Stability of End-to-End Base Stacking Interactions in Highly Concentrated DNA Solutions

Abstract

Positionally ordered bilayer liquid crystalline nanostructures formed by gapped DNA (GDNA) constructs provide a practical window into DNA–DNA interactions at physiologically relevant DNA concentrations; concentrations several orders of magnitude greater than those in commonly used biophysical assays. The bilayer structure of these states of matter is stabilized by end-to-end base stacking interactions; moreover, such interactions also promote in-plane positional ordering of duplexes that are separated from each other by less than twice the duplex diameter. The end-to-end stacked as well as in-plane ordered duplexes exhibit distinct signatures when studied via small-angle X-ray scattering (SAXS). This enables analysis of the thermal stability of both the end-to-end and side-by-side interactions. We performed synchrotron SAXS experiments over a temperature range of 5–65 °C on GDNA constructs that differ only by the terminal base-pairs at the blunt duplex ends, resulting in identical side-by-side interactions, while end-to-end base stacking interactions are varied. Our key finding is that bilayers formed by constructs with GC termination transition into the monolayer state at temperatures as much as 30 °C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. By modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes, we estimate the stacking free energies in DNA solutions of physiologically relevant concentrations. The free-energies thereby determined are generally smaller than those reported in single-molecule studies, which might reflect the elevated DNA concentrations in our studies.

Liquid Crystalline Layering and Divalent Cations Cooperatively Enhance DNA Condensation

“…The diffraction from the 0 mM Mg 2+ sample (Fig. 2E) is again consistent with a bilayer smectic-A phase; however, the bilayer peak survives to a higher temperature than in the corresponding AT-AT sample, confirming the end-to-end stacking interactions are stronger in AT-GC sample compared to AT-AT sample, in agreement with published reports (30,(33)(34)(35)(36). Also similar to AT-AT samples, the introduction of divalent cations induces lateral positional ordering of the duplexes and enhances its thermal stability up to 30 mM Mg 2+ concentration and 53°C temperature, with a reversal in this trend developing between 30 and 100 mM (Fig.…”

“…On this basis, we refer to them separately as AT-AT, AT-GC, or GC-GC (Fig.1A). As we demonstrated earlier (30), 48-20T-48 GDNA constructs, suspended at ∼280 mg/ml concentration in 150 mM Na + solution, exhibit bilayer smectic ordering whose stability against temperature differs significantly within the range 35-64 °C. We thereby established a clear correlation between the identity of the terminal base pairs and the thermal stability of the bilayer structure.…”

“…The double-Hill function (resulting in characteristic melting temperatures Tm1 and Tm2) provides a better description of the data than the single-Hill function (resulting in a single Tm), although in several cases data are clearly lacking in the temperature range where the "kink" in the double-Hill fit occurs. The analysis with two Hill functions implies a multi-step melting process, whose possible mechanism we discussed previously (30) for the bilayer structure sketched in Fig. 1B.…”

“…GDNA Synthesis: Details of the GDNA synthesis process, sample loading process into thinwalled borosilicate capillaries, and the details of the SAXS/WAXS measurements carried out are described in previous publications (29,30,32). Briefly, the GDNA constructs, denoted as 48-20T-48, consist of two symmetric 48-base pair duplexes bridged by a single-strand gap of 20 Thymine bases (Fig.…”

“…Remarkably, LC formation may occur via an initial end-to-end stacking of ultrashort duplexes (20)(21)(22)(23)(24)(25), producing longer aggregates whose aspect ratio (L/d, where L is the length and d the diameter) satisfies the Onsager criterion (L/d > 4.7) for orientational ordering of semi-rigid spherocylindrical particles (26,27). Incorporating a flexible 'gap' of single-stranded DNA between two fully-paired duplexes (with L below the ~50 nm persistence length) further enriches the LC phase diagram through the appearance of previously unobserved layered (smectic) phases (28)(29)(30). Two different smectic layer structures are observed: a "bilayer" stacking with ~2 duplex periodicity, prevailing for longer gap lengths (e.g., 20 bases) and a "monolayer" stacking (~1 duplex periodicity, occurring for gaps of 4 -10 bases in length (29).…”