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. 2023 Sep 21;127(37):7907-7924.
doi: 10.1021/acs.jpcb.3c03538. Epub 2023 Sep 8.

Structure and Dynamics of DNA and RNA Double Helices Formed by d(CTG), d(GTC), r(CUG), and r(GUC) Trinucleotide Repeats and Associated DNA-RNA Hybrids

Ashkan Fakharzadeh  1 , Jing Qu  1 , Feng Pan  2 , Celeste Sagui  1 , Christopher Roland  1
Affiliations

Structure and Dynamics of DNA and RNA Double Helices Formed by d(CTG), d(GTC), r(CUG), and r(GUC) Trinucleotide Repeats and Associated DNA-RNA Hybrids

Ashkan Fakharzadeh et al. J Phys Chem B. .

Abstract

Myotonic dystrophy type 1 is the most frequent form of muscular dystrophy in adults caused by an abnormal expansion of the CTG trinucleotide. Both the expanded DNA and the expanded CUG RNA transcript can fold into hairpins. Co-transcriptional formation of stable RNA·DNA hybrids can also enhance the instability of repeat tracts. We performed molecular dynamics simulations of homoduplexes associated with the disease, d(CTG)n and r(CUG)n, and their corresponding r(CAG)n:d(CTG)n and r(CUG)n:d(CAG)n hybrids that can form under bidirectional transcription and of non-pathological d(GTC)n and d(GUC)n homoduplexes. We characterized their conformations, stability, and dynamics and found that the U·U and T·T mismatches are dynamic, favoring anti-anti conformations inside the helical core, followed by anti-syn and syn-syn conformations. For DNA, the secondary minima in the non-expanding d(GTC)n helices are deeper, wider, and longer-lived than those in d(CTG)n, which constitutes another biophysical factor further differentiating the expanding and non-expanding sequences. The hybrid helices are closer to A-RNA, with the A-T and A-U pairs forming two stable Watson-Crick hydrogen bonds. The neutralizing ion distribution around the non-canonical pairs is also described.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(a) Schematic of sequences considered in this study (for both DNA and RNA): r(CUG), r(CUG)4, d(CTG), and d(CTG)4. Structures for r(GUC) and d(GTC) are similar and may be obtained by interchanging G and C in the illustrated sequences. (b) χ angle of the U base is the O4′-C1′-N1-C2 dihedral (similar for T in DNA). (c) Schematic view of the center-of-mass pseudodihedral angle Ω (for U14 in r(CUG)).
Figure 2
Figure 2
Different conformations of U·U mismatches in the local minima of r(CUG) free energy landscape. Here, A conformations are all anti–anti, while B conformations represent anti–syn and C is the syn–syn conformation. The H-bonds are also shown.
Figure 3
Figure 3
Different conformations of T·T mismatches in the local minima of d(CTG) free energy landscape. Here, A represents the anti–anti conformation, B represents the anti–syn, and C represents the syn–syn conformations. Observed H-bonds are shown.
Figure 4
Figure 4
Free energy maps for single mismatches in r(CUG)(a–c) and d(CTG)(d–f) with different choices of collective variables. (a,d):(Ω14, χ14); (b,e):(χ5, χ14); and (c,f):(Ω5, Ω14). The values in the bar are given in kcal/mol. Each contour line approximately shows 1 kcal/mol.
Figure 5
Figure 5
Free energy maps for single mismatches in r(GUC)(a–c) and d(GTC)(d–f) with different choices of collective variables. (a,d):(Ω14, χ14); (b,e):(χ5, χ14); and (c,f):(Ω5, Ω14). The values in the bar are given in kcal/mol. Each contour line approximately shows 1 kcal/mol.
Figure 6
Figure 6
