All-Atom MD Predicts Magnesium-Induced Hairpin in Chemically Perturbed RNA Analog of F10 Therapeutic

Abstract

Given their increasingly frequent usage, understanding the chemical and structural properties which allow therapeutic nucleic acids to promote the death of cancer cells is critical for medical advancement. One molecule of interest is a 10-mer of FdUMP (5-fluoro-2'-deoxyuridine-5'-O-monophosphate) also called F10. To investigate causes of structural stability, we have computationally restored the 2' oxygen on each ribose sugar of the phosphodiester backbone, creating FUMP[10]. Microsecond time-scale, all-atom, simulations of FUMP[10] in the presence of 150 mM MgCl₂ predict that the strand has a 45% probability of folding into a stable hairpin-like secondary structure. Analysis of 16 μs of data reveals phosphate interactions as likely contributors to the stability of this folded state. Comparison with polydT and polyU simulations predicts that FUMP[10]'s lowest order structures last for one to 2 orders of magnitude longer than similar nucleic acid strands. Here we provide a brief structural and conformational analysis of the predicted structures of FUMP[10], and suggest insights into its stability via comparison to F10, polydT, and polyU.

Figure 1

Nucleotides of the 10mer 5-fluorouridine-5′-monophosphate (FUMP[10]) is produced computationally by replacing a thymidine monophosphate’s methyl group with fluorine and adding a hydroxyl group at C2′. Throughout this article, we use the sugar numbering, base numbering and atom colors shown here.

Figure 2

For each macrostate, (A) folded, (B) partially folded, and (C) extended, the structure with the smallest RMSD from the average structure is shown as solid with shows representing one standard deviation (by RMSD). These shadows serve as a conformation space uncertainty sphere, revealing the variety of conformations in each macrostate.

Figure 3

Most common base interactions across and within all macrostates are phosphate interactions wherein an oxygen atom in one phosphate group accepts a hydrogen bond from a nucleotide. The numbered residues are those referenced in Table 2.

Figure 4

Starting from any initial state (i.e., folded, partially folded, or extended), these transition probabilities converge within 40 steps (200 ns) to steady state populations of macrostates that is within 0.1% of the populations derived from RMSD clustering on the MD trajectories. This recovery of kinetics via Markov analysis reveals that the transitions folded< – > partially folded and unfolded< – > partially folded are more likely than direct transitions between folded and unfolded. Based on the transition probabilities shown here, the expected transition times are 13 ns for extended to partially folded, 75 ns for partially folded to folded, and 78 ns for extended to folded. On the other hand, the unfolding times for the fully and partially folded states are 101 and 49 ns, respectively.

Figure 5

A combination of RMSD clustering, RGYR-based histograms, and visual inspection indicates 3 macrostates: folded, partially folded, and extended. Free energy calculations show that while folded and partially folded are equally likely there is a large energy barrier to transitioning between the two states. Furthermore, while moving from partially folded to extended is not favorable, moving from extended to partially folded is favorable. This apparent asymmetry comes from our simplified presentation of the pathway as unidimensional.

Figure 6

(A) Little correlated motion occurs between atoms other self-residue interactions. (B) In the partially folded macrostate, some interactions occur among neighboring residues. (C) Neighboring residue interactions and interactions across the hairpin conformation seen in Figure 2A appear in the folded macrostate. The difference in partially folded and folded correlations is shown in (D) revealing that the greatest differences occur in residue interactions across the hairpin structure. That is, the greatest differences occur along and near the antidiagonal elements of the folded and partially folded correlation matrices.

Figure 7

PolydT forms stable but not structured conformations (A) that have lifespans on the order of 80 ns. PolyU’s long-lived states (B) have lifespans on the order of 10 ns, compared to F10s macrostates (Figures 2 and 3), which last for microseconds at a time. (C) F10 shows both short medium range interactions with magnesium ions. (D) PolydT has the tightest binding with magnesium. (E) PolyU shows the lowest propensity to bind closely with magnesium.