THE JOUIWAL OP BIOLOGICAL CHENISTRY Vol.245, No. 10, Issue of Mny 25, pp. 2633-2630, 19iO

Printed in U.S.A.

Specific Acylation of the Guanine Residues of Ribonucleic Acid*

(Received for publication, January 2, 1970)

ROBERT SHAPIRO, BERTRAM I. COHEN,~ AND DONALD C. CLAGETT

From the Department of Chemistry, New York Univekty, New York, New YOTk 1000.5’

SUMMARY

Treatment of yeast RNA with Kethoxal (P-ethoxy-oc-keto- butyraldehyde), followed by periodate cleavage, led to the specific introduction of oc-ethoxypropionyl groups onto the guanine amino groups of the RNA. The extent of nitrous acid deamination of the guanines of the acylated RNA was reduced by two-thirds when compared to unmodified RNA. The presence of the acyl groups in the nucleic acid was shown by nuclear magnetic resonance spectroscopy. The acyl nucleotide, N2-a-ethoxypropionylguanosine 5’-phosphate (V) was isolated from a venom phosphodiesterase hydrolysate of the RNA.

The use of glyoxal in place of Kethoxal in the above pro- cedure led to a nonspecific formylation of the RNA. A re- duction in the extent of deamination by nitrous acid of adenine, cytosine, and guanine was noted. This contrasts with the results obtained with the same conditions at the base or deoxynucleoside level, at which specificity for guanine was observed. Treatment of adenine with glyoxal and periodate in the presence of acid, however, afforded N6- formyladenine (IVa).

Glyoxal and certain substituted glyoxals react specifically with guanine derivatives to yield adducts of Structure I, Scheme I (l-3). This property has made the reaction useful for the

0

I SCHEME 1

modification of nucleic acids (1, 4-7). Its usefulness has been limited, however, by the instability of the adducts at pH 7 and above, in the absence of the excess glyoxal derivative (2, 5). One objective of our research has been to devise a blocking group,

* This research was supported by Grant GM-11437 from the United States Public Health Service.

19:9; t’ a ional Institutes of Health Predoctoral Fellow, 1967 to

stable to nitrous acid, to protect the guanine amino groups of nucleic acids. Adducts of type I, unfortunately, are deaminated by nitrous acid to xanthine derivatives. We recently reported, however, that periodate cleavage of these adducts converts them into the more stable acylguanines (II) (3). We now wish to report the successful application of this method to effect the specific acylation of the guanine amino groups in yeast RNA. This reaction has been shown by several independent and novel methods.

EXPERIMENTAL PROCEDURE

Methods and 111ateriaZsBases, nucleosides, and nucleotides were purchased from Schwarz BioResearch, and were found to be chromatographically homogeneous. The methods used in obtaining melting points, pH readings, and infrared, ultraviolet, and nuclear magnetic resonance spectra and in conducting paper, thin layer, and ion exchange column chromatography have al- ready been described (3, 8). The solvent systems used in thin layer and paper chromatography were: Solvent 1, 1-but.anol- water (86: 14); SoIvent 2, isoamyl alcohol-O.35 M NatHP04 (1 :l); Solvent 3, CH&N-water (8 :2); Solvent 4, acetone-water (9 : 1); Solvent 5, 2-propanol-concentrated HCl-water (65 : 16.7 : 18.3) ; Solvent 6, 1-butanol-water-concentrated NH,OH (86 : 14 : 1); Solvent 7, 13.8 g of NaH2P04. Hz0 are dissolved in 900 ml of water, the pH is adjusted to 6.8 with H3P04, the volume is diluted to 1000 ml with water, and (NH&SO4 (200 g) and l-propanol (20 ml) are added; Solvent 8, l-butanol-water-acetic acid (86 : 14:2). The nuclear magnetic resonance spectra of RNA were taken with the aid of a Ovarian Associates Cl024 time-averaging computer. Microanalyses were performed by the Spang Microanalytical Laboratory, Ann Arbor, Michigan. Sephadex G-25M was obtained from Pharmacia and prepared according to the manufacturer’s recommendation.

