Differential Effects of Strand Asymmetry on the Energetics and Structural Flexibility of DNA Internal Loops

Abstract

Internal loops within structured nucleic acids disrupt local base stacking and destabilize neighboring helical domains; however, these structural motifs also expand the conformational and functional capabilities of structured nucleic acids. Variations in the size, distribution of loop nucleotides on opposing strands (strand asymmetry), and sequence alter their biophysical properties. Here, the thermodynamics and structural flexibility of oligo-T-rich DNA internal loops were systematically investigated in terms of loop size and strand asymmetry. From optical melting experiments, a thermodynamic prediction model is proposed for the energetic penalty of internal loops that accounts for diminishing enthalpic and increasing entropic contributions due to loop size and strand asymmetry for bulges, asymmetric loops, and symmetric loops. These single-stranded domains become less sequence-dependent and more polymeric as the loop size increases. Single-molecule fluorescence resonance energy transfer studies reveal a gradual transition in conformation and structural flexibility from an elongated domain to an increasingly flexible bend that results from increasing strand asymmetry. The findings provide a framework for understanding the thermodynamic and conformational effects of internal loops for the rational design of functional DNA nanostructures.

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

“…This result contrasts with previous findings in which purine/pyrimidine bulge substitutions did not affect the conformer state; however, these junctions had lower GC content for the terminal base pairs in the core than the current study [27]. Considering that thermodynamic studies on duplexes show that A2 bulges are more destabilizing than T2 bulges [20,57] and that unpaired purines have higher intrastrand stacking potential than thymines in single-stranded loops [58], the observed differences in the folding behavior for J8 and J11 suggest that the stabilities of the core and loop conformation may be additional factors that influence conformer selection. Lastly, the EFRET values for the fully complementary J12, which lacks a dinucleotide bulge, do not change appreciably from the Mg 2+free value and are intermediate between the values observed for the two stacked conformers, indicating that it remains primarily in the trigonal planar form.…”

“…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]. Bulges within helical domains can induce a flexible hinge conformation in which the helical domains becomes increasingly bent as the size of the bulge expands [24][25][26].…”

Modeling DNA-Strand Displacement Reactions in the Presence of Base-Pair Mismatches

“…To model bubble formation, we considered a combined macrostate composed of a closed state with two isolated mismatches and the bubble state. The free energy for bubble formation comprises an initiation penalty for disrupting the helix phase, the disruption of the base pairs energy of Δ G BP / b p and an entropic loop penalty for confining the ssDNA strands of Δ G L / b p . We estimated the initiation penalty combined with the free energy for disrupting the first base pair to be on the order of Δ G MM .…”

“…The free energy for bubble formation comprises an initiation penalty for disrupting the helix phase, the disruption of the base pairs energy of ΔG BP /b p and an entropic loop penalty for confining the ssDNA strands of ΔG L /b p . 37 We estimated the initiation penalty combined with the free energy for disrupting the first base pair to be on the order of ΔG MM . The free energies of the two states relative to the perfectly matching invader are then given as…”

Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop

“…Previous reports on small internal loops in DNA show that the free energy contribution of the loop depends on the nucleotide sequence, with a penalty of 3.2 kcal/mol for internal loop of 3 nt being utilized in the nearest neighbor models (SantaLucia and Hicks, 2004;Tran and Cannon, 2017). Noncanonical T•T base pair formations have been shown to reduce the penalty for DNA loop motifs (Tran and Cannon, 2017).…”