Μ. A. Surzhik, Α. L. Timkovsky AFFINITY SORPTION ANALYSIS OF THE STRUCTURE OF POLY(G) .POLY(C) COMPLEX OBTAINED BY MEANS OF TEMPLATE-DEPENDENT SYNTHESIS

The analysis of the structure of poly(G)-poly (C) complexes obtained by the enzymatic synthesis on poly(C) template was carried out using the method of affinity sorption. This latter was used also for the purification of the synthesized so far complexes from. the redundant template as well as for the investigation of the structure of poly(G)X Xpoly(C) complexes obtained by the «traditional» mixing of complementary ho mo polynucleotides. It Lν as shon that the degree of template transcription depends on the quality of RNA polymerase and the incompleted transcription is connected with the inhibition of the elongation of poly(G) chain.

The analysis of the structure of poly(G)-poly (C) complexes obtained by the enzymatic synthesis on poly(C) template was carried out using the method of affinity sorption.This latter was used also for the purification of the synthesized so far complexes from.the redundant template as well as for the investigation of the structure of poly(G)X Xpoly (C) complexes obtained by the «traditional» mixing of complementary ho mo polynucleotides.
It Lν as shon that the degree of template transcription depends on the quality of RNA polymerase and the incompleted transcription is connected with the inhibition of the elongation of poly(G) chain.
Introduction.The preparation of double-stranded polyribonucleotide complex poly(G)-poly(C) by means of poly(G) synthesis from GTP on the poly (C) "template with the use of bacterial DNA-dependent RNA polymerase as well as the evidence of its high structural regularity had been described by us earlier [1].But it was shown in the course of followinginvestigations that the degree of radioactive GTP incorporation into the macromolecular fraction depended on the used lot of commercial RNA polymerase.This may be the effect of either the action of contaminating RNAses partially hydrolized the template in the course of synthesis or the incomplete template transcription.This work is devoted to the analysis by means of affinity sorption of the structure of poly(G)-poly(C) complexes obtained in the course of template-dependent synthesis.This method was applied also for the purification of resulting complexes from redundant unused template as well as for structural investigation of «traditional» poly(G)-poly(C) complexes obtained by the mixing of complementary polynucleotides in solution [2].Materials and methods.Commercial preparations of DNA-dependent RNA polymerase from Escherichia coli (Scientific Research Design-Technology Institute of Biologically Active Sabstances, Berdsk, Novosibirsk •district, Russia) were used.
Preparative scale synthesis of poly(G)-poly(C) with the use of RNA polymerase was performed on poly(C) template as described earlier [1].
Our own modification of the described in the literature method of DNA immobilization on cellulose powder [4] was used for the preparation of poly(G)-cellulose (PGC) which was used as affinity adsorbent for poly(C).Microcrystalline cellulose, qualification «LK -for column chromatography» was purified from fine components by repeated decanting of its water suspension and washed with 0,5 N NaOH on paper filter till the absence of yellow colour of filtrate.Then it was washed by small aliquotes of 96 % EtOH and air-dried overnight at room temperature.Poly(G) was dissolved at a concentration of 2 mg/ml in 1 mM EDTA with 10 mM NaCl.Purified dry cellulose was added with continuous stirring to the poly(G) solution -1 g per 15-20 mg of poly(G).

C; M. A. Surzhik, A. L. Timkovsky, 1993
^SSN 0233-7G57.БИОПОЛИМЕРЫ И КЛЕТКА.1993.Т. 9. № З After thorough mixing on magnetic stirred for 1 hr the mixture was dried in rotational evaporator and suspended in 7-10 ml of 96 % EtOH per 1 g of cellulose.This suspension was UV irradiated with vigorous stirring at 4 °С for 18 hrs at a distance of 15 cm from 3 parallel sterilizing 15 W UV-Iamps BUV-15, then dried on paper filter, suspended in 2M NaCl solution containing 10 mM EDTA, stirred for 30-60 min and washed with the same solution at paper filter till the disappearance of the optical density at 260 nm in filtrate.Final steps were washing with water, then with 96 % EtOH and air-drying.
