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Fluorescence of nucleic acids with ethidium bromide: An indication of the configurative state of nucleic acids
210
SHORT COMMUNICATIONS
periments were performed several times and concentration factors of 4 9-fold were obtained. DAVIDSON11 has reported the concentration of rRNA cistrons in E. coli by CsC1 density-gradient centrifugation. Recently, COLLI A~,'D OISHI~2 concentrated the rRNA cistrons in B. subtilis 4o-fold by sedimentation of D N A - r R N A hybrids on Cs2S0~-Hg density gradient. I also concentrated the rRNA cistrons in 13. subtilis about 3o-fold in double-stranded state by CsC1 density-gradient centrifugation performed with an angle rotor s. The concentration procedure described here offers advantages of rapidity and technical simplicity as compared to that used by the above authors. The author wishes to thank Professors Y. Ikeda and H. Saito for their interest in and support of this investigation.
Institute o~ Applied Microbiology, University o~ Tokyo, Tokyo (Japan) i 2 3 4 5 6 7 8 9 IO II 12
HIDEO TAKAHASHI
b2. MIURA, Biochim. Biophys. AcIa, 55 (1962) 62. J. K. MIDGLEY, Biochim. Biophys. Acta, 61 (1962) 513 . H. TAKAHASHI, H. SAITO AND Y. IKEDA, J. Gen. Appl. Microbiol., 15 (1969) 2o9. I. SMITH, D. DUBNAU, P. MORELL AND J. MARMUR, J. 3101. Biol., 33 (1968) 1-' 3 . M. OISHI AND N. SUEOKA, Pro& Nail. Acad. Sci. U.S., 54 (1965) 483 . A. P. NYGAARD AND ]3. D. HALL, Biochem. Biophys. Res. Commm¢., 12 (1963) 98. W. G. FLAMM, E. BOND AND H. E. BURR, Biochim. Biophys. dcla, 129 (1966) 31o. M. TAKAHASHI, in p r e p a r a t i o n . H. TAKAHASttI, H. SAITO AND Y. IKEDA, Biochim. Biophys. dcla, [34 (t967) r24. D. GILLESPIE AND S. SPIEGEL1MAN, J . l"~Iol. Biol., 12 (1965) 829. P. F. DAVIDSON, Science, 152 (1966) 5o9. ~V. COLLI AND M. OISHI, Federation Proe., 28 (I969) 531.
Received May i9th, 1969 Biochim. Biophys. dcta, 19o (1969) 214-216
BBA 93445
Fluorescence of nucleic ocids with ethidium bromide: configurotive stote of nucleic acids
An indicotion of the
Ethidium bromide binds to configuration-specific regions of nucleic acids to give fluorescent complexes 1 a. As the rate of binding depends on the conformation of tile nucleic acid, it was attempted to find out whether the fluorescent behaviour of a certain nucleic acid reflects its structural features. Tobacco mosaic virus (TMV) RNA was extracted from TMV with phenol and sodium dodecyl sulphate, precipitated with ethanol and washed twice with salt-free ethanol before use. Highly polymerised DNA, ethidium bromide and tRNA were obtained from Calbiochem. A coleman Model-i 4 spectrophotometer equipped with a mercury light source and devised for fluorimetric measurements was used. The mercury line of 365 my A b b r e v i a t i o n : TMV, t o b a c c o m o s a i c virus.
