Formation of large DNA aggregates induced by spermidine

JOURNAL OF BIOSCIENCEAND BIOENGINEERING Vol. 92, No. 2, 183-185.2001

Formation of Large DNA Aggregates Induced by Spermidine HIDETOSHI K U R A M O C H I 1. AND Y A S U O Y O N E Z A W A 1 SVBL, Graduate School of Science and Engineering, Ibaraki University, 4-12-1 Nakanarusawa, Hitachi, Ibaraki 316-8511, Japan ~ Received 19 February 2001/Accepted 11 May 2001

The formation of large DNA aggregates induced by spermidine was investigated by UV absorptiometry and polarizing microscopy. The present results reveal that it is stepwise and involves the following morphological variations: fiber, fiber bundles, and a highly condensed phase. Furthermore, the influence of DNA concentration on not only the spermidine concentration required for the DNA aggregation but also the concentration of free spermidine during the aggregation is analyzed. [Key words: DNA aggregation, formation, morphological variation, spermidine] Recently, interest in the development o f an efficient process for in vitro transcription and cell-free protein synthesis has been aroused due to some o f advantages o f in vitro biosynthesis in terms o f biotechnological and medical applications. Although the trivalent cation spermidine is often used to stimulate R N A or protein synthesis, it is also known that the addition o f excess spermidine above a critical concentration can induce two types o f D N A aggregation: smalldispersed-particle and large-precipitate aggregations, both o f which are attributed to the binding o f positively charged spermidine to negatively charged phosphates in DNA. Since the aggregated forms o f D N A have the potential to accelerate enzymatic reactions using D N A as a substrate (1, 2), it may be a possible strategy to employ them to improve in vitro R N A and protein synthesis. This approach is also helpful in elucidating the effect of the higher-order structure of D N A on gene expression. In large-mass application, large-precipitate aggregation is used instead o f small-dispersed-particle aggregation, which occurs in a limiting dilute D N A concentration o f less than 10 ~tg/ml. In early studies on the latter (3-6), the microscopic structure o f the large aggregates, concerning its liquid crystalline property, and the precipitation]redissolution conditions o f D N A during addition o f spermidine were mainly investigated. Thus, experimental studies on the formation and morphology o f the aggregate are rare. However, information on these features should be available prior to its application to in vitro biosynthesis. In this study, using X phage D N A (linear double strand, 48.5 kbp) as a model DNA, we reported the formation o f spermidine-induced D N A aggregates o f more than 10 lag/ml D N A on the basis o f the amount o f D N A precipitate and its morphology, and further discussed the effect of DNA concentration on the aggregation. Our findings provide useful

information for the optimization o f the conventional R N A synthesis in vitro as well as the design o f R N A synthesis using the aggregate, particularly, with respect to the quantitative relationship o f DNA-spermidine complex formation.

0.g II I

Z~ 0.6

~

CseD~m~ 12 gg/mlDNA

0.4 [I

/ CsPD'thresh'

Jx

~'~ ........ CSPD.thresh' 12 ttg/mlDNA

~

~

b--

Ot

0

~ -- ' ' ' 0.2 0.4 0.6 0.8 Spermidine concentration [mM]

1

FIG. 1. Precipitation of DNA induced by spermidine at various concentrations. The amount of DNA precipitate is calculated as the ratio of precipitated DNA to total DNA, (A°E6o-A26o)/A°26o,where, A°260 and A26o denote the absorbance of the DNA solution at 260 nm in the absence of spermidine and that in the presence of spermidine, respectively. From these plots, CsvD-thresh • . and Csvr~o~_ P. are denoted by the . dotted and solid arrows, respectwely. The experimental procedure is briefly described as follows: L DNA, purchased from Takara Shuzo Co. Ltd., was dissolved in the TE solution (10 mM Tris, 1 mM EDTA at pH 8.0) and used without further purification. The spermidine solution, as the aggregation agent, was prepared by dissolving spermidine trihydroehloride (Nacalai Tesque Inc.) in the same TE buffer. DNA aggregation was induced by addition of the spermidine solution to an aliquot of the DNA solution. After vortexing for 5 s, the sample was equilibrated for 1 h at room temperature (about 293 K), and then centrifuged for 7 min at 11,000x g with a Beckmann GS-15R microeentrifuge. For the determination of the amount of the precipitate, the absorbance of the supematant at 260 run was determined with a Beckmann DU700 spectrometer. Symbols: open circle, 12 ~tg/ml DNA; asterisk, 24 btg/ml DNA; closed triangle, 48 btg/ml DNA; open square, 90 ~g/ml DNA.