Results from 1 μs MD simulation for r(CUG)4 helices. Shown are angle χ (left) and number of hydrogen bonds (right) associated with the internal mismatches of the helices. Initial conformations for individual panels (top to bottom) are (a) anti–anti; (b) anti–syn; and (c) syn–syn. Each individual panel, (a), (b), or (c) is divided in two rows. The upper row corresponds to U5·U20 while the lower row corresponds to U8·U17. χ5 and χ8 are black, and χ20 and χ17 are red. On the right, different colors represent different numbers of hydrogen bonds (0, 1, or 2).
Figure 7
Figure 7
Results of the PCA analysis for the U·U mismatch in anti–anti conformation for r(CUG). (a) Histogram of projections on the first (cyan) and second (grey) eigenvectors. (b) Fluctuations of conformation along the second eigenvector. Cyan and red structures highlight the A1 and A1′ (mirror image of A1) structures as in Figure 2.
Figure 8
Figure 8
Results of PCA analysis for the T·T mismatch in anti–anti conformation for d(CTG). (a) Histogram of projections on the first (cyan) and second (grey) eigenvectors. (b) Fluctuations of conformation along the first eigenvector. Cyan and red structures show the two conformations with the largest RMSD.
Figure 9
Figure 9
Conformational fluctuations around the first eigenvector direction based on the PCA analysis of the backbone of (a) r(CUG)4, (b) d(CTG)4, (c) r(GUC)4, and (d) d(GTC)4.
Figure 10
Figure 10
Comparison of step parameters for homoduplexes and WC helices. Average twist, inclination, bend (formula image), rise, slide, and Zp for middle base-pair steps of homoduplexes r(CUG)4, r(GUC)4, d(CTG)4, and d(GTC)4 (all with mismatches in anti–anti conformation); and of standard helices r(CUG:CAG)4, r(GUC:GAC)4, d(CTG:CAG)4, and d(GTC:GAC)4. Colors are used as follows. (a) Red: d(CTG:CAG)4; orange: r(CUG:CAG)4; blue: d(CTG)4; green: r(CUG)4; and (b) red: d(GTC:GAC)4; orange: r(GUC:GAC)4; blue: d(GTC)4; green: r(GUC)4. Data were averaged over the last 200 ns. Here, ‘X’ in the horizontal label indicates either T, U, or A, depending on the sequence being considered.
Figure 11
Figure 11
(a–c) Average ion occupancy around U·U mismatches in r(CUG)4 (left) and r(GUC)4 (right). Blue: bases in the first strand. Red: bases in the second strand. Different panels are for different initial conformations of the mismatches. The averages are based on data taken from the last 200 ns of the simulations.
Figure 12
Figure 12
(a–c) Average ion occupancy around T·T mismatches in d(CTG)4 (left) and d(GTC)4 (right). Blue: bases in the first strand. Red: bases in the second strand. Different panels are for different initial conformations of the mismatches. The averages are based on data taken from the last 200 ns of the simulations.
Figure 13
Figure 13
Illustration of two main Na+ ion binding sites for U·U and T·T mismatches. U·U/T·T mismatches are highlighted in cyan color, and Na+ ions are represented by orange spheres. (a) For r(CUG) in anti–anti conformation, there is a binding site in the major groove, where the ion binds to the O4 atoms in each U base of the mismatch, and to the neighboring G:O6 atom. (b) For d(CTG) in anti–anti conformation, there is a binding site in the minor groove where Na+ binds to T14:O2 and neighboring G15:N3.
Figure 14
Figure 14
(a–c) Comparison of step parameters for hybrid and standard helices. Average twist, inclination, bend (formula image), rise, slide, and Zp for middle base-pair steps of hybrid r(CUG)4:d(CAG)4, and r(CAG)4:d(CTG)4 helices and standard A-RNA and B-DNA. Colors are used as follows. Red: d(CTG:CAG)4 (B-DNA); orange: r(CUG:CAG)4 (A-RNA); blue: r(CUG)4:d(CAG)4 initially B-DNA; green: r(CUG)4:d(CAG)4 initially A-RNA; yellow: r(CAG)4:d(CTG)4 initially B-DNA; purple: r(CAG)4:d(CTG)4 initially A-RNA. Data were averaged over the last 200 ns. ‘X’ in the x-axis labels stands for either T, U, or A, according to the sequence.
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