Biochim. Biophys. dcta, i9o (z969) 216-219
SHORT COMMUNICATIONS
217
served for excitation, and filter selected tile emitted light above 56o m#. Ethidium bromide was added to a given solution of nucleic acid to make IO/~g/ml. This is above binding saturation for DNA concentrations below ioo/~g/ml. Fluorescence intensity was calculated as percentage increment above a blank of ethidium bromide in the tested medium, as compared to a given standard considered as IOO %. General consideration. Three distinctive types of nucleic acids, exhibiting different configurations, were selected for this work: (a) TMV RNA, a highly polymerised RNA molecule. This is a flexible molecule with a certain degree of secondary structure and, like other RNA molecules of its size, has a rod-shaped configuration resulting from hairpin overfolding of its basic secondary structure 4. This RNA exhibits an easy configurative transition upon changing of the ionic environment 5. (b) tRNA, a family of small RNA molecules, having a high degree of secondary structure. However, being small in size, their ability to form tertiary structure is somewhat limited 4. (c) DNA, having the highest possible degree of secondary structure, being helical to its entire length. In spite of its size, it is a rigid inflexible molecule unable to fold back on itself, and therefore lacks any tertiary structure in its general sense. The three configurative types of nucleic acids were exposed to conditions that influence their structural state, and the resulting fluorescent behaviour under the various conditions was studied in the following manner. Speci/ic /luorescence increment. Results of the fluorescence of various concentrations of the different types of nucleic acids with ethidium bromide are presented in Fig. I. These results confirm previous reports 1,6, showing linear increase in fluorescence incremerJ~ with increase of nucleic acid concentration. Additionally they demonstrate a steeper slope for the more configurative structures. Thus DNA exhibited the steepest slope while TMV RNA the least steep one. It is also shown
#l
J
l 21
/J
t~
"0'
tRNA
c~
m
I l
10
20 30 CONCENTRATION (~ug JmL )
40
50
0.02
0.0Z*
0.06 [NaCt](M)
00B
0~1
Fig. i. Fluorescence of different t y p e s of nucleic acids w i t h e t h i d i u m bromide, as c o m p a r e d to a c o m m o n s t a n d a r d (5 ° / ~ g / m l D N A ) . All solutions (except where noted) were m a d e in I mM NaC1. Fig. 2. Fluorescence of different nucleic acids (25 # g / m l ) at m o d e r a t e salt concentrations. The fluorescence for each species of nucleic acid at o.i M NaC1 w a s considered as IOO % intensity.
Biochim. Biophys. Acts, 19o (1969) 216-219
218
SHORT COMMUNICATIONS
that TMV RNA largely compensates for its limited secondary structure by its extensive tertiary one, since increased salt concentration (IO mM) of the TMV-RNA solutions brought about a slope similar to that of tRNA. Fluorescence profiles at diHerent salt concentrations. The configurative state of TMV RNA highly depends on Na + concentration. Thus, at low ionic strength it stretches out to give an open structure of about 3 S but folds up rapidly with increased Na + concentrations to ultimately give a compact structure of about 3 ° S at o.I M NaC1 (ref. 5). However, molecules with a high degree of secondary structure are less dependent on the environmental ionic strength. Hence for example it is difficult to obtain a hyperchromic effect with t R N A b y merely lowering the ionic strengtM. This is well expressed in terms of fluorescence at moderate salt concentrations (Fig. 2). As expected fluorescence rates of DNA and t R N A vary only slightly with different salt concentrations at that range, but TMV-RNA fluorescence is increased considerably with increased salt concentrations. However, the drop of the t R N A curve at low salt and this of the DNA at high salt concentrations led to further examination at a wider range of salt concentrations (Fig. 3). Basically two types of curves were obtained: a trapeze-shaped curve for these species of nucleic acids which largely have helical structures, and a linear rising curve for TMV RNA with its flexible random coiled structure. The increase in fluorescence rates of TMV RNA and of the initial part of the t R N A curve clearly involve two distinctive mechanisms demonstrated by the break in the rising curve at I mM salt. This is in full agreement with WARING2 and with LE PECQ AND PAOLETTI3 who reported two different binding mechanisms of ethidium bromide to nucleic acids. The continuous rise of the TMV-RNA curve is well explained b y its easy configurative transition allowing overfolding into a compact tertiary structure and resulting in better binding of the ethidium bromide. As for the trapeze-shaped curves, it might be assumed that the initial increase in salt concentrations causes a certain tightening of duplex structures through reducing repulsion forces, and this •2°,
-
,
r
~/o~
.~
~00 •
i
o
\
~/
g
-
90~
i
-40°
-~o%
[Noc~]
50 °
60 ° TEMPERATURE
70 °
80~
Fig. 3. Fluorescence of nucleic acids at a wide range of salt concentrations. Fluorescence int e n s i t y of each species of nucleic acid at o.i M was considered as i o o ~o. Fig. 4- Absorbance ( O - G ) a n d fluorescence ( O - Q ) expressions of the melting curves of D N A (5 ° / , g / m l in o.I mM NaC1).