Preparation of N6-Formyladenine (ZVa)-A solution contain- ing 0.5 g (3.7 mmoles) of adenine and 1.2 g (16 mmoles) of glyoxal monohydrate (British Drug Houses) in 50 ml of 4 M

sodium acetate buffer, pH 4.7, was heated, with stirring, at 70” for 1 hour. The solution was cooled to O”, and 4.2 g (20 mmoles) of sodium metaperiodate were added in one portion. The reac- tion mixture was stirred for 30 min (some precipitation was seen) and then added to 200 ml of 2-propanol. The acid present was neutralized by addition of sodium bicarbonat,e, and the solids (inorganic salts) were removed by filtration. The filtrate was reduced t,o 10 ml under vacuum and deposited 78 mg (13%) of iVG-formyladenine as a pale pink, amorphous solid. An analyti- cal sample, a hygroscopic white powder, was prepared by three reprecipitations from a 2-propanol-water (9 : 1) mixture: m.p.

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2634 Specific Acylation of Guanine Residues of RNA Vol. 245, No. 10

293-295” with decomposition; infrared absorption (KBr) at 3.22, 3.33, 3.53, 5.86, 6.24, 6.95, 12.30, and 12.51 p; ultraviolet maximum (pH 6.6) at 274 rnp (E 10,300), shoulder at 255 rnp, minimum at 232 rnp; ultraviolet maximum (pH 11) at 280 rnp, minimum at 244 rnp; nuclear magnetic resonance (CDISOCDB), 7 0.26, singlet (CHO), 1.40 and 1.51, singlets (adenine H-2 and H-8); mass spectrum, molecular ion at m/e 163; thin layer chro- matography, RF 0.52 in Solvent 1; RF 0.45 in Solvent 2.

CsH5NjO. *Ha0

Calculated: C 41.86, H 3.51, N 40.68 Found: C 41.87, H 3.12, N 40.47

Reaction of Deoxyademsine with Glyoxal and Periodate-This was run in the same manner as the adenine reaction, above, with 0.10 g (0.4 mmole) of deoxyadenosine and 0.91 g (12 mmoles) of glyoxal monohydrate in 50 ml of 4 M sodium acetate buffer, pH 5.1. The amount of sodium metaperiodate subsequently added was 3.44 g (16 mmoles). After removal of inorganic salts by precipitation with 2-propanol, as in the adenine reaction, the solution was examined by paper chromatography in Solvent 4. A single new spot (RF 0.86) was observed, in addition to deoxy- adenosine. Upon elution with HzO, the former showed an ultraviolet maximum (pH 6.5) at 264 rnp. The substance, presumably Wformyldeoxyadenosine (IVb), decomposed upon attempted isolation by thin layer or column chromatography. Ne-Formyladenine, identified via its ultraviolet spectrum and RIP values, was obtained. Upon elution of IVb from a thin layer chromatogram with dilute NHIOH solution, pH 10, a substance was produced with the ultraviolet spectra (at pH 2, 6.8, and 10) of deoxyadenosine.

Reaction of Deoxyguanosine with Glyoxal and Periodate-The reaction and workup were conducted in the same manner as in the adenine reaction, with 0.2 g (0.75 mmole) of deoxyguanosine, 0.48 g (6.3 mmoles) of glyoxal monohydrate, and 1.68 g (7.9 mmoles) of sodium metaperiodate. After the removal of inorganic salts by precipitation with 2-propanol, the filtrate was evaporated to dryness under vacuum. The white solid residue was dissolved in 25 ml of methanol and applied to a dry column (9), 5 x 22 cm, of Avicel microcrystalline cellulose (American Viscose Company, Marcus Hook, Pennsylvania). The column was eluted with Solvent 4, and eluted fractions were examined via paper chromatography in the same solvent system. Frac- tions containing the presumed Wformyldeoxyguanosine (II, R = H, R’ = &deoxy-n-ribofuranosyl) (RR 0.78) free of deoxyguanosine (RF 0.68) were combined and concentrated to IO ml. 2-Propanol was added, and the solution was held at 10” to precipitate 10 mg (4.5y0) of N2-formyldeoxyguanosine. The sample was homogeneous upon paper chromatography in Sol- vents 2 (RF 0.14), 4 (RF above), and 3 (RF 0.30); m.p. 180” with decomposition; ultraviolet maximum (pH 6.9) at 250 rnp, shoul- der at 275 rnp, minimum at 226 rnp, e275:@50 = 0.58; infrared absorption (KBr) at 2.90 to 4.10 (broad), 5.55, 5.79, 5.86, 6.24, 6.42, and 12.90 p.

