Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution

Abstract

Limited tools exist that are capable of monitoring nucleic acid conformations, fluctuations, and distributions in free solution environments. Single molecule free solution hydrodynamic separation enables the unique ability to quantitatively analyze nucleic acid biophysics in free solution. Single molecule fluorescent burst data and separation chromatograms can give layered insight into global DNA conformation, binding interactions, and molecular distributions. First, we show that global conformation of individual DNA molecules can be directly visualized by examining single molecule fluorescent burst shapes and that DNA exists in a dynamic equilibrium of fluctuating conformations as it is driven by Poiseuille flow through micron-sized channels. We then show that this dynamic equilibrium of DNA conformations is reflected as shifts in hydrodynamic mobility that can be perturbed using salt and ionic strength to affect packing density. Next, we demonstrate that these shifts in hydrodynamic mobility can be used to investigate hybridization thermodynamics and binding interactions. We distinguish and classify multiple interactions within a single sample, and demonstrate quantification amidst large concentration differences for the detection of rare species. Finally, we demonstrate that these differences can resolve perfect complement, 2 bp mismatched, and 3 bp mismatched sequences. Such a system can be used to garner diverse information about DNA conformation and structure, and potentially be extended to other molecules and mixed-species interactions, such as between nucleic acids and proteins or synthetic polymers.

Figure 1

CICS optical detection platform and the hydrodynamic separation principle. (a) Schematic of the CICS optical detection platform with the cylindrical lens (CL) providing the beam expansion over the burned viewing window of the mounted separation capillary. Further description of the optical components is provided in the Methods section. (b) Schematic of the hydrodynamic separation principle: larger molecules are more excluded from the slower moving flow regimes near the wall, resulting in a faster average velocity for larger molecules than smaller molecules. (c) Separation of a 50 bp ladder with single molecules detected for each fragment size. The inset provides a closer look at the 9 detected molecules of the largest fragment size. (d) Single molecule bursts from the raw trace are summed over a larger bin size to form a single molecule chromatogram where each peak is a subpopulation of uniform fragment sizes. Every chromatogram peak in (d) corresponds to a fragment species present in the ladder, but only enough species are labeled to orient the reader.

Figure 2

Single molecule bursts of 23 kbp species separated in high salt conditions (25 mM NaCl) display a large variety in burst shapes ranging from stretched (1) to condensed (4), as characterized by the packing density (burst height divided by burst width).

Figure 3

A sample comprised of 13 DNA fragments ranging in size from 19 nt to 48 kbp is separated in the same 1.6 μm diameter capillary under both low ionic strength (20 mM EB buffer) and high salt conditions (20 mM EB + 25 mM NaCl). (a) Single molecule chromatograms of both separation conditions reveal that adding salt to the separation buffer induces relative mobility changes for small and large fragments. (b) Comparison of the effect of the added salt on ratiometric changes in relative mobility and packing density of the dsDNA fragments suggests a positive correlation between the two metrics.

Figure 4

The effects of both sodium chloride (blue) and magnesium chloride (red) on the packing density of double stranded and single stranded DNA is probed by comparing their relative mobilities in the same 1.6 μm diameter capillary. (a) HindIII digested λ dsDNA is separated in a buffer with added ionic strengths of 0.01 mM (Diamond), 0.1 mM (square), 0.5 mM (triangle), and 10 (star). (b) ssDNA oligos (19 nt, 24 nt, 50 nt, and 90 nt) are separated in added ionic strengths of 0.01 mM (Diamond), 0.1 mM (square), 0.5 mM (triangle), and 1 mM (star). Error bars represent the width of each fragment’s elution peak, which was calculated as 4 times the standard deviation of the fitted Gaussian peak.

Figure 5

Denaturation of a double stranded DNA duplex into single strands results in a relative mobility shift. (a) A sample consisting of three single stranded species (19 nt, 50 nt, and 90 nt) and one double stranded species (24 nt – 26 nt duplex*) is separated in a 1.6 μm capillary at room temperature (top) and after heating the sample to 95 C for 5-10 min (bottom). (b) The average relative mobilities from the chromatograms in (a) is plotted against species size to illustrate that only the duplex mobility is affected by heating. (c) High pH environments (top – 1 mM NaOH; bottom 10 mM NaOH) also exhibit denaturation-induced mobility shifts for the duplex species only. Error bars represent the width of each fragment’s elution peak, which was calculated as 4 times the standard deviation of the fitted Gaussian peak.

Figure 6

Hybridization of a fluorescently tagged single stranded probe to a complementary oligo is accompanied by a relative mobility shift. (a) Equal ratios of four complementary targets (lengths 22 nt, 48 nt, 108 nt, and 160 nt) are combined with a 22 nt ssDNA probe at five-times higher concentration and separated. (b) The hybridization efficiency of the competing hybridization reactions in (a) is calculated by comparing the quantity of a detected assemblies (determined by single molecule counting) with the number predicted (determined from the input ratios and the total number of fluorescent molecules present). (c) Separation and detection of the 160 nt complement/probe assembly (inset) in 2000-fold excess concentration of labeled probe. (d) The raw APD trace of the single fluorophore bursts that correspond to the hybridized 160 nt complement/probe assembly.

Figure 7

Mimic sequences from the Let-7 microRNA family are used to investigate the sensitivity of hybridization-induced mobility shifted peaks to sequence complementarity and bond stability. (a) The length and complementarity of the oligo combinations used in the separations shown in (b)-(e) are listed: i and g are fluorescently tagged mimics of Let-7i and Let-7g respectively; M, N, O, and P are unlabeled oligos of varying complementarity. The species present in the chromatograms shown in (b)-(e) are labeled pictorially with probe i and its complementary oligos in green, probe g and its complements in blue, and oligo O, which does not have a fully complementary probe, in gold. Red stars designate the fluorescently labeled species: probes i and g and the free alexa dye. (b) Separation chromatogram of probe i combined with perfectly complementary oligos P (160 nt) and N (100 nt) shows distinct mobility shifted peaks for each complementary oligo. (c) No 100 nt peak is observed in the separation chromatogram when oligo P is replaced with oligo O (3 bp mismatch). (d) Separation chromatogram of probe i combined with oligos P (perfect complement) and M (73 nt oligo with 2 bp mismatch). (e) Separation chromatogram of probe g with the same M and P oligos used in (d), making M a perfect complement and P a 2 bp mismatch.