Interaction of methylene blue with transfer RNA — a spectroscopic study

Interaction of methylene blue with transfer RNA — a spectroscopic study

ELSEVIER Chemico-Biological Interactions 97 (1995) 199-214 Interaction of methylene blue with transfer RNA - a spectroscopic study Thomas Antony, M...

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ELSEVIER

Chemico-Biological Interactions 97 (1995) 199-214

Interaction of methylene blue with transfer RNA - a spectroscopic study Thomas Antony, M. Atreyi*, M.V.R. Rao Department of Chemistry, University of Delhi, Delhi-l 10007, India

Received 22 April 1994; revision received 9 January 1995; accepted 13 .ianuary 1995

Abstract The binding of methylene blue (MB) with tRNA was studied using absorption, fluorescence and circular dichroic spectroscopy. In the spectral titration of MB with tRNA, hypochromism was observed in the absorption maximum of the dye in the visible region till P/D = 4 and thereafter the intensity increased with a red shift at P/D > 9, indicating electrostatic and intercalative binding at low and high P/D ratios, respectively. Analysis of absorption data, following Schwa& procedure, showed that the electrostatic binding is cooperative in nature (cooperatively parameter q = 50) with a binding constant K = 7.77 x lo3 M-l. A non-linear Scatchard plot was observed for the intercalative binding (at P/D > 4), probably due to a difference in the spectral characteristics of the dye intercalated between the base pairs and that between the bases in the single stranded domains. Quenching of fluorescence was observed for both the binding processes. In the circular dichroism spectra of tRNA-MB complexes at high P/D (- 30), nonconservative positive ICD bands were seen at 620 and 680 nm while at low P/D (- 2), two conservative negative CD bands at 300 and 660 nm and two bisignate bands with cross avers at 565 and 605 nm were observed. The short wavelength component of the bisignate band at 565 nm is negative while that of the 605-nm band is positive, indicating that the former arises from left handed and the latter from the right handed helical disposition of dye molecules along the tRNA backbone. The changes in the CD spectrum of tRNA on dye binding could be due to a conformational change of the nucleic acid or a negative CD being induced at that region. Keywords:

Methylene blue; Transfer RNA; UV fluorescence; Circular dichroism; ICD; Binding constant

l

Corresponding author, Tel: +91 I1 7257794.

0 1995 Elsevier Science Ireland Ltd. All rights reserved 0009-2797/95/.$09.50 SSDI 0009-2797(95)03616-T

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1. Iotroductioo

Methylene blue (MB) (Fig. I), is a polyaromatic cationic dye used in the staining of nucleic acids and for the diagnosis of diseases including carcinoma [l-4]. Binding of MB to transfer RNA is shown to have a pronounced effect on the aminoacylation activity of the nucleic acid [5]. Electrophoresis of tRNA, prestained with MB, showed many bands in the profile at D/P (dye/phosphate) ratios ~0.16 indicating the formation of a variety of complexes. This was attributed to tRNA-MB complexes which differ in the extent of dimerisation/aggregation of bound dye molecules. Based on these studies, Murthy et al. [5] suggested that the enhancement of aminoacylation activity of the nucleic acid is probably due to the change in its structure on dye binding. In the present work, a detailed study of the interaction of MB with tRNA was undertaken using UV, fluorescence and circular dichroism (CD) spectroscopy. 2. Materials and methods Methylene blue was purchased from GL Chemical Testing Lab. (India) and was purified by the method of Bergmann et al. [6]. Baker’s yeast tRNA was a gift from Dr M.R.V. Murthy, University of Laval, Canada. The dyes, ethidium bromide (EB) and proflavine (PF), were purchased from Sigma Chemical Co. (St. Louis, MO) and a&dine orange (AO) from SDS Lab. Chem. (India) and used without further purilication. The concentrations of various solutions were determined using the molar absorptivity values listed below. AO: e&2 = 54 ooo M-’ EB: ~480 = mo

M-l

171

Cm-'

Cm-'

PI

PF: Ed = 41 000 M-’ cm-’

[91

MB: eW = 76 000 M-’ cm-’

