Thin-layer chromatography of synthetic polydeoxyribonucleotides. Part III

ANALYTICAL

RIOCHEMISTRY

49,

3i%392

Thin-Layer

(1972)

Chromatography

of Synthetic

Polydeoxyribonucleotides. S. A. I’?ARANG Biochemist,

y Laboratory,’ Ottawa Received

AND

Part

Ill1

.J.
National Reseal ch Council RlA ORG. Canada December

of Canada,

28. 1971

During the synthesis of a gene, the chemical synthesis of intermediate oligonucleotide fragments (6-10 units long) involves the most timeconsuming part of the whole synthetic work. This is mainly due to tedious and time-consuming fractionation of synthetic polynucleotides on the DEAE-cellulose column, followed by characterization on conventional paper chromatography. In order to simplify this procedure, we recently introduced gel-filtration column technique on superfine Sephadex for the easy purification and isolation of synthetic polynucleotides (1,21. With our ultimate goal of gene synthesis, we considered it essential to develop new, simple, and more precise analytical techniques in the microscale fractionation and unambiguous characterization of polynucleotides. As a result, we have now investigat.ed the thin-layer chromatographic technique on Avicel-cellulose plates. Previously, mixture of oligonucleotides had been separated on layers of PEI-cellulose (3j and cellulose (4-6). In this paper, we wish to report’ the successful application of Avicel-cellulose plates for: (i) the rapid and efficient fractionation of complex chemically polymerized thymidine 5’-phosphate reaction mixture (on 5-10 absorbance units at 260 rnp scale) ; (ii) characterization and in situ quantitative analysis of the oligonucleotides on TLC plate by reflectant spectrophotometry; (iii) in situ identification of the common mononucleosides and mononucleotides by double-scanning technique, and, finally, (iv) fractionation of oligonucleotides on thicklayer Avicel-cellulose plates on a preparative scale. A part of this work has already appeared in a preliminary communication (7). MATERIALS

AND

METHODS

TLC Plates. Analytical precoated layers of Avicel-cellulose powder (0.1 mm thickness) on aluminum sheets (20 X 20 cm) were purchased from Brinkmann Instruments Ltd. Preparative uniplates of Avicel’ Part II ’ N.R.C.C.

(reference 2). Part No. 12576.

I (reference

1). 379

Copyright @ Ail

rights

197’2 by of reproduction

Academic Press, Inc. in any form reserved.

380

NARANG

AND

MICHNIEWICZ

cellulose (1.0 mm thickness) on glass (20 X 20 cm) were supplied by Analtech, Inc. Solvent Systems. The following solvent systems were used: Solvent I, isopropanolJ5% ammonium hydroxide (2: 1 v/v). Solvent II, isobutyric acid/l M ammonium hydroxide/O.1 M EDTA (100: 60: 1.6 v/v). Solvent III, isobutyric acid/l M ammonium hydroxideJO.1 M EDTA (75:60: 1.6 v/v). Solvent IV, Lelvoir pH 7.5, 1 M ammonium acetate/ethyl alcohol (7:3 v/v). Photographic

Records of TLC Plates. A Polaroid Land camera U-5, equipped with filters (No. 54 and 4A) and loaded with Polaroid film type 104 was used. The TLC plate was illuminated with three ultraviolet lamps (Fisher Scientific UVS-12) in a dark room and exposed for 20 to 30 sec. Scanning of the TLC Plates. A Zeiss chromatogram

scanner (by Stahl) attached with a digital integrator (Vidar 6300 Autolab) was employed with the following settings: slitwidth 0.5 mm, chart and motor drive speeds 40 mm/min and 50 mm/min, respectively. Polymerization of Thymidine 5’-Phosphate. The chemical polymerization of thymidine 5’-phosphate was carried out in anhydrous pyridine solution under the following conditions : (i) mesitylenesulfonyl chloride (10 molar equivalents) ; (ii) mesitylenesulfonyl chloride (1.0 molar equivalent) ; (iii) dicyclohexylcarbodiimide (10 molar equivalents) according to the procedures reported previously (8,9). Two-Dimensional TLC of Polymerized Thymidine 5’-Phosphate. The polymerized mixture (5-10 absorbance units at 267 rnp) was applied as a small round spot at one corner (1.5 cm distant from each side) on a 20 X 20 cm Avicel-cellulose plate. The plate was then chromatographed in Solvent I by the ascending technique. After drying thoroughly, it was next rechromatographed in Solvent II or III perpendicular to the original direction. Partial Degradation with Snake Venom Phosphodiesterase. The oligonucleotide (0.5-1.0 absorbance unit at 260 mp) in 0.01 ml of ammonium bicarbonate buffer, pH 8.5, was incubated with 0.01 ml solution of snake venom phosphodiesterase (W.orthington) (5 mg dissolved in 5 ml of water) for 10 min at room temperature (23-25’). The solution was directly applied on the TLC plate and chromatographed in the appropriate solvent system. Elution of Compounds from TLC Plates. The cellulose powder containing the desired spot was loosened from the plate with a spatula and sucked into a cotton-plugged disposable pipet by applying water-pump vacuum at the narrow end of the tube. Next, the cellulose powder was washed with isopropanol/anhydrous methanol (85: 15 v/v) (2 x 2 ml)