* Corresponding author, e-mail: [email protected] phone: +81-(0)298-50-2841 Present address: Research Center for Material Cycles and Waste Management, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan. 183

184

KURAMOCHIAND YONEZAWA

J. B I O S C I . B I O E N G . ,

For several different DNA concentrations (12-90 Ixg/ml), precipitation of DNA was examined by UV absorptiometry. Figure 1 shows the amounts of precipitate calculated from the absorbance data at 260 nm. Moreover, the spermidine concentrations, CspDthreshand CspD comp,which correspond to the concentration o~; added s p e ~ l d i n e for the threshold of DNA aggregation and that for the completion of DNA aggregation, respectively, were determined and are shown in Fig. 1. This wide-range measurement up to 90 p,g/ml showed that the precipitation process was not continuous but stepwise. In addition, this process was dependent on DNA concentration: CspD.... p was shifted from 0.45 to 0.60 mM by an increase in DNA concentration. This dependence on DNA concentration is discussed below. To understand the aforementioned stepwise behavior, the morphology of the DNA aggregate was observed along the precipitation curve at 48 pg/ml DNA by polarizing microscopy. A polarizing microscope is used to observe liquid crystals and order phases with birefringence. Since spermidine-induced DNA aggregates have ordered phases (3-5, 7), the morphology of the aggregate becomes more distinct. The morphologies are shown in Figs. 2a-d. They varied significantly between 0.45 and 0.55 mM spermidine. At 0.45 mM, where a visible aggregate could be first recognized, a long fiber (Type I) was observed as shown in Fig. 2a. Figure 2b shows that several fiber bundles (Type II) were formed from Type I at 0.50 mM. Type II implies that many cross-link points exist between fibers. As shown in Fig. 1, the amount of DNA aggregate at 0.45 mM was roughly equal to that at 0.50 mM. Therefore, the 0.05 mM increment of spermidine concentration from 0.45 to 0.50 mM is seldom consumed for fiber formation. This finding suggests that the increment binds to the residual negatively

(a)

(b) / ~

z

charged phosphates on Type I, resulting in the formation of a fiber linker. After the formation of Type I, the formation of fiber bundles (Type II), due to the increase in the concentration of binding spermidine, may be thermodynamically preferred to the further formation of Type I. At 0.55 mM above which the DNA concentration of aggregate no longer changed, many fiber bundles were so tightly associated that a highly condensed phase (Type III) was formed as shown in Fig. 2c. The density of Type III was significantly different from those of the others. It is interesting whether such significant difference has any influence on the enzymatic reactions such as transcription and replication. Study on this influence will be necessary for further biotechnological application. Moreover, it may lead to clarify the effect of the higher-order structure of DNA on gene expression. From the series of observations, we suggest the following process accompanied by morphological variations as an aggregation process: the formation of long fibers, bundling of the fibers resulting from an increase in the number of fiber linkers, and finally the formation of a highly condensed phase. From the point of view of using the aggregate, it is also important to determine whether the aggregation process is reversible or not. The resolubilization of Type III was carried out by the dilution of spermidine. Figure 2d demonstrates that the aggregation process is reversible, but has a hysteresis, that is, there is a difference in aggregation behavior between the addition and dilution directions. Since this morphological switching can be controlled by varying the spermidine concentration while monitoring the aggregated form of DNA, the variation of aggregate morphology is expected to be a switch for activating or inactivating in vitro gene expression. To analyze the effect of DNA concentration on the aggregation, in addition, the CSPD/CDNA.P ratio which is the ratio of concentration of the added spermidine to that of the phosphates of the added DNA, and the concentration of free spermidine Cfr~.SpD were calculated at the threshold and completion of aggregation with varying DNA concentration. In Fig. 3, CSPD/CDNA.P is plotted as a function of DNA 15

200 p.m

(c)

bundles

~

100 Ixm 12

(d)

9 6 200 ~m

100 ~tm

FIG. 2. Polarizing microscopy images of DNA aggregate at various spermidine concentrations. The aggregate recovered from sample tube was observed between crossed polars under an Olympus BX60 microscope. (a) Fiber (Type I) at 0.45 mM spermidine, (b) fiber bundles (Type II) at 0.50 mM spermidine, (c) a highly condensed phase (Type III) at 0.55 mM spermidine, and (d) disaggregation of Type III by the dilution of spermidine concentration to 0.35 mM. Even at spermidine concentration lower than 0.45 mM, the aggregated form similar to Type I is observed.

3

conditions

Non-aggregatin; • - . conditions

0 0

° ° "•

I

h

I

I

20

40

60

80

100

DNA concentration [Ixg/ml] FIG. 3. Ratio of CSPD/CDNA_Prequired for the DNA aggregation. Symbols: closed circle, CSPD_t~h/CDnA.P at the threshold of aggregation; open triangle, CSpO_comp/CDsA.Pat the completion of aggregation.