[lOI

tRNA: e260= 6900 M-’ cm-’

[71

All titrations were performed at room temperature (25 f 2°C) in 1.0 mM sodium chloride/O.1 mM sodium citrate (SSC) buffer, pH 7.0. UV spectra were recorded on a Gilford 2600 spectrophotometer attached to a Hewlett-Packard printer-plotter. Fluorescence spectra were recorded on a Hitachi 650-60 spectrofluorimeter equipped with a Hitachi 056 recorder. A 5-nm slit width was used for recording the excitation as well as the emission spectra. Cells with a 1.O-cm path length were used for absor-

Fig. 1. Structure

of methylene

blue.

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bance and fluorescence studies. Circular dichroic (CD) spectra were recorded on a Jasco J500-A spectropolarimeter attached to a DP 5OONdata processor. A l.O-cm path length cylindrical cell was used for CD measurements. A sensitivity of 1 m’/cm and a scan speed of 50 nm/min were used. Since it is convenient to express the molar ellipticity in terms of the nonvarying parameter, the values are expressed as deg cm* dmol-’ in terms of tRNA concentration (per phosphate) for ‘direct titration’ in which MB was added to tRNA and in terms of dye concentration for ‘reverse titration’. The data are expressed in terms of phosphate to dye (P/D) ratios for reverse titations and D/P for direct titrations. 3. Results The absorption spectrum of MB in the visible region has a X,,, of 664 nm with 0.600

0.540

0.480

0.420

0.36C

t

I-

0.300

E C

0.24C

;

I-

0 :: a 0.

I BC )-

)-

0

06C

l-

I

525

1

I

575

1

I

625

I

1

675

I

1

725

X(nm)-

Fig. 2. Absorption spectra of tRNA-MB complexes at different P/D ratios in 1.1 mM SSC buffer, pH 7.0. P/D ratios as follows: (1) 0, (2) 4.7, (3) 35.2. Concentration of MB = 8.8 x 10m6M.

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a shoulder at 610 nm. When tRNA was added, the intensity of both these bands decreased until P/D = 4 and remained without any further change till P/D = 9. On further addition of tRNA, the intensity of both bands increased with a red shift; saturation was observed in the absorption spectra at P/D = 186. The red shift of the band maximum at this P/D ratio was 15 nm. Typical absorption spectra of tRNAMB complexes at three P/D values are depicted in Fig. 2. MB is a weak fluorophore and its emission spectrum in 1.1 mM SSC buffer has a hem,,, at 681 nm on excitation at 600 nm (cont. of MB = 1 x 10e6 M). Upon addition of tRNA, the fluorescence emission intensity decreased quite sharply below

I

1

I

70

60 t

Fig. 3. Fluorescence emission spectra of tRNA-MB complexes at different P/D ratios in 1.1mM SSC buffer, pH 7.0. P/D ratios as follows: (1) 0.0, (2) 7.2, (3) 21.5, (4) 50.1, (5) 107.3, (6) 280.0. Concentration of MB = 1.0 x 10e6 M; XeX= 600 nm, slit width = 5 nm.

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P/D of 5 and then gradually, with a blue shift in the XemmaX, which was noticeable at P/D 1 36. Saturation in the fluorescence spectra was observed at P/D = 280. The shift in the hem,, at this P/D ratio was 13 nm (Fig. 3). In 1.1 mM SSC buffer, tRNA has a positive CD band at 260 nm ((0) = I1 000 deg cm* dmol-‘) and a weak negative band at 240 nm (((3)= 3 000 deg cm’ dmol-‘). When MB was added to tRNA, the intensity of the positive CD band of RNA decreased and simultaneously a negative band developed around 300 nm. The molar ellipticity of the 260~nm CD band decreased from 11 000 to 2000 deg cm* dmol-’ when D/P was increased to 0.6. In the visible region, a nonconservative induced circular dichroism (ICD) band was observed at 590 nm at D/P = 0.06. On further addition of the dye, a conservative band was induced with a positive maximum at 590 nm and a negative extremum at 620 nm with the crossover at 605 nm. At higher D/P values (2 0.12), a new negative band was seen about 550 nm. The CD spectra of tRNA-MB complexes at different D/P values are given in Fig. 4 and the variation of ellipticity of CD bands with D/P is depicted in Fig. 5.