TLC

OF

POLYDEOXYNUCLEOTIDES

381

followed by anhydrous ether (2 ml), then air-dried. The nucleotidic component was then eluted with 25% aqueous pyridine (3 X 1 ml). The eluent was collected in a centrifuge for evaporation to dryness. RESULTS

AND

DISCUSSION

In the present studies, we desired mainly to exploit the TLC technique for an efficient and quick fractionation and characterization of reaction products in the chemical synthesis of polynucleotides. For this purpose, we have particularly selected one of the most complex reaction mixture (chemically polymerized thymidine 5’-phosphate) as a model system. Such a reaction mixture generally contains all the components commonly encountered in a typical reaction mixture. These include the desired linear oligonucleotides containing natural 3’ + 5’ phosphodiester bonds, cycle- and pyrophosphate compounds, C-pyridinium oligonucleotides containing terminal phosphomonoester group, and various other unidentified side-products. The fractionation of such a complex reaction mixture has been previously attempted by column chromatography on DEAE-cellulose column (9). This method is generally very time-consuming and the recovery of oligonucleotidic components is quite a tedious procedure. Now we have achieved the fractionation of such a reaction mixture simply by two-dimensional TLC using only 5-10 absorbance units at 267 rnp of the reaction mixture. The mapping diagrams of polymerized thymidine 5’-phosphate with mesitylenesulfonyl chloride (10 molar equivalents), dicyclohexylcarbodiimide (10 molar equivalents), and mesitylenesulfonyl chloride (1.0 molar equivalent) are shown in Figs. lA, lB, and 2, respectively. Characterixation of Thymidine Oligonucleotides. The characterization of each spot was carried out by their usual sequence of bacterial alkaline phosphomonoesterase treatment, TLC, spleen phosphodiesterase and snake venom phosphodiesterase treatments, and again TLC in Solvent III. All the compounds located in the right-hand row in Fig. 2 were found to be resistant toward bacterial alkaline phosphomonoesterase and spleen phosphodiesterase treatment, whereas the compounds in the middle row were completely digested with spleen enzyme after BAP3 treatment. The chain length of each component in the middle row was determined by partial digestion with snake venom phosphodiesterase at room temperature for 10 min. The product from each digestion was chromatographed on TLC plate in Solvent III. Figure 3 shows the photographs of each digestion whereas Fig. 4 represents the scanning of each digested mixture. Thus, in Fig. 2, spot 6 (dinucleotide) on partial digesa BAP,

bacterial

alkaline

phosphomonoesterase.

382

NARANG

AND

MICHNIEWICZ

FE. 1. Two-dimentional 1LC fractionation of chemically 5’-phosphate reaction mixture on Avicel-cellulose plate (20 X ncas) : (A) with mesitglenesulfonyl chloride (MS), 10 molar dicyc!ohexylcarbodiimide, 10 molar equivalents. Solvent I dimension and Solvent II in the second dimension.

polymerized thymidin\: 20 cm) (0.1 mm thickequivalents; (B) with was used in the first

components (mono- and dinucleotides), spot 7 (trigave three components (mono-, di-, and trinucleotides) , spot 8 (tetranucleotide) gave four components (mono-, di-, tri-, and tetranucleotides) , spot 9 (pentanucleotide) yielded mono-, tri-, tetra-, and pentanucleotides and spot 10 (hexanucleotide) gave mono-, tri-, tet,ra-, penta-, and hexanucleotides. The absence of dinucleotide from

tion yields nucleotide)

two

TLC

OF

POLY~~XY~UCLEOTID~S

383

RG. 2. Two-dimensional TLC fractionation of chemically polymer&d thymidine 5’-phosphate reaction mixture with mesitylenesulfonyl chloride (1.0 molar equivalent), on Rvicel-cellulose plate (20 X 20 cm) (0.1 mm thickness). Solvent I was used in the first dimension and Solvent III in the second dimension. Spots 1-4 constitute the right-hand row.