The degree of poly(G) fixation was checked in the following manner: 100 mg of PGC were suspended in 4 ml of 0,4 N NaOH and incubated at 37 °С for 48 hrs, then 4 ml of 2,5 % HClO 4 were added for 10 min in the ice bath.After centrifugation the UV absorbance spectrum in supernatant was registered and the amount of alkaline-splitted GMP was calculated on the basis of molar spectral data.The amount of immobilized poly(G) was equal usually to 1,0-1,1 μ moles per 100 mg of PGC.The interaction of poly(C) or complexes poly(G) -poly(C) with PGC was performed at 20 °С with stirring The interaction of poly(G)-poly(C) complexes after template-dependent synthesis was performed as follows.To 0,42 ml of the reaction mixture containing 0,15 mM poly(C), 1,5 mM 3 H-GTP, and RNA polymerase [1], 25 μ 1 of 4 M NaCl and H 2 O till 1 ml were added.Then 50 mg of PGC containing 0,5-0,55 μ moles of poly(G) were added and incubation was proceeded for 4 hrs at 20 °С.Radioactivity of acid-insoluble fraction in the supernatant was measured [1] before and after incubation with PGC.
Column gel chromatography of samples on Sepharose 2B was performed in usual manner [5].
The method of determining free poly(C) concentration in solution using the differential pulse polarography at the mercury dropping electrode was described earlier [6].Automated polarographic recorder model 174 A (PARC, USA) was used.
The radioactivity of samples was measured in the liquid scintillation counter SL-30 («Intertechnique», France).
Results and discussion.First of all the control experiments were carried out in order to check the binding of poly(C) with PGC in the presence of complex poly (G) -poly (C).Fig. 1 shows UV absorbancy spectra of the complex and its mixture with added extra poly(C) both before and after the interaction with PGC.We see that the presence of complex did not prevent full binding of free poly(C) with PGC.
Then we checked that PGC could bind not only the excess of free poly(C) but also the imperfect complex molecules bearing sites of poly (C) uncoupled with poly(G). in table 1 the composition of poly(G) and poly(C) mixtures prepared for complex formation is shown (column 1), in parallel with the degree of changes in the optical density after the contact of resulting complexes with PGC (column 2).In addition to the equimolar ratio poly(G)/poly(C) we used also the excess of poly(C), up to 70 % in molar concentration.All absorbance spectra were almost identical in their form to the spectrum of complex at the equimolar ratio of poly(G) to poly(C), with the maximum of OD at 260-261 nm, so we do not give them here.The decrease in total absorbancy after PGC treatment was found to be proportional to the molar excess of poly (C).As far as the mechanical addition of poly(C) to the finished complex resulted in drastic change of the form of summary absorbance spectrum (cf.Fig. 2. Gel-chromatographic pattern on Sepharose 2B of the complex poly (G) • poly (C) obtained with the 70% molar excess of poly(C) (tabl.2, sample 4): 1 -before interaction with poly(G)-cellulose; 2-after interaction with poly(G)-cellulose Fig. 3. Gel-chromatographic pattern on Sepharose 2B of the product of incomplete template-dependent synthesis: 1 -before interaction with poly(G)-cellulose; 2-after interaction with poly(G)-cellulose Fig. 1), we conclude that in the samples 2, 3, and 4 almost all redundant poly(C) is involved into the interaction with poly(G).This interaction is incomplete, but absolutely free molecules of poly(C) are apparently absent.
In Fig. 2 gel chromatography patterns of the sample N 4 with 70 % molar excess of poly(C) before and after its interaction with PGC is shown.Most real explanation of their features consists in the extraction from this sample in the course of affinity sorption of imperfect complex molecules with the sections of uncoupled poly(C).
After ,this the analysis of the products of template-dependent synthesis of poly(G)-poly(C) was performed.The presence of 3 H-GTP in incubation mixtures allowed us to determine the degree of the synthesis on the basis or radioactive label incorporation into the acid-insoluble fraction.In fig. 3 the results of gel chromatography on Sepharose 2B of the sample with the degree of completeness of the synthesis near 50 % from maximal level before and after the treatment with PGC is shown.The sample was polydisperse before the treatment with PGC and contained high amount of relatively low-molecular-weight components, but af- or "they consist of poly (C) molecules that had not contacted with RNA polymerase and remained free.* We are sure that the results of following experiments answer this question.We used now the specific curcumstance that radioactive 3 H-Iabel is present in the macromolecular component in the poly(G) strand only.It is obvious therefore that combining the measurements of OD and radioactivity distributions permits the discrimination of completely free poly(C) molecules from free poly(C) mojeties in the imperfect poly(G)X Xpoly(C) complexes.The results are presented in table.2. We see that incubation with PGC of reaction mixture with about 50 % level of po-Iy(G) synthesis resulted in two-fold decrease of 3 H amount in the acidinsoluble fraction.At the same time the amount of radioactive label in the analogous fraction of the product of complete template-dependent synthesis did not change after the incubation with PGC.