I

260

1

I

340

I

I

I

I

420 h(nm)

500

I

580

I

I

660

--

Fig. 4. CD Spectra of tRNA-MB complexes at different D/P ratios in 1.1 mM SSC buffer, pH 7.0. DIP ratios as follows: (1) 0.0, (2) 0.12, (3) 0.18, (4) 0.24, (5) 0.30, (6) 0.36, (7) 0.42, (8) 0.50, (9) 0.60. Concentration of tRNA = 2.4 x 10e5 M.

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20-

16-

-0”: ; z E I? E ” m 0,

6-

4-

?.? ‘0

WI260

x

0

aI

0 2’0

3

04

05

C

4-

D/ P

Fig. 5. Variation of molar ellipticity with D/P of tRNA-MB complexes at different wavelengths in the direct titration.

Circular dichroic spectra were also obtained using a titration procedure identical to the one used in UV absorbance and fluorescence studies, that is, the macromolecule was added to the dye. At a low P/D ratio (P/D = 2.4), features of the ICD band were similar to those observed at equivalent D/P ratios (Fig. 6). However, with an increase in tRNA concentration, the semiconservative band with crossover at 605 nm became a positive band with a maximum about 610 nm and at the same time there was a sign reversal for the 660-nm nonconservative negative band. In the UV region, the intensity of the negative ICD band centered around 300 nm of tRNA-MB complex obtained at low P/D ratios, gradually decreased with the addition of tRNA and completely vanished at P/D = 8.5 (Fig. 7). The effect of NaCl on binding of MB to tRNA was studied at two P/D ratios (P/D = 1.4 and 27) where the binding is predominantly electrostatic (P/D = 1.4) or intercalative (P/D = 27) in nature (see section 4, Discussion). The ICD of tRNA-MB

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540

Inreracrions 97 (1995) 199-214

580

620

660

205

700

h(nm)

Fig. 6. CD spectra of tRNA-MB complexes at high P/D ratios in 1.1 mM SSC buffer, pH 7.0. P/D ratios as follows: (1) 2.4, (2) 7.3, (3) 19.5, (4) 24.2, (5) 34.1. Concentration of MB = 9.5 x 10e6 M.

complex at P/D = 1.4 gradually decreased with the addition of NaCl at the molar ellipticities of the CD bands at 300, 590 and 620 nm became almost zero in 300 mM NaCl indicating nearly complete dissociation of the complex. However, for the complex at P/D = 27, the ICD band at 680 nm increased initially on the addition of NaCl and decreased only after 40 mM NaCl in the solution, the other band at 610 nm decreased with the first addition itself. A decrease of about 70% was observed in the molar ellipticities of these bands (610 and 680 nm) in 300 mM NaCl. The variation of (0) at different wavelengths of tRNA-MB complexes (P/D = 1.4 and 27) with the addition of NaCl is depicted in Figs. 8 and 9. The hypochromism followed by hyperchromism with red shift observed for the titration of MB with tRNA clearly indicates two modes of binding of the dye. Since there was no change in the absorption spectra between P/D = 4 and 9, assuming that, at low P/D (<4), only one binding process is taking place and that the second process becomes operative at high P/D values (see section 4, discussion), the binding constant, for the first process which could be deemed to arise on account of electrostatic interaction, was calculated following Schwarz’s method [ 11,121. This binding mode is cooperative in nature (cooperativity parameter q = 50) with a binding constant of 7.77 x lo3 M-‘. Assuming that the electrostatic binding is nearly complete at P/D = 4, where a saturation was observed in the absorption spectra and the electrostatically bound dye molecules intercalate between the base pairs/bases with further addition of tRNA,

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50

40

rr) ‘0 x

1

0

z l-d

IO (--) 20

I

220

260

340

300 ?I

tnm)