the partial digestion of penta- and hexanucleotides is due to the limited digestion occurring in 10 min at room temperature. If the digestion of hexanucleot,ide was carried out at 37” for 10 min it did yield mono-, di-, t,ri-, and tetranueleotides, whereas it was eom~letely digested to mononucleotide at 37” for 1 hr. Thus this technique offers a powerful tool for characterizing the oligonucleotide on a very small amount (0.51.0 absorbance unit). This method is also under extensive investigation for the sequence determination of deoxyoligonucleotides of heterosequence (10). From these characterization studies (see Table l), a unique pattern of the position of various spots on the chromatogram has been observed. For example in Fig. lA, the cyclohomologs from spots 1-4 fall on the lowermost row. Similarly, the linear components from 5-11 occupy a middle row in the chromatogram. Finally, a third homologous series containing C-pyridinium thymidine and terminal phosphate group (spots 12-18) fall in the uppermost row. In addition to these, some minor components were also noted. Thus, the too-dimensional TLC on Avicel-celtu-

FIG. 3. ‘Partial digestion with snake venom phosphodiesterase of various components from Fig. 2: (A) Partial digestion of spots 5, 6, 7, and 8, and TLC in Solvent III. (B) Partial digestion of spot 9 and two-dimensional TLC in Solvent I and III. (C) Partial digestion of spot 10 for 10 mm at room temperature and two-dimensional TLC in Solvent I and III. (D) Partial digestion of spot 10 for 10 min at 37” and two-dimensional TLC in Solvent I and III.

TLC

OF

POLYDF;OXYNUCLEOTIDES

FIG. 4. Scanning of TLC chromatograms at 260 rn$ of partial digestion snake venom phosphodiesterase of various components from Fig. 3.

385

with

386

NARANG

Characterization spot No. 1 2 3 4 5 6 7 8 9 10 11 12

of Oligonucleotides Identification Fig. 1A

Cycle-pT Cycle-pTpT Cycle-pTpTpT Cycle-pTpTpTpT c&2 (PT), (PT),

MICHNIEWICZ

TABLE 1 from “Maps”

Identification Fig. 2

Cycle-pT Cycle-pTpT Cycle-pTpTpT Cycle-pTpTpTpT Cycle-pTpTpTpTpT PT (pT)t

Cycle-pT Cycle-pTpT Cycle-pTpTpT Cycle-pTpTpTpT

(P% (pV4 (pT)b

(P’&

(P’&

C-Pyridinium thymidine containing phosphomonoester High homologs showing C-pyridinium thymidine UV spectra and also containing phosphomonoester groups

Shown in Figs. lA, lB, and 2

Identification Fig. 1B

(P% (P%

grOUP

13-18

AND

&z (pT!s (p’U4 (P’J% (pT)s (P’VT

C-Pyridinium thymidine containing phosphomonoester grow

lose not only offers an efficient and rapid fractionation procedure but also facilitates identification of the various spots due to the alignment of each type ,of homolog in each row. This pattern was then used to compare the extent and nature of polymerization by using mesitylenesulfonyl chloride (10 molar equivalents) with dicyclohexylcarbodiimide (DCC) (10 molar equivalents). As shown in Fig. 1A and lB, excess of mesitylenesulfonyl chloride is indeed a powerful reagent which also yields extensive degradation products, especially the C-pyridinium compounds containing a free phosphomonoester end group (spots K&18), whereas DCC gives a much simpler mixture. However, when thymidine 5’-phosphate was polymerized with only one equivalent of mesitylenesulfonyl chloride, the two-dimensional TLC pattern (Fig. 2) showed a much simpler and extensively polymerized product. The major products were the homologous linear compounds containing 3’ + 5’ phosphodiester bonds with the absence of third uppermost row containing the C-pyridinium homologs. Thus, the polymerization of nucleotide with mesitylenesulfonyl chloride (1 .O-1.5 molar equivalent) and then fractionation of the product on TLC by two dimensions offers a very easy method

