Spermine Condenses DNA, but Not RNA Duplexes

Abstract

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA.

Figure 1

Fraction of 25 bp nucleic acid duplexes remaining in solution, measured by UV absorption, as a function of the added spermine concentration. Individual spermine molecules are shown in addition to the nucleic acid structures. To see this figure in color, go online.

Figure 2

(a and b) Representative snapshots of mixed-sequence (a) DNA and (b) RNA duplexes simulated with spermine. Spermine molecules are shown in magenta. On average, ∼11 of the 12 spermine molecules are bound to the nucleic acids in each snapshot. These frames were taken from Movies S1 and S2 of the simulations. (c) Distribution of the charge of bound spermine counterions around 25 bp mixed-sequence DNA and RNA duplexes derived from 500 ns of MD trajectories. Interestingly, this plot qualitatively resembles the distributions computed for CoHex (Fig. 3 of Ref. (23)). (d) Spermine charge distributions around RNA simulated with no NaCl (for comparison with condensation data) and with 24 NaCl per simulation box (80 mM, for comparison with WAXS). The distributions are qualitatively unchanged by the addition of NaCl. To see this figure in color, go online.

Figure 3

(a and b) Distribution of spermine charge around (a) mixed-sequence DNA and (b) mixed-sequence RNA with the four-point OPC water model (34) and three-point TIP3P model. To see this figure in color, go online.

Figure 4

(a–c) CD spectra for (a) mixed-sequence DNA, (b) mixed-sequence RNA, and (c) poly(rA):poly(rU) RNA with and without added spermine. The wavelengths of the peaks and valleys shift slightly when spermine is added, suggesting a geometrical change. Panels (b) and (c) highlight the differences between the structures of the mixed-sequence duplex (specified in Materials and Methods) and the poly(rA):poly(rU) duplex. To see this figure in color, go online.

Figure 5

(a and b) WAXS profiles for (a) mixed-sequence DNA and (b) mixed-sequence RNA with and without spermine. Larger changes are observed in the profile of the RNA. To see this figure in color, go online.

Figure 6

(a and b) Differences in the scattering profiles for (a) mixed-sequence RNA and (b) DNA with and without spermine. Differences were taken from the logs of the intensities of the original profiles to allow easier examination of differences at higher q-values. The noisier (red) profiles are experimental data, while the smoother (blue) profiles show the average of profiles generated with CRYSOL from 200 different MD snapshots. The RNA plot also shows the experimental curve multiplied by a factor of 5 to emphasize the locations of extrema. To see this figure in color, go online.