Fig. 7. CD spectra tRNA-MB complexes at high P/D ratios in I.1 mM SSC buffer, pH 7.0 (in the UV region). P/D ratios as follows: (1) 1.2, (2) 3.6, (3) 8.5, (4) 13.4. Concentration of MB = 1.9 x lo-’ M.

the concentration of the bound dye molecules at high P/D ratios was calculated. The resultant Scatchard plot is shown in Fig. 10. The nonlinear nature of Scatchard plot indicates that there is more than one binding mode at these P/D values. This is probably due to the difference in the spectral behaviour of the dye bound between the base pairs and that between the bases in the single stranded domains of tRNA. 4. Discussion

tRNAs are polyribonucleotides with over 75 units arranged in the shape of a clover leaf that contains four stem regions and three loop regions (Fig. 11). In the three dimensional structure, the acceptor and T stem form a continuous RNA double helix while the D stem and the anticodon stem are stacked upon each other to

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R 20

(+) I6 n

12 -

I 40

I 80

1 I20

I 160

[NoCI]

I 200

I 240

I 200

320

(mM)

Fig. 8. Variation of molar ellipticity of tRNA-MB complexes (P/D = I .4) at different wavelengths, as a of NaCl concentration.

I

I

20

I

I

60

I

I

100

I

N

140

I

I

180

I

I

220

(NaCI] (mM) -+ Fig. 9. Variation of molar ellipticity at 610 and 680 nm of tRNA-MB complexes (P/D = 27) with NaCl concentration.

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4 I_-

O iL_..~*_~-l---10.007

I-

0.009

0.011

dOI5

L_LL_L-._ 0.015

0.017

1/--

Fig. 10. Scatchard

plot for tRNA-MB

system at high P/D ratios (from absorption

Extra

Anticodon Fig. 11. Cloverleaf

Loop

Loop structure

of tRNA.

studies).

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form another double helix [14]. Although only about 40 bases are involved in Watson-Crick type of base pairing, most of these bases are stacked [ 151.In the binding of aminoacridines, which are structurally similar to MB, with various ribo- and deoxyribonucleic acids, it had been shown that at low P/D ratios (< 4), binding is by electrostatic interaction between the cationic dye and the negatively charged phosphates assisted by the stacking tendency of the dye molecules, while at high P/D ratios (> lo), intercalation of dye molecules between the nucleic acid bases is the preferred [9,15] mode. In many dye-nucleic acid systems, hypochromism and blue shift of the dye spectrum were observed for external association and a red shift with or without hyperchromism for intercalation [9,15,16]. The hypochromism observed at low P/D ratios (< 4) for the tRNA-MB system in the present work suggests electrostatic external binding at these P/D ratios. On the other hand, a red shift in the absorption spectrum has also been reported for many groove binders like Hoechst 33258 and Netropsin in their complexes with DNA [17-191, making it difficult to distinguish groove binding from intercalation, by absorption studies alone. In the present study, the dye chromophore is a planar molecule unlike the groove binders, which are associated with bent structures and also have functional groups that can give additional stability to the binding in the groove. Hence, any binding entirely in the groove is unlikely for a molecule like MB and changes in the absorption spectra suggest the possibility of intercalation of the dye between the base pairs in the double helical regions or stacking between the bases in the single stranded domains. The absence of any isosbestic points in the absorption spectra could arise from the following reasons. The molar absorptivity of electrostatically bound MB is lower than that of free MB and the difference between their X,,, is very small (Fig. 2). Hence, electrostatic binding of MB to tRNA will not show any isosbestic point. The intercalated MB has a much higher molar absorptivity and &,,,,. The absence of any clear isosbestic point in the absorption spectra at higher P/D ratio (> 4) shows that the transition from the electrostatic to the intercalative mode of binding is not a unique equilibrium between the two forms. This could be due to the overlap of the spectral behaviour of MB intercalated between the base pairs and the bases in the single stranded domains. Quenching of fluorescence was found to be associated with both the binding modes, but the extent of the decrease in intensity was much larger for the first binding process. Again, a blue shift MB hem,, is seen at high P/D ratios as observed in the absorption spectra. MB is known to readily dimerise and aggregate [6,20] and it is reported that dimerisatiomaggregation of the dye molecules is associated with a decrease in the fluorescence emission intensity. The large decrease in the emission intensity at the initial additions of tRNA indicates that the dye molecules bind to tRNA as dimers or higher aggregates. The quenching of fluorescence observed at high P/D ratios, where intercalation of the dye between the nucleic acid bases is indicated in absorption studies also, could be due to the electron/H atom transfer from the nucleic acid bases to the intercalated dye molecules [21,22]. Transient absorption and luminiscence studies of the complexes of MB with poly(dG-dC) and poly(dAdT) have shown that the rapid electron transfer from the purine bases to the singlet excited state of MB leads to the formation of the reduced form of MB and hence