TLC

OF

POLPDE!OXPNUCLEOTIDES

387

for the supply of small amount of oligonucleot~idic suhstrate for enzymic studies. Fractionation and in Situ C:haracterimtion of dfixt,ure of Monotleoxynzrcleosides and MonorleorynlLcleotidex. This TT,C technique lnw also been applied in the easy fractionarion and charar;erization of a m:sture of commonly known mononucleosides and mononucleotides on the TLC plate. The two-dimensional mapping of a mixLure of all four mononucleosidcs and mononucleotides into well-resolved spots on Avicelcellulose in Solvent IV and Solvent III is shown in Fig. 5. Finally, the in Gllc identification of each spot was achicvctl hy the double scann;ng (i.e., 260 m~,/280 mp) tcclmique and the results arc shown in Fig. 6. Tmc to their characteristic peak rat&, a quick teclm:clue for in situ idciitification of each mononucleoside and mUnonucleotide without thrir isolation from the TLC plate is thus provicled (we Fig. 6). Quantitative Analysis of the TLC Piate. The quantitative analysis of the compounds on the TLC plates has hecn carried out hy two procedures: (i) spectrophotometry after rlution ant1 (ii) in situ reflectance spectrophotometry of the plates: (i 1 Spectrophotovzetry after elrction. The nucleetidic cemponcnt~ was

FIG. 5. Two-dimensional fraciionation of mixlure of four deoxyribonucleositlcs and four deoxyribonucleotides on Avicel-cellulose plate. Solvent IV: Lelvoir, pH 7.5. Solvent II: isobutyric acid/l M ammonium hydroxide/O.1 M EDTA (160:60: 1.6).

Fh. 6. Double scanning at 260 rnp and 280 nyl of d-PA, pT, d-pG, d-pC and d-A, pT, d-G and d-C from Fig. 5.

extracted from cellulose with 0.01 N hydrochloric acid until the extract had no absorbancy at 260 mp, followed by centrifugation. The clear solution was read in a spectrophotometer with the extract from an equivalent area of cellulose containing no sample as a blank. The compound can also be extracted with 25% aqueous pyridine. For the spectrophotometric determination aqueous pyridine was evaporated in the presence of 2 N ammonium hydroxide to remove the pyridine. (ii) In situ analysis of the TLC plate by reflectance spectrophotometry. Recently it was demonstrated that the reflectance spectra of the

TLC

OF

389

POLYDEOXYNUCLEOTIDES

substances on the TLC plates can be used for their identification as well as their quantitative determination (llJ2). A comparison with spectra in solution shows the bathochromic shift of the X,, value on the TLC plate (12). Reflectance spectrophotometric analysis of the TLC plate usually involves the use of a calibration curve prepared by measuring the reflectance of samples containing known amounts of the substance. In order to obtain more accurate data, it is important to select a suitable concentration range for the analysis which gives a linear relationship between reflectance and concentration. A linear relation between the peak area and the square root of the applied amount (up to 0.5 absorbance unit at 260 mp) has been observed for all four mononucleotides by the in situ reflectance measurement (Fig. 7). The departure from linearity observed at higher concentrations may be ascribed to be approaching saturation of the absorbent surface by first monomolecular layer of the adsorbed species. Thus the quantitative estimation of each nucleotide could be carried out by using an internal standard and the standard curves. This technique has been successfully applied in the enzymic characterization of oligonucleotides. For example, Fig. 8 shows the results of snake venom phosphodiesterase degradation of d-pTpApC. The area under each peak was recorded by using digital integrator. Preparative TLC. Preparative-scale separation up to 1000 absorbance units at 260 rnp of oligonucleotides on Avicel-cellulose (1.0 mm thickness) can readily be accomplished in any of the commonly used solvents (I to IV) as shown by some of the typical results (Fig. 9). The desired compound was isolated by removing the cellulose containing the appro-

FIG. 7. Standard spectrophotometry.

curves

of d-PA,

T,

d-pG,

and

d-pC

at ‘260 ma

by

reflectance

390

NARANG

AND

MICHNIEWICZ

FIG. 8. Scanning of TLC chromatogram of d-pApTpC digestion with snake venom phosphodiesterase. Solvent II : isobutyric acid/l M ammonium hydroxide/O.1 M EDTA (100:60:1.6).

