Sensing DNA–DNA as Nanosensor: A Perspective Towards Nanobiotechnology

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Sensing DNA – DNA as nanosensor: a perspective towards nanobiotechnology Ralf Metzler∗ and Tobias Ambj¨ornsson†

arXiv:cond-mat/0508490v1 [cond-mat.soft] 20 Aug 2005

NORDITA - Nordic Institute for Theoretical Physics, Blegdamsvej 17, DK-2100 Copenhagen Ø, Denmark (Dated: 2nd February 2008) Based on modern single molecule techniques, we devise a number of possible experimental setups to probe local properties of DNA such as the presence of DNA-knots, loops or folds, or to obtain information on the DNA-sequence. Similarly, DNA may be used as a local sensor. Employing single molecule fluorescence methods, we propose to make use of the physics of DNA denaturation nanoregions to find out about the solvent conditions such as ionic strength, presence of binding proteins, etc. By measuring dynamical quantities in particular, rather sensitive nanoprobes may be constructed with contemporary instruments. Key words: DNA, DNA breathing, single molecule spectroscopy, nanosensors, fluorescence correlation spectroscopy, fluorescent resonance energy transfer PACS numbers: 87.15.-v, 82.37.-j, 87.14.Gg

I.

INTRODUCTION

Single molecule techniques allowing both the manipulation and probing of single molecules, have come of age. Optical tweezers, atomic force microscopes, or single molecule tracking and optical detection methods (for instance, fluorescence correlation spectroscopy, FCS, or fluorescence (F¨ orster) resonance energy transfer, FRET) have become standard methods in laboratories. By means of these techniques having access to scales in the nanometre domain allows us to obtain quantitative information about the physical properties of molecules without being masked by the inevitable ensemble averaging inherent in bulk measurements. Even though typical single molecule data are more noisy than bulk signals, the gain of individual molecular behaviour by far outweighs this disadvantage. In certain cases, single molecule experiments can reveal information, that is not accessible to bulk measurements, for instance, the recent experiments on the characteristics of single-stranded DNA-binding proteins1 , or the measurements of the passage of single biopolymers through nanopores2,3 . Moreover, one may even extract information from the single molecule noise; for example, on the nature of such known phenomena as Brownian motion4 . This progress is essential to recent advances in a number of fields like biological and soft matter physics, or nanobiotechnology. The small system sizes also make it possible to test fundamental physical theories such as the Jarzinsky relation connecting measurements of the nonequilibrium work needed, e.g., to stretch an RNA segment5 , to the difference in the corresponding thermodynamic potential6 ; or the entropy production along single trajectories exposed to stochastic forces7 . In what follows, we devise a number of potential experimental setups probing on scales down to the nanolevel, both the physical behaviour of DNA itself as well as different ways to employ DNA as a nanosensor. A certain emphasis is put on methods where theoretical models are available so the physical parameters of the DNA and its

surroundings may be quantitatively extracted from experimental data. These setups should be well within reach of the state of the art techniques and may be used to obtain important new information on DNA, or prompt new technologies based on DNA. As the DNA molecule is the main ingredient for our exposition, we start with a primer on the physical properties of DNA, before embarking for setups to probe (some of) these properties on the single molecule level and propose several possibilities to use DNA as a sensor.

II.

DNA-PHYSICS

DNA has a number of remarkable properties. Made up of two chemically very stable individual molecules that wind around each other to produce the double-helix, it carries, embedded in its core, the entire genetic code of an organism. Modern gene technology is able to produce custom-designed DNA molecules with any given sequence. There exists proteins (”biological glue”) by which DNA can be attached to microbeads, that, in turn, can be manipulated by optical tweezers or microbeads. These properties make DNA an ideal object for single molecule experiments. DNA consists of a backbone of sugar and phosphate molecules suspending the base-pairs in its core, see Figure 1. This ladder structure in 3D forms the spiral staircase structure (see Figure 1 on the right) originally predicted by Watson and Crick8 . The Watson-Crick double-helix, or, more precisely, its B-form, is the thermodynamically stable configuration of a DNA molecule under physiological and a large range of in vitro conditions. This stability is effected first by Watson-Crick H-bonding, that is essential for the specificity of base-pairing (”key-lock principle”). Base-pairing therefore guarantees the high level of fidelity during replication and transcription. The second, major, contribution to DNA-helix stability comes from base-stacking between neighbouring base-pairs, through hydrophobic interactions between the planar aromatic

