Conformational Heterogeneity in a Fully Complementary DNA Three-Way Junction with a GC-Rich Branchpoint

Abstract

DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.

Sub‐Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single‐Molecule FRET

“…[17] We recently used smFRET to probe the structureo fa3WJ [18] and found that there were unpaired bases at the branchpoint of af ully-complementary 3WJ, resulting in an anoscale cavity. [18][19] This supported earlier accounts that 3WJs could bind supramolecular complexes at the branchpoint. [20] It also agreedw ith chemical footprinting experiments, which demonstrated that thymine bases at the 3WJ branchpoint werer eactive to osmium tetroxide, [21] and to diethyl pyrocarbonate.…”

“…Three‐ and four‐way junctions are among the archetypal building blocks of DNA nanoscience, and play important roles in vivo . We recently used smFRET to probe the structure of a 3WJ and found that there were unpaired bases at the branchpoint of a fully‐complementary 3WJ, resulting in a nanoscale cavity . This supported earlier accounts that 3WJs could bind supramolecular complexes at the branchpoint .…”

“…The products of strand displacement would be unlabeled dsDNA and a two‐stranded forked DNA molecule (2SF). The 3WJ studied here has been reported previously by us, and consists of three 50mer strands designed to form a fully‐complementary 3WJ after annealing (see Figure S1). The experiment involved incubating the pre‐formed 3WJ (≈0.6 μ m ) with a six‐fold excess of a 50mer ssDNA strand (Tris buffer, pH 7.5, 10 m m MgCl 2 ) at 37 °C for up to 24 hours.…”

“…The mismatched bases in the 3WJ studied here are 24 or 25 bases away from the blunt‐end of the duplex where the reaction initiates. Since we believe that the bases at the 3WJ branchpoint are, at least transiently, unpaired, mismatches at the branchpoint should be even less of a barrier.…”

Detection of Mg2+-dependent, coaxial stacking rearrangements in a bulged three-way DNA junction by single-molecule FRET

“…Fully complementary 3WJs primarily adopt a static, trigonal planar conformation without coaxial stacking [15] and display some flexibility in the relative positioning of the junction arms [16][17][18]. The presence of single-stranded domains, such as single mismatches, tandem mismatches, bulges, and internal loops, locally destabilize neighboring helical structure [19][20][21][22][23].…”