Upon standing in 4 RI sodium acetate buffer, pH 5, at 25”, Wformyldeoxyguanosine was observed, by paper chromatog- raphy, to decompose to a substance with the Rr of deoxyguano- sine (Solvents 3 and 4). The half-life was approximately 6 hours.

Reaction of Deoxyguanosine, Deoxyaderwsine, Deoxycytidine, and N2-Formyldeoxyguanosine with Nitrous Acid-Reactions were run in 4.5 ml of 4 M sodium acetate buffer at pH 5 and 32”,

with 0.09 mmole of the nucleoside and 0.31 g (4.5 mmoles) of NaN02. The reactions were followed by paper chromatography in Solvent 1. Deoxycytidine (RF 0.20) yielded only deoxy- uridine (RF 0.31). Deoxyadenosine (RF 0.35) yielded initially only deoxyinosine (RF 0.19) but, after 24 hours, some hypo- xanthine as well. The deoxyguanosine reaction mixture gave rise, after 24 hours, to a precipitate identified by its RF in Sol- vent 5 and its ultraviolet spectra as xanthine. Examination of the ultraviolet spectrum of the reaction mixture after 3 hours of reaction indicated that deoxyxanthosine, rather than xan- thine, was present. (The ultraviolet spectrum changed between pH 4.5 and 6.5, but not between 6.5 and 8.4. Xanthine has a pK, at 7.5. Deoxyxanthosine, like xanthosine, presumably has a pK, near 5.7 (lo).) Attempts to demonstrate deoxy- xanthosine by paper chromatography were unsuccessful, and it presumably decomposed to xanthine during the process. The deoxyguanosine reaction mixture had a yellow color, and a faint yellow spot (RF 0.09) observed in Solvent 3 suggested that 2-nitrodeoxyinosine or 2-nitrohypoxanthine (8) was also formed in the reaction. N2-Formyldeoxyguanosine was found to hydrolyze to deoxyguanosine, which then reacted with nitrous acid. The presence of both N2-formyldeoxyguanosine and deo,xyguanosine was shown by paper chromatography in Solvent 1, followed by elution of the spot of RF 0.16 and rechromatog- raphy in Solvent 3 or 4.

A kinetic study was made of the reactions, under the above conditions, with the general methods already described for ribonucleosides (8). Only the first 4 hours were followed, to avoid complications due to the hydrolysis of purine N-glycosyl bonds, In the reaction of N2-formyldeoxyguanosine, each aliquot was treated for 15 min with dilute NH,OH, pH 9.4, at 32”, to convert remaining N2-formyldeoxyguanosine to deoxy- guanosine. This procedure resulted in the generation of addi- tional ultraviolet absorbances at the wave lengths used, and the measured absorbances were corrected for these, as determined in a blank run. The equations used to calculate the relative amounts of each deoxynucleoside and its deamination product, with corrected absorbances at the wave lengths indicated, were: deoxyadenosine = (1.82 A 265 - A&/IO.3 and deoxyinosine = (1.03 A254 - A&/4.27; deoxycytidine = (3.12 AZ*0 - A2&/13.2, and deoxyuridine = (A2G0 - 1.12 ATg0)/6.42; deoxyguanosine = (A260 - A&/5.70, and deoxyxanthosine = (2.1 A286 - A2&/8.46.

Ion Exchange Chromatographic Analysis of Yeast RNA- Sodium yeast ribonucleate (Schwarz BioResearch) (200 mg) was purified by HCIOl precipitation at 5”, followed by washing with 95% ethanol and then ether. The RNA was suspended in 50 ml of water and brought into solution by addition of NaHC03. The final solution was at pH 6.8, and contained 1.85 mg of RNA per ml. The RNA had an optical density at 260 mp of 23.3 units per mg.

