Stabilization of nucleic acids by unusual polyamines produced by an extreme thermophile, Thermus thermophilus

Abstract

Extreme thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in thermophile cells.

Figure 1. The 16 polyamines present in the cells of T. thermophilus

The number of carbon atoms separating the amino or aza groups is shown in parentheses. The nine underlined polyamines were used in this study.

Figure 2. Secondary structure of a 14-mer ssDNA (S1)

Figure 3. Excess heat capacity curves observed for thermal denaturation of a 14 bp dsDNA in the absence (broken line) and presence (solid line) of various amounts of tetrakis(3-aminopropyl)ammonium

Curves are shifted on the y-axis for illustrative purposes. From left to right: all melting temperature experiments were conducted with tetrakis(3-aminopropyl)ammonium at concentrations ranging from 0.05 to 0.3 mM, in steps of 0.05 mM.

Figure 4. Effects of various polyamine concentrations on the melting temperature (T_m) of a 14 bp dsDNA

(A) Effects of polyamine concentrations on the melting temperature (T_m) of a 14 bp dsDNA. Variations in T_m values for norspermidine (○), spermidine (△), thermine (◇), spermine (●), caldopentamine (□), caldohexamine (▲), mitsubishine (◆) and tetrakis(3-aminopropyl)ammonium (■) are shown. (B) Relationship between T_m and number of nitrogen atoms of polyamines. Line A, polyamines containing a butyl group; line B, polyamines containing only propyl groups as carbon backbone. T_m values in the presence of branched polyamines are in parentheses. The upside-down open triangle is homocaldopentamine. The relationship between T_m and number of nitrogen atoms is determined at a polyamine concentration of 0.2 mM.

Figure 5. Temperature dependence of enthalpy change associated with the thermal melting of dsDNA in the presence of various polyamines

○, Norspermidine; △, spermidine; ◇, thermine; ●, spermine; □, caldopentamine; ▲, caldohexamine; ◆, mitsubishine; and ■, tetrakis(3-aminopropyl)ammonium.

Figure 6. Excess heat capacity curves of 0.2 mM tRNA^Phe

Experiments were conducted in the absence (A) and in the presence of Mg²⁺ (B), thermine (C), spermine (D), caldopentamine (E), caldohexamine (F), mitsubishine (G), tetrakis(3-aminopropyl)ammonium (H).

Figure 7. Decomposition of the observed melting curves of tRNA^Phe

Experiments were conducted in the absence (A) and in the presence of Mg²⁺ (B), thermine (C), spermine (D), caldopentamine (E), caldohexamine (F), mitsubishine (G), tetrakis(3-aminopropyl)ammonium (H). In each plot, the dark lines represent the minimum number of non-two-state component transitions required to fit the experimental curve. The thin line is the sum of the component transition peaks.

Figure 8. Temperature dependence of the enthalpy change of melting of tRNA^Phe

Experiments were conducted in the absence (A) and in the presence of Mg²⁺ (B), thermine (C), spermine (D), caldopentamine (E), caldohexamine (F), mitsubishine (G), tetrakis(3-aminopropyl)ammonium (H).