Salt Has a Biphasic Effect on the Higher-Order Structure of a DNA−Protamine Complex

Abstract

We observed single DNA molecules by fluorescence microscopy to clarify the effect of protamine on their higher-order structure. With an increase in the protamine concentration, the conformation of DNA molecules changes from an elongated coil state to a compact state through an intermediate state. Furthermore, the long-axis length of DNA gradually decreases while maintaining a distribution profile with a single peak. Such behavior is markedly different from the conformational transition of DNA induced by small polyamines such as spermidine and spermine, where individual DNA molecules exhibit an all-or-none transition from a coil to a globule state and the size distribution is characterized by twin peaks around the transition region. Next, we examined the effect of salt on the conformation of the DNA-protamine complex. Interestingly, at a fixed concentration of protamine, DNA tends to shrink with an increase in the NaCl concentration up to 300 mM, and then swells with a further increase in the NaCl concentration, that is, biphasic behavior is generated depending on the salt concentration. For comparison, we examined the effect of salt on DNA compaction induced by the trivalent polyamine spermidine. We confirmed that salt always has an inhibitory effect on spermine-induced compaction. To clarify this biphasic effect of salt on protamine-induced DNA compaction, we performed a numerical simulation on a negatively charged semiflexible polyelectrolyte in the presence of polycations with relatively large numbers of positive charges by taking into account the effect of salt at different concentrations. The results showed that salt promotes compaction up to a certain concentration and then tends to unfold the polyelectrolyte chain, which reproduced the experimental observation in a semiquantitative manner. This biphasic effect is discussed in relation to the specific shielding effect that depends on the salt concentration.

Divalent cation shrinks DNA but inhibits its compaction with trivalent cation

“…3(b) in a good agreement with earlier studies. 6,29,30 It is now clear that divalent cations, Mg(2+) and Ca(2+), as well as a monovalent cation, Na(+), inhibit spermidine-induced DNA compaction. The difference between divalent and monovalent cations is that the former condense DNA, whereas the latter have almost no apparent condensing effect.…”

“…We have experimentally confirmed that the fluorescence dye, Gelgreen, at the adapted concentration exhibits negligible effect on the conformation of DNA molecules, referring to the past studies on the higher-order structure of DNA. [25][26][27][28][29][30][31] All observations were carried out at room temperature (24 • C).…”

Nanoscale structure of protamine/DNA complexes for gene delivery

“…Although it is not doubted that the electrostatic interaction between protamine and DNA could produce compact structures, 6,12 detailed investigation of their local organization has not been reported so far. Among factors affecting protamine/DNA complexation process, DNA size is particularly relevant because it largely influences the DNA-polypeptide interaction.…”

A nanofluidic device for single molecule studies with in situ control of environmental solution conditions

“…30 The extent of the compaction depends on the concentration of protamine and the ionic strength of the supporting buffer medium. 1,31 Inside regular, rectangular nanochannels with a cross section of 300 × 200 nm 2 , we have verified that T4-DNA can be compacted with 0.4 µM of protamine in 1×T buffer. With the addition of 10 mM NaCl to the 1×T buffer, the threshold for compaction decreased about tenfold to a protamine concentration of 0.03 µM.…”