Blob size $\xi$ of the DPD polymer. The dashed line shows the scaling predicted by Eq. (3).

Figure 5

Spooling tethered DNA strands. (a) Simulations of the critical rotation rate for which deformation of tethered DNA occurs. The solid line shows the predicted scaling of ${\mathrm{\Omega }}_{\mathrm{Wi}}^{*}\sim N^{-3\nu }$. (b) Conformation of a single $\lambda$-phage DNA strand in the period immediately after the microwire has stopped rotating at $\mathrm{\Omega }=130\mathrm{rpm}$, showing the relaxation of the polymer (see Movie 2 in Ref. [16]). The end of the polymer that appears towards the top of the image is tethered to the wire. The conformational size is observed to increase steadily away from the tethering point, as predicted for a tethered polymer in the shofar configuration. (c) French-horn conformations seen via DPD blob size $\xi$ as a function of segment index from the free end. Various rotation rates are collapsed according to Eq. (8), with blob size normalized by the free-end blob size ${\xi }_1$. The dotted line shows the scaling prediction $\xi \sim n^{-\nu /(3\nu -1)}\sim n^{-0.77}$. Diamonds ($\diamond$) denote the predicted transition from shofar to stem at $n^{*}\simeq {({\tau }_b\mathrm{\Omega })}^{-1}$. Inset: Wrapping angle from the free end, collapsed via Eq. (7). The dashed line shows the scaling $\varphi \sim n^{2\nu /(3\nu -1)}\sim n^{1.54}$.