VOL. 92, 2001

NOTES

0.6

Aggregating conditions ~ ~ ' ~ ~)....,~ .......

0.4

& 8

......

AZ ~ Aggregation process /

.........................

0.2

Non-aggregating conditions

0

20

40

60

80

100

DNA concentration [p.g/ml] FIG. 4. Estimation of the concentration of free spermidine Cfrec.SpD at the threshold and completion of the DNA aggregation assuming that 0 is fixed at a given value. Above 20 pg/ml DNA, Cfr~.SpD vaFies significantly between the threshold and the completion. The dotted and solid curves correspond to the limitation of non-aggregating condition, or the threshold, and the completion, respectively. Symbols: closed circle, 0=0.8 at the threshold; open circle, 0=0.4 at the threshold; closed triangle, 0 =0.9 at the completion; open triangle, 0 =0.5 at the completion. concentration. It reveals that an increase in DNA concentration decreases both CSeD.thr~sh/CDNA.e and CSPD~omp/CDNA.P under the experimental condition used. This indicates that the DNA aggregation is facilitated by an increase in DNA concentration. However, we note that a higher DNA concentration affects the molar ratio to a lesser degree, namely, an attenuation behavior. Above 100 ~tg/ml DNA, the ratio may be kept constant according to the report on spermineinduced DNA aggregation by Raspaud et al. (6). Cfree.SPD Was estimated from CSPD, CDNA.P~and the fraction of phosphate neutralized by spermidine. Fraction 0 is defined as: 0 = 3 Cbo~d.seo/CoNg.e

(1)

where Cbound.SPD denotes the molarity of bound spermidine. 3Cbound_SPo means that three negative phosphates are neutralized by one spermidine molecule. Using the above equation, Cf~.sv D was calculated as follows: Cf~e.SPD= CspD- Cbo~d.SPO= CspD- 0 (CDNA.p/3)

(2)

To examine the effect of 0 on Cf~e~.svo,0 was varied from 0.4 to 0.9. Here, 0.9 was employed as the maximum value

185

according to the Manning counterion condensation theory (8). The estimation results are shown in Fig. 4. Even if 0 changes significantly, it has a little effect on Cf~o.svD. In any case, Cf~.spD at the threshold declines with an increase in DNA concentration. On the other hand, Cf~.spD at the completion increases. Hence, the present estimation suggests that C~e.spD strongly depends on DNA concentration, and also that Cf~e.SPD, as well as the aggregate morphology, varies significantly between the threshold and the completion of aggregation at high DNA concentration as shown in Fig. 4. In the case of using high DNA concentration, we assume that both the variations and the dependence on DNA concentration revealed in this study are key parameters for the design of in vitro RNA synthesis using the DNA aggregate. Furthermore, dotted curves in Figs. 3 and 4 also show the critical conditions of CSPD/CDNA.P and Cfree_SPDfor DNA to exist in a random coil. Therefore, the figures provide useful information for the optimization of the conventional RNA synthesis, which is performed in the absence of aggregated form of DNA. REFERENCES

1. Krasnow, M.A. and Cozzarelli, N.R.: Catenation of DNA rings by topoisomerases. Mechanism of control by spermidine. J. Biol. Chem., 257, 2687-2693 (1982). 2. Baeza, I., Gariglio, P., Rangei, L.M., Chavez, P., Cervantes, L., Arguello, C., Wong, C., and Montanez, C.: Electron microscopy and biochemical properties of polyamine-compacted DNA. Biochemistry, 26, 6387-6392 (1987). 3. Sikorav, J.-L., Pelta, J., and Livolant, E: A liquid crystalline phase in spermidine-condensed DNA. Biophys. J., 67, 1382-1392 (1994). 4. Pelta, J., Durand, D., Doucet, J., and Livolant, F.: DNA mesophases induced by spermidine: structural properties and biological implications. Biophys. J., 71, 48-63 (1996). 5. Pelta, J., Livolant, F., and Sikorav, J.-L.: DNA aggregation induced by polyamines and cobalthexamine. J. Biol. Chem., 271, 5656-5662 (1996). 6. Raspaud, E., Oivera de la Cruz, M., Sikorav, &-L., and Livolant, F.: Precipitation of DNA by polyamines: a polyelectrolyte behavior. Biophys. J., 74, 381-393 (1998). 7. Lin, Z., Wang, C., Feng, X., Liu, M., Li, &, and Bai, C.: The observation of the local ordering characteristics of spermidine-condensed DNA: atomic force microscopy and polarizing microscopy studies. Nucleic Acids Res., 26, 32283234 (1998). 8. Manning, G.S.: The molecular theory of polypeptide solutions with applications to the electrostatic properties of polynucleotides. Quart. Rev. Biophys., 11, 179-246 (1978).