Biochim. Biophys. tlcta, 19o (1969) 216-219
SHORT COMMUNICATIONS
219
brings about a better geometrical fitness for the intercalated dye and a certain increase in fluorescence. Further increase in salt concentrations could no more affect the structure of single DNA or t R N A molecules but might still permit intermolecular interactions forming various types of 'aggregates' such as multistranded structures which reduce fluorescence intensity a. Low salt concentrations alone could not break highly helical structures; therefore fluorescence values at o.I mM NaC1 dropped only to about 60 °/o with DNA and to 20 % with tRNA. TMV RNA, however, could stretch out its coiled structure and approach near zero fluorescence values. Fluorescent expression o/ thermal transitions. If the secondary structure of nucleic acids is the major configurative factor allowing ethidium bromide binding and fluorescence, then a decrease in fluorescence values should be obtained upon 'melting'. To check this point nucleic acids were made in o.I mM NaC1 to minimize coiling of the separated strands. Absorbance at 260 m# was read at different temperatures, and the samples were cooled rapidly in solid ice. Then, ethidium bromide was added, and their fluorescent intensities were measured. The results obtained with DNA are included in Fig. 4. TMV RNA and t R N A gave constant fluorescence values, although absorbance melting curves could be obtained. I t seems as if once the separated strands of DNA fold to give a coiled structure, the process is not easily reversed. However, with the RNA molecules, renaturation involves transitions in the very same strands which are easily reversed to give back their original fluorescence values. A close analysis of the fluorescent melting curve of DNA reveals dynamic changes at temperature levels (4o-55 °) where no absorbance changes could be observed. This is in agreement with our former hypothesis that ethidium bromide binding requires a certain tightness of the duplex structure. Hence, looser structure, although still helical to its entire length, resulted in a decrease in fluorescence intensities. However, once completely separated, the single strands give lower but constant fluorescence values.
Virus Laboratory, The Hebrew University o/ Jerusalem, Faculty o/ Agriculture, Rehovoth (Israel)
ILAN SELA
J. B. LE PECQ AND C. PAOLETTI, Anal. Biochem., 17 (1966) ioo. M. J. WARING, Biochim, Biophys. Acta, 114 (1966) 234. J. ]3. LE PECQ AND C. PAOLETTI, J. Mol. Biol., 27 (1967) 87. A. S. SI'IRIN, in J. N. DAVIDSON AND W. E. COHN, Progress in Nucleic Acid Research, Voh i, Academic Press, N e w Y o r k - L o n d o n , 1963, p. 3Ol. 5 H. BOEDTKER, J. Mol. Biol., 2 (196o) 171. 6 K, VAN DYKE AND C. SZUSTKIEWICZ, Anal. Biochem., 23 (1968) lO9. I 2 3 4
Received May 5th, 1969 Biochim. Biophys. Acta, 19 ° (1969) 216-219
SHORT COMMUNICATIONS
periments were performed several times and concentration factors of 4 9-fold were obtained. DAVIDSON11 has reported the concentration of rRNA cistrons in E. coli by CsC1 density-gradient centrifugation. Recently, COLLI A~,'D OISHI~2 concentrated the rRNA cistrons in B. subtilis 4o-fold by sedimentation of D N A - r R N A hybrids on Cs2S0~-Hg density gradient. I also concentrated the rRNA cistrons in 13. subtilis about 3o-fold in double-stranded state by CsC1 density-gradient centrifugation performed with an angle rotor s. The concentration procedure described here offers advantages of rapidity and technical simplicity as compared to that used by the above authors. The author wishes to thank Professors Y. Ikeda and H. Saito for their interest in and support of this investigation.
Institute o~ Applied Microbiology, University o~ Tokyo, Tokyo (Japan) i 2 3 4 5 6 7 8 9 IO II 12
HIDEO TAKAHASHI
b2. MIURA, Biochim. Biophys. AcIa, 55 (1962) 62. J. K. MIDGLEY, Biochim. Biophys. Acta, 61 (1962) 513 . H. TAKAHASHI, H. SAITO AND Y. IKEDA, J. Gen. Appl. Microbiol., 15 (1969) 2o9. I. SMITH, D. DUBNAU, P. MORELL AND J. MARMUR, J. 3101. Biol., 33 (1968) 1-' 3 . M. OISHI AND N. SUEOKA, Pro& Nail. Acad. Sci. U.S., 54 (1965) 483 . A. P. NYGAARD AND ]3. D. HALL, Biochem. Biophys. Res. Commm¢., 12 (1963) 98. W. G. FLAMM, E. BOND AND H. E. BURR, Biochim. Biophys. dcla, 129 (1966) 31o. M. TAKAHASHI, in p r e p a r a t i o n . H. TAKAHASttI, H. SAITO AND Y. IKEDA, Biochim. Biophys. dcla, [34 (t967) r24. D. GILLESPIE AND S. SPIEGEL1MAN, J . l"~Iol. Biol., 12 (1965) 829. P. F. DAVIDSON, Science, 152 (1966) 5o9. ~V. COLLI AND M. OISHI, Federation Proe., 28 (I969) 531.