2 Sugar− phosphate backbone

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Figure 1: Left: Schematic view of the chemical structure of the DNA molecule, showing the bases suspended by the outer Sugar-Phosphate scaffold. Right: Reproduction of the original graph of the proposed double-helical structure of DNA. Reprinted with permission from Reference8 , Watson c 1953, Nature. and Crick, N ature 171, 737 (1953).

bases, that overlap geometrically and electronically9,10 . The relevant length scales of DNA span several orders of magnitude10,11,12,13 . The distance between neighbouring base-pairs is approximately 3.4 ˚ A, while the hard core diameter of DNA is 2 nm. One full turn of the double-helix is made up of 10.5 base-pairs. The persistence length, i.e., the distance over which the tangenttangent correlations decay, is of the order of 50 nm (340 base-pairs), more than an order of magnitude larger than the diameter. Locally, double-stranded DNA (dsDNA) therefore appears stiff. In contrast, single-stranded DNA (ssDNA) has a persistence length of a few nm, depending on solvent conditions and sequence. Finally, the overall length of naturally occurring DNA ranges from several µm in viruses, over some mm in bacteria, to tens of centimetres in higher organisms. The South American lungfish hosts 35 m of DNA per cell10 . An important feature of double-stranded DNA (dsDNA) is the ease with which its component chains can come apart and rejoin, without damaging the chemical structure of the two daughter-strands. This unzipping of the H-bonds between base-pairs is crucial to many physiological processes such as replication and transcription. Classically, the melting and reannealing behaviour of DNA has been studied in solution in vitro by increasing the temperature, or by titration with acid or alkali. Such equilibrium measurements are described by the ZimmPoland-Scheraga model based on the following physical parameters of DNA14,15,16,17 : (i) the statistical weight u = exp(−βǫ) (with β = 1/(kB T ), where kB is the Boltzmann constant, and T the temperature), associated with

the free energy ǫ of breaking a single base-pair. Note that ǫ is smaller for AT than for GC bonds9,10,18 . u also depends on ambient salt concentration, applied torques and forces; (ii) the non-universal prefactor σ0 ≪ 1 that measures the loop initiation energy associated with breaking the stacking interactions15,16,18,19 ; (iii) and the loop closure exponent c that stems from the entropy loss due to the closed loop structure of the ssDNA bubble, compare15,17,18 . While the double-helix is the thermodynamically stable configuration of the DNA molecule below the melting temperature (or at non-denaturing pH), even at physiological conditions there exist local denaturation zones, socalled DNA-bubbles, predominantly in AT-rich regions of the genome15,16 . A DNA-bubble is a dynamical unit, whose size varies by thermally activated zipping and unzipping of successive base-pairs at the two zipper forks where the ssDNA-bubble meets the intact double-helix. This DNA-breathing is possible due to the fact that on bubble formation the enthalpy cost and entropy gain, despite each being significant amounts in terms of kB T , almost cancel and the unzipping of a base-pair involves a free energy cost of the order of a kB T . We will in the subsequent sections discuss different possible experimental setups that allow for the measurement of the properties of DNA and its surroundings.

III. SENSING DNA: NANO-SETUPS MEASURING THE PHYSICAL PROPERTIES OF THE MOLECULE OF LIFE AND ITS ENVIRONMENT

In this section, we propose a number of arrangements by which physiological processes and the fundamental physical properties of DNA can be monitored. Apart from measuring the characteristics of DNA itself, microand nanosetups are suggested for obtaining information about its topological state or the solution conditions.

A.

Melting and monitoring a nanoregion of DNA

The local stability of DNA can be probed as sketched in Figure 2. Here, a linear stretch of DNA is held in place by two microbeads, and a local denaturation zone is monitored by fluorescence of a fluorophore at the bubble position, for instance, by fluorescence correlation spectroscopy20 . Recent developments in the theoretical description of DNA breathing dynamics21,22,23,24 relate measurable dynamical quantities to the ZimmPoland-Scheraga physical parameters discussed in the previous section, as well as to the properties of the surroundings21,22 . In particular the fluorescence correlation could be quantified and shown to depend on (i) the local statistical weights u, i.e, temperature, salt concentration, twist, as well as the local DNA sequence; (ii) the bubble in...

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