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to the quenching of the fluorescence of the dye molecules [21,22]. The absence of any isoemissive point in the fluorescence spectra is probably due to the low quantum yield of electrostatically bound and intercalated MB (Fig. 3) and the presence of more than one type of intercalation as described earlier. The decrease in the ellipticity of the tRNA CD band at 260 nm with the addition of MB (Fig. 4) could be due either to a conformational change in tRNA on dye bind-

I

n

IO

T;

6

E D “E w 0,

4

5 (+I VI ‘0 2 X

z

0

(2 200

240

2!30

320

Fig. 12. CD spectra of tRNA in 1.1 mM SSC buffer, pH 7.0, as a function of temperature: (1) 3O”C,(2) SOT, (3) 7O”C, (4) 90°C.

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ing, similar to the decrease in the ellipticity on temperature induced unwinding (Fig. 12), or to a negative CD being induced in the 260-nm region, or both. The decrease in the ellipticity of tRNA on dye binding is much higher than that observed at 80-90°C where the tertiary structure of tRNA is disrupted. Hence, conformational change associated with unwinding alone cannot account for the observed change, which in turn supports the overlapping of a negative ICD band with the CD of tRNA. The changes in the 260 nm tRNA CD band are associated with the appearance of a negative extremum around 300 nm. In order to ascertain whether this band is due to a new conformation of tRNA, like that of the left handed nucleic acids, which exhibits a negative extremum around 290 nm, or any CD induced from dye transitions, the binding to tRNA of three planar dyes proflavine (PF), acridine orange (AO) and ethidium bromide (EB) was compared. All three dyes exhibit ICD around 300 nm (EB: +ve, 300 nm; PF: -ve, 285-nm shoulder; AO: -ve, 290~nm shoulder). Clearly the negative ICD of tRNA-PF and tRNA-A0 systems partially overlap that of the native CD of tRNA and alters its profile in a characteristic way similar to MB, but in the case of EB, the ICD band is positive and has a maximum at 300 nm and the overlap is minimal (Fig. 13). All the same, the tRNA band intensity is decreased in all four systems. If the 300-nm negative CD band observed in tRNA-MB complexes at high D/P ratios ( > 0.12) is due to a new conformation of tRNA, identical CD spectra should have been observed for the complexes of MB, AO, PF and EB with tRNA, irrespective of the &,,,, of these dyes in the UV region. However, the wavelength and intensity of the CD band extremum are different in the various dye-tRNA systems as shown in Fig. 13. Further, the tRNA-EB complex, unlike others, has instead a positive band at 300 nm. These observations clearly suggest that the 300~nm CD band of tRNA-MB complexes must arise from the coupling of dye transitions with each other or with that of the bases and cannot be attributed to any new conformation of tRNA. The ICD bands observed in the visible region also reveal interesting features. The intense positive ICD band of 590 nm flanked by two negative bands around 550 and 620 nm in the CD spectra (Fig. 4) can be considered to be a result of the overlap of two conservative bands with isodichroic points at 565 and 605 nm. The conservative nature of the ICD band with crossover at the absorption maximum of dimers of MB, 605 nm, indicates that the band originates from the coupling of the transitions of the dyes bound externally to the neighbouring phosphates. The appearance of the CD band (short h +ve, long X -ve) implies that this conservative band originates from dye molecules arranged in a left-handed helical fashion along some portions of the tRNA molecule [lo]. Such an orientation is also reported for CT DNA-MB complexes [lo]. In single stranded poly(rA)-MB complexes, however, the dye molecules are arranged in a right handed fashion as evident from the appearance of the CD band (short X -ve, long h +ve) [23]. These observations suggest that the arrangement of externally bound MB on the tRNA molecule is likely to be in the double stranded region. The other conservative band centered around 565 nm is relatively weak and ap-