Received May i9th, 1969 Biochim. Biophys. dcta, 19o (1969) 214-216
BBA 93445
Fluorescence of nucleic ocids with ethidium bromide: configurotive stote of nucleic acids
An indicotion of the
Ethidium bromide binds to configuration-specific regions of nucleic acids to give fluorescent complexes 1 a. As the rate of binding depends on the conformation of tile nucleic acid, it was attempted to find out whether the fluorescent behaviour of a certain nucleic acid reflects its structural features. Tobacco mosaic virus (TMV) RNA was extracted from TMV with phenol and sodium dodecyl sulphate, precipitated with ethanol and washed twice with salt-free ethanol before use. Highly polymerised DNA, ethidium bromide and tRNA were obtained from Calbiochem. A coleman Model-i 4 spectrophotometer equipped with a mercury light source and devised for fluorimetric measurements was used. The mercury line of 365 my A b b r e v i a t i o n : TMV, t o b a c c o m o s a i c virus.
Biochim. Biophys. dcta, i9o (z969) 216-219
SHORT COMMUNICATIONS
217
served for excitation, and filter selected tile emitted light above 56o m#. Ethidium bromide was added to a given solution of nucleic acid to make IO/~g/ml. This is above binding saturation for DNA concentrations below ioo/~g/ml. Fluorescence intensity was calculated as percentage increment above a blank of ethidium bromide in the tested medium, as compared to a given standard considered as IOO %. General consideration. Three distinctive types of nucleic acids, exhibiting different configurations, were selected for this work: (a) TMV RNA, a highly polymerised RNA molecule. This is a flexible molecule with a certain degree of secondary structure and, like other RNA molecules of its size, has a rod-shaped configuration resulting from hairpin overfolding of its basic secondary structure 4. This RNA exhibits an easy configurative transition upon changing of the ionic environment 5. (b) tRNA, a family of small RNA molecules, having a high degree of secondary structure. However, being small in size, their ability to form tertiary structure is somewhat limited 4. (c) DNA, having the highest possible degree of secondary structure, being helical to its entire length. In spite of its size, it is a rigid inflexible molecule unable to fold back on itself, and therefore lacks any tertiary structure in its general sense. The three configurative types of nucleic acids were exposed to conditions that influence their structural state, and the resulting fluorescent behaviour under the various conditions was studied in the following manner. Speci/ic /luorescence increment. Results of the fluorescence of various concentrations of the different types of nucleic acids with ethidium bromide are presented in Fig. I. These results confirm previous reports 1,6, showing linear increase in fluorescence incremerJ~ with increase of nucleic acid concentration. Additionally they demonstrate a steeper slope for the more configurative structures. Thus DNA exhibited the steepest slope while TMV RNA the least steep one. It is also shown
#l
J
l 21
/J
t~
"0'
tRNA
c~
m
I l
10
20 30 CONCENTRATION (~ug JmL )
40
50
0.02
0.0Z*
0.06 [NaCt](M)
00B
0~1
Fig. i. Fluorescence of different t y p e s of nucleic acids w i t h e t h i d i u m bromide, as c o m p a r e d to a c o m m o n s t a n d a r d (5 ° / ~ g / m l D N A ) . All solutions (except where noted) were m a d e in I mM NaC1. Fig. 2. Fluorescence of different nucleic acids (25 # g / m l ) at m o d e r a t e salt concentrations. The fluorescence for each species of nucleic acid at o.i M NaC1 w a s considered as IOO % intensity.
