K+ promotes the favorable effect of polyamine on gene expression better than Na+

Abstract

Background Polyamines are involved in a wide variety of biological processes including a marked effect on the structure and function of DNA. During our study on the interaction of polyamines with DNA, we found that K + enhanced in vitro gene expression in the presence of polyamine more strongly than Na +. Thus, we sought to clarify the physico-chemical mechanism underlying this marked difference between the effects of K + and Na +. Principal findings It was found that K + enhanced gene expression in the presence of spermidine, SPD(3+), much more strongly than Na + , through in vitro experiments with a Luciferase assay on cell extracts. Single-DNA observation by fluorescence microscopy showed that Na + prevents the folding transition of DNA into a compact state more strongly than K +. 1 H NMR measurement revealed that Na + inhibits the binding of SPD to DNA more strongly than K +. Thus, SPD binds to DNA more favorably in K +-rich medium than in Na +-rich medium, which leads to favorable conditions for RNA polymerase to access DNA by decreasing the negative charge. Conclusion and significance We found that Na + and K + exhibit markedly different effects through competitive binding with a cationic polyamine, SPD, to DNA, which causes a large difference in the higher-order structure of genomic DNA. It is concluded that the larger favorable effect of Na + than K + on in vitro gene expression observed in this study is well attributable to the significant difference between Na + and K + on the competitive binding inducing conformational transition of DNA.

Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions

“…The difference in physico-chemical properties between the shrunken state and compact state of DNA has also been recently revealed experimentally [48]. Interestingly, for experiments on in vitro gene expression, the promotion and inhibition of gene expression are observed for the shrunken state and compact globule states, respectively [39,[41][42][43][44].…”

“…Recently, it was found that, through in vitro experiments with cell-free gene expression assay, polyamines promote gene expression just below the concentration to induce tight compaction but inhibit gene expression completely at higher concentrations [39][40][41][42]. It was revealed that such biphasic effect of polyamines neatly concerns with transition of higher order structure of DNA molecules [39,[41][42][43][44]. It would be highly expected that such characteristic function of polyamine on the structure and function of DNA play an important role in living cellular media accompanied with microphase separation.…”

Longer DNA exhibits greater potential for cell-free gene expression

“…Cell-free in vitro transcription/translation of Luc -gene was performed with a TnT T7 Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions and previous reports 9 – 11 . Reaction mixtures containing the reporter DNAs were incubated for 90 min at 30 °C on a Dry Thermo Unit (TAITEC, Saitama, Japan).…”

“…In this regard, cell-free gene expression systems are one of the most widely used techniques in molecular biology 1 – 3 . The efficiency of cell-free gene expression is known to be affected by various factors such as vector type 4 – 7 , DNA topology 4 – 7 , and reaction components 8 – 11 , as well as the template DNA concentration 12 – 14 . It is noted that there exists a longstanding controversy regarding the topological effect of supercoiling, or linking number, in circular DNAs on their genomic activity 4 – 7 .…”

Water‐in‐Water Droplets Selectively Uptake Self‐Assembled DNA Nano/Microstructures: a Versatile Method for Purification in DNA Nanotechnology

“…The selective uptake of DNA by dextran‐rich droplets was explored for the separation of single‐ and double‐stranded DNAs, high‐ and low‐molecular‐weight DNAs, genomic DNA from a cell lysate, and even chromosomes [15–19] . We also observed that genomic DNA and DNA hydrogels were concentrated in the dextran‐rich droplets, whereas short ssDNA were distributed in both dextran‐rich droplets and a PEG‐rich continuous phase [20–22] . We expected small ssDNA and large DNA structures would have different partitions and be separable by W/W‐droplet fractionation (Figures 1B and 1 C).…”