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L

220

260

300

340

380

Nnm)

Fig. 13. CD spectra in 1.I mM SSC buffer, pH 7.0, of tRNA and its complexes with A0 (2), PF (3) and EB (4) in UV region. D/P ratios as follows: (2) 0.83, (3) 0.75, (4) 0.89.

pears only at DIP > 0.12. The appearance of this band (short X -ve, long X +ve), opposite in disposition to the other bisignate band centered around 605 nm, suggests that a different binding mode is responsible for this band. The conservative nature of this ICD band implies that this also arises from dye-dye coupling. Aggregates of MB are shown to absorb at this wavelength [24] and the dimers bound to double helical DNA are shown to have a left handed helical arrangement and, therefore, the conservative band centered about 565 nm could be due to the right handed arrangement of aggregated dye molecules along the single stranded domains of tRNA. The transitions at 290, 610 and 664 nm of MB are long-axis polarized [lo] and

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it is noteworthy that the ICD bands around 660 nm and 300 nm are nonconservative in nature and only the 610~nm band leads to a type of exciton spectrum. This only means that the bound dye molecules may not be suitably oriented to result in an effective coupling of these transitions (300 and 660 nm) in two adjacent dye molecules to result in a conservative ICD band. At high P/D ratios, where intercalation of the dye molecules was indicated from the absorption studies, the features of the ICD observed are different from those at low P/D ratios; the reversal of sign of the CD band with a shift in the &,,,, upon addition of tRNA (Fig. 6) indicates a change in the binding process. Again, the absence of any conservative ICD band around 605 nm, suggests that there is no external association of the dye molecules at these P/D ratios. The non-conservative nature of the CD bands with extrema at 610 nm also indicates that these CD bands arise from the coupling of intercalated dye molecules with the nucleic acid bases. A reversal of the sign of the CD band with a red shift in the extremum is also observed for A0 [25] and PF (present work) in their titration with tRNA. In the UV region, the induced CD at 300 nm gradually disappears with the addition of tRNA which suggests that the ICD, if any, is different at higher binding ratios and is overlapped by the strong tRNA CD band. The CD spectra also do not indicate any major conformational change of tRNA on intercalation of the dye molecules. The CD data also support the observation from the absorption studies that at low P/D, electrostatic binding, stabilized by the stacking of the dye molecules, is favoured and at high P/D, intercalation of the dye between the bases/base pairs is the predominant binding mode. The tRNA-MB complex at P/D = 1.4 dissociates almost completely in 300 mM NaCl, as indicated by the disappearance of the ICD bands, while the decrease in the molar ellipticities of the ICD bands of tRNA-MB complex at P/D = 27 is only about 70% in 300 mM NaCl implying that ionic interaction is not the sole contributor to the intercalative binding mode. A decrease in the electrostatic as well as intercalative binding is reported for DNA-MB complexes also with the addition of NaCl [lo]. The present study clearly shows that the mode of binding MB to tRNA is a function of P/D (or D/P) ratios and the tRNA appears to undergo a conformational change at high D/P ratios. As suggested earlier, the enhancement in the aminoacylation activity might be due to the likely change in the secondary structure of tRNA, on dye binding, particularly at D/P ratios >0.12. Acknowledgements

The authors thank Professor M.R.V. Murthy for generating interest in this work. One of the authors (T.A.) is grateful to the University Grants Commission, India, for the award of a Senior Research Fellowship. References [I] L.C. Malhotra, M.R.V. Murthy and K.D. Chaudhary, Separation and purification of small quantities of specific RNA species by polyacrylamide gel electrophoresis using prestained RNAs as markers, Anal. B&hem., 86 (1978) 363-370.

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