Biochim. Biophys. Acts, 19o (1969) 216-219
218
SHORT COMMUNICATIONS
that TMV RNA largely compensates for its limited secondary structure by its extensive tertiary one, since increased salt concentration (IO mM) of the TMV-RNA solutions brought about a slope similar to that of tRNA. Fluorescence profiles at diHerent salt concentrations. The configurative state of TMV RNA highly depends on Na + concentration. Thus, at low ionic strength it stretches out to give an open structure of about 3 S but folds up rapidly with increased Na + concentrations to ultimately give a compact structure of about 3 ° S at o.I M NaC1 (ref. 5). However, molecules with a high degree of secondary structure are less dependent on the environmental ionic strength. Hence for example it is difficult to obtain a hyperchromic effect with t R N A b y merely lowering the ionic strengtM. This is well expressed in terms of fluorescence at moderate salt concentrations (Fig. 2). As expected fluorescence rates of DNA and t R N A vary only slightly with different salt concentrations at that range, but TMV-RNA fluorescence is increased considerably with increased salt concentrations. However, the drop of the t R N A curve at low salt and this of the DNA at high salt concentrations led to further examination at a wider range of salt concentrations (Fig. 3). Basically two types of curves were obtained: a trapeze-shaped curve for these species of nucleic acids which largely have helical structures, and a linear rising curve for TMV RNA with its flexible random coiled structure. The increase in fluorescence rates of TMV RNA and of the initial part of the t R N A curve clearly involve two distinctive mechanisms demonstrated by the break in the rising curve at I mM salt. This is in full agreement with WARING2 and with LE PECQ AND PAOLETTI3 who reported two different binding mechanisms of ethidium bromide to nucleic acids. The continuous rise of the TMV-RNA curve is well explained b y its easy configurative transition allowing overfolding into a compact tertiary structure and resulting in better binding of the ethidium bromide. As for the trapeze-shaped curves, it might be assumed that the initial increase in salt concentrations causes a certain tightening of duplex structures through reducing repulsion forces, and this •2°,
-
,
r
~/o~
.~
~00 •
i
o
\
~/
g
-
90~
i
-40°
-~o%
[Noc~]
50 °
60 ° TEMPERATURE
70 °
80~
Fig. 3. Fluorescence of nucleic acids at a wide range of salt concentrations. Fluorescence int e n s i t y of each species of nucleic acid at o.i M was considered as i o o ~o. Fig. 4- Absorbance ( O - G ) a n d fluorescence ( O - Q ) expressions of the melting curves of D N A (5 ° / , g / m l in o.I mM NaC1).
Biochim. Biophys. tlcta, 19o (1969) 216-219
SHORT COMMUNICATIONS
219
brings about a better geometrical fitness for the intercalated dye and a certain increase in fluorescence. Further increase in salt concentrations could no more affect the structure of single DNA or t R N A molecules but might still permit intermolecular interactions forming various types of 'aggregates' such as multistranded structures which reduce fluorescence intensity a. Low salt concentrations alone could not break highly helical structures; therefore fluorescence values at o.I mM NaC1 dropped only to about 60 °/o with DNA and to 20 % with tRNA. TMV RNA, however, could stretch out its coiled structure and approach near zero fluorescence values. Fluorescent expression o/ thermal transitions. If the secondary structure of nucleic acids is the major configurative factor allowing ethidium bromide binding and fluorescence, then a decrease in fluorescence values should be obtained upon 'melting'. To check this point nucleic acids were made in o.I mM NaC1 to minimize coiling of the separated strands. Absorbance at 260 m# was read at different temperatures, and the samples were cooled rapidly in solid ice. Then, ethidium bromide was added, and their fluorescent intensities were measured. The results obtained with DNA are included in Fig. 4. TMV RNA and t R N A gave constant fluorescence values, although absorbance melting curves could be obtained. I t seems as if once the separated strands of DNA fold to give a coiled structure, the process is not easily reversed. However, with the RNA molecules, renaturation involves transitions in the very same strands which are easily reversed to give back their original fluorescence values. A close analysis of the fluorescent melting curve of DNA reveals dynamic changes at temperature levels (4o-55 °) where no absorbance changes could be observed. This is in agreement with our former hypothesis that ethidium bromide binding requires a certain tightness of the duplex structure. Hence, looser structure, although still helical to its entire length, resulted in a decrease in fluorescence intensities. However, once completely separated, the single strands give lower but constant fluorescence values.
Virus Laboratory, The Hebrew University o/ Jerusalem, Faculty o/ Agriculture, Rehovoth (Israel)
ILAN SELA
J. B. LE PECQ AND C. PAOLETTI, Anal. Biochem., 17 (1966) ioo. M. J. WARING, Biochim, Biophys. Acta, 114 (1966) 234. J. ]3. LE PECQ AND C. PAOLETTI, J. Mol. Biol., 27 (1967) 87. A. S. SI'IRIN, in J. N. DAVIDSON AND W. E. COHN, Progress in Nucleic Acid Research, Voh i, Academic Press, N e w Y o r k - L o n d o n , 1963, p. 3Ol. 5 H. BOEDTKER, J. Mol. Biol., 2 (196o) 171. 6 K, VAN DYKE AND C. SZUSTKIEWICZ, Anal. Biochem., 23 (1968) lO9. I 2 3 4
Received May 5th, 1969 Biochim. Biophys. Acta, 19 ° (1969) 216-219