priate band and transferring it into a sintered-glass funnel. The compound was isolated by washing the cellulose with 257%aqueous pyridine (after preliminary washing with isopropanol/anhydrous methanol (85: 15 v/v) and ether in the case of Solvent II or III). In conclusion, present studies have successfully demonstrated the application of Avicel-cellulose TLC plates for the efficient and easy fractionation and characterization of polydeoxyribonucleotides. These techniques offer the following advantages over conventional paper and column chromatographic separation methods: (i) They provide a very quick (30 min to 4 hr) analytical tool to check the purity of the oligonucleotides by using as low as 0.1 absorbance unit at 260 rnp on Avicel-cellulose plate. (ii) Separation of individual components into well-defined spots or bands is generally more well defined. (iii) In situ quantitative analysis and characterization of each spot by reflectance spectrophotometry are possible. (iv) Recovery of the compounds from the TLC plate is much easier and faster. SUMMARY

Thin-layer chromatography on Avicel-cellulose plates has been developed for (i) efficient and easy fractionation of complex chemical reaction mixture such as polymerized thymidine 5’-phosphate, (ii) characterization of oligonucleotides, (iii) in situ quantitative analysis

FIG. 9. (A) Fractionalion of misturc of plot&cd mono- and dinucleotidea (CH,OC,H,NHCO-CHCH,OpT and CH:,0C,H,SHC0.CH,CH20pTpT) (350 ahsorbance units at 255 rnp) on Avicel-cellulose plate (20 X 20 cm) (0.1 mm t,hickness), Solvent I. (B) Fractionation of mixture of protected di- and trinucleotidcs (2-nitro-4-chloro-CoH:O-pTpT and 2-nitro-l-chloro-CaHnO-pTpTpT) (320 absorbanc:, units at 267 mu) on Avicel-cellulose plate (20 X 20 cm) (1.0 mm thickness), Solvent I. (C) Fractionation of mixture of pT, (pT),, and (pT)$ (400 absorbance units at 267 mp) on Svicel-cellulose plate (20 X 20 cm) (1.0 mm thickness), Solvent II. (D) Fractionation of mixture of protected trinucleotidc and hexanucleotidrs (2nitro4-chloro-CoHaO-pTpTpT and 2-nitro-4-chloro-CaH!O-pT(pT)s) (360 absorbance units at 267 rnp) on Avicel-cellulose plate (20 X 20 cm) (1.0 mm thickne=), Solvent II.

by reflectant spectrophotometry, (iv) in situ characterization of all four mononucleosides and mononucleotides by double scanning technique, and, finally, (v) fractionation of oliganucleotides on TLC plates on t,he preparative scale. REFERENCES 1. NARANG, S. A., MICKNIEWICZ, J. J., AND DHEER, S. K., J. Amer. Chem. Sot. 91, 939 (1969). 2. NARANG, S. A., AND DHEER, S. K., Biochemistry 8, 3443 (1969). 3. WEIMANN, G., AND RANDERATH, K., Ezpetientia 19, 49 (1963). 4. BERGQVIST, P. L., J. Chromatogr. 19, 615 (1965). 5. GASSEN, H. G., J. Chromatogr. 39, 147 (1969).

392

NARANG

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MICHNIEWICZ

6. AUQUSTI-TOC~O, G., CARESTIA, C., PARISI, E., AND SCARANO,F., B&him. Biophys. Acta 155, 8 (1968). GRIPPO, P., IACCARINO, M., ROSSI, M., AND SCARANO, E., B&him. Biophys. Acta 95, 1 (1965). 7. NARANG, S. A., BHANOT, 0. S., DHEER, S. K., GOODCHILD, J., AND MICHNIEWI~Z, J. J., B&hem. Biophys. Res. Commun. 41, 1248 (1970). 8. NARANQ, S. A., JACOB, T. M., AND KHORANA, H. G., J. Amer. Chem. Sot. 89, 2167 (1967). 9. KHOEANA, H. G., AND VIZSOLYI, J. P., J. Amer. Chem. Sot. 83, 675 (1961). 10. NABANa, S. A., AND MICHNIEWICZ, J. J., unpublished work. 11. WENDLANDT, W. M., AND HECHT, H. G., “Reflectance Spectroscopy.” Interscience, New York, 1966. 12. PATAKI, G., AND NIEDERWIE~~ER, J. Chromatogr. 29, 133 (1967).