We conclude that after the incomplete template-dependent synthesis the reaction mixture contains considerable amount of imperfect complex molecules resulting from the incomplete template transcription and having rather lengthy free poly(C) sequences.These imperfect complex molecules just bind with the affinity sorbent.Our results show also that the method of affinity sorption permits the purification of poly(G)-po-Iy(C) preparations, both from template dependent synthesis and traditional, from contaminating imperfect molecules.
It was shown by us earlier [6] that differential pulse polarography (DPP) at a mercury dropping electrode is a sensitive quantitative method for measuring the amount of free poly(C) sequences in the molecules of polyribonucleotide complexes.This method was applied now in order to obtain additional evidence of the purification of our samples from the redundant free poly(C).The results of polarographic current measurements for model complex preparations before and after their interaction with PGC are shown in table 1 (columns 3 and 4).They confirm that free poly(C) completely disappeared after treatment with PGC.The analogous measurements for the products of incomplete template-dependent synthesis gave identical results which by this reason aren't shown here.
We are sure therefore that the affinity sorbent is capable to extract from the samples described here both unused poly(C) template and the imperfect complex molecules.Their possible remainder is obviously behind the frames of the sensitivity of the methods used here.In order to estimate additionally the completeness of this extraction we could use the results of our previous work where the hydrolysis of poly (G) -poly (C) complexes by human blood nucleases was investigated.We used complexes obtained in template-dependent synthesis as well as by traditional mixing and standardized them in accordance with the criteria elaborated by us earlier [5].It was shown that complexes which had in gel chromatographic pattern on sepharose 2B only one peak eluted directly after void volume had not been by no means destroyed by serum nucleases.We concluded that they did not contain sufficient amounts of one-stranded contaminants and regions which are much more sensitive to RNAses than double-stranded RNA [8].As far as we see in sepharose 2B after PGC treatment only one peak without retardation in the gel, this could serve as additional evidence of full extraction of one-stranded regions and molecules.
The main conclusion of this work is that the affinity sorption on poly (G)-cellulose allows us to analyze the structure of complexes poly(G)x Xpoly(C) and to isolate the most regular part from their preparations.As for the incompleteness of the template-dependent synthesis in case of some lots of RNA polymerase, it is clear now that it is connected rather with the inhibition of poly (G) chain elongation than with the decreasing

Fig
Fig. 1.UV absorption spectra, / -complex poly(G) • poly(C); 2 -complex poly(G) • poly(C) with added free poly(C) before interaction with poly(G)-cellulose; 3 -complex poly(G) •poly(C) with added free poly(C) after interaction with poly(G)cellulose for 4 hrs in 5 mM sodium phosphate, pH 7,5, containing 100 mM NaCLThe interaction of poly(G)-poly(C) complexes after template-dependent synthesis was performed as follows.To 0,42 ml of the reaction mixture containing 0,15 mM poly(C), 1,5 mM 3 H-GTP, and RNA polymerase [1], 25 μ 1 of 4 M NaCl and H 2 O till 1 ml were added.Then 50 mg of PGC containing 0,5-0,55 μ moles of poly(G) were added and incubation was proceeded for 4 hrs at 20 °С.Radioactivity of acid-insoluble fraction in the supernatant was measured [1] before and after incubation with PGC.Column gel chromatography of samples on Sepharose 2B was performed in usual manner[5].The method of determining free poly(C) concentration in solution using the differential pulse polarography at the mercury dropping electrode was described earlier[6].Automated polarographic recorder model 174 A (PARC, USA) was used.The radioactivity of samples was measured in the liquid scintillation counter SL-30 («Intertechnique», France).Results and discussion.First of all the control experiments were carried out in order to check the binding of poly(C) with PGC in the presence of complex poly (G) -poly (C).Fig.1shows UV absorbancy spectra of the complex and its mixture with added extra poly(C) both before and after the interaction with PGC.We see that the presence of complex did not prevent full binding of free poly(C) with PGC.Then we checked that PGC could bind not only the excess of free poly(C) but also the imperfect complex molecules bearing sites of poly (C) uncoupled with poly(G). in table 1 the composition of poly(G) and poly(C) mixtures prepared for complex formation is shown (column 1), in parallel with the degree of changes in the optical density after the contact of resulting complexes with PGC (column 2).In addition to the equimolar ratio poly(G)/poly(C) we used also the excess of poly(C), up Ct Ю. И. Губский, Е. JT.Левицкий, Р. Г. Примак, А. Г. Горюшко, JI.К. Ленчевская, В. А. Матюшкин, И. Е. Вистунова, Л. Г. Саченко, 1993 ^SSN 0233-7G57.БИОПОЛИМЕРЫ И КЛЕТКА.1993.Т. 9. № З