For determination of base composition, 2.7 ml of stock solution were further purified by two passages through Sephadex G-25M columns (0.8 x 55 cm), eluting with wat,er. The ultraviolet- absorbing peak that rapidly emerged was collected. The RNA solution was concentrated to 10 ml and hydrolyzed with 10 ml of 1 N KOH at 37” for 24 hours. The reaction mixture was neutralized to pH 6.5 by the addition of HClO, and kept at 4” for 16 hours. The solution was filtered to remove KClO, and analyzed by anion exchange chromatography. A column of Amberlite CG-400 resin (formate) was used, wit,h an exponential gradient (11) in formate. Initially, 200 ml of water weie used

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I I jC, 1 A, G, I, X, U,

I

I I t I I 200 400 600 800 1000 1200 1400

FIG. 1. Separation of nucleotides from an alkaline hydrolysate of nitrous acid-treated yeast RNA by anion exchange chromatog- raphy on Amberlite CG400 resin, with an exponential gradient in formate. The vertical dotted lines mark the places where the formate gradient was changed. Cp, cytidine 2’(3’)-phosphate; Ap, adenosine 2’(3’)-phosphate (two peaks); Gp, guanosine 2’(3’)-phosphate; Ip, inosine 2’(3’)-phosphate; Xp, xanthosine 2’(Y)-phosphate; Up, uridine 2’(3’)-phosphate. The rising base line is caused by the increasing concentration of formic acid in the eluate.

in the mixing chamber and 300 ml of 0.75 N formic acid in the reservoir. When the reservoir emptied, it was filled with 250 ml of 1.0 N formic acid. When this had, in turn, emptied, the reservoir was refilled with 1000 ml of 15 N formic acid. The peaks t.hat emerged (nucleoside 2’(3’)-phosphates) were pooled, concentrated to about 5 ml, and brought to 10 ml with water. They were identified by their ultraviolet spectra at pH 1, 7, and 11 and their RF on paper chromatography in Solvent 5. The order of elution is as illustrated in Fig. 1, except that inosine 2’(3’)-phosphate and xanthosine 2’(3’)-phosphate were not present. The amounts of nucleotides present were determined quantitatively by the following parameters (wave length, ex- tinction coefficient x 10M3, pH) : adenosine 2’(3’)-phosphate (267, 12.1, 2); cytidine 2’(3’)-phosphate (278, 12.7, 1); guanosine 2’(3’)-phosphate (272, 8.4, 1); uridine 2’(3’)-phosphate (272, 7.63, 2.5). Wave lengths above 265 rnE.c were utilized to avoid interference by residual formate.

Reaction of RNA with Nitrous Acid-The RNA stock solution (2.7 ml) was passed through one Sephadex column (see “Ion Exchange Chromatographic Analysis”) and was then concen- trated to 2.7 ml. To this were added 4.5 ml of 4 M sodium ace- tate buffer, pH 5.0, and 0.5 g of NaN02. The reaction mixture was stirred for 5 hours (10 hours in one run) at 37” and then brought to pH 6.5 by the addition of KOH solution. The mix- ture was again passed t.hrough a Sephadex column, eluting with HZO. The RNA peak eluted before nitrite ion (as detected by acidic starch iodide paper) and was collected. The RNA solu- tion was concentrated, hydrolyzed, and analyzed on an anion exchange column as described in the above section. A typical column elution pattern is illustrated in Fig. 1. The new de- amination products were determined quantitatively with the following parameters (wave length, extinction coefficient x 10d3, pH): inosine 2’(3’)-phosphate (265, 7.1, 0); xanthosine 2’(3’)- phosphate (277.5, 9.35, 7.5).

Reaction of RNA with Glyoxal and Periodate-To 2.7 ml of RNA stock solution were added 2.7 ml of sodium phosphate buffer (1.46 M, pH 6.5) and 3.2 mg of glyoxal monohydrate. The reaction mixture was stirred at 55” for 1 hour, during which time the absorbance ratio, 269:254, in the ultraviolet changed from 0.83 to 0.79, and then remained constant. A shift of the emax from 258 to 256 rnp and an increase of intensity of the maximum were also observed. The reaction mixture was cooled to 0”,

I I r 2 4 6 8 IO

FIG. 2. Nuclear magnetic resonance spectrum in DzO of or-eth- oxypropionyl-RNA. Peak A is composed of aromatic hydrogens from the heterocycles of the RNA (see “Results” for details). Peak B represents the ethoxy CH2 of the modifying group and Peak C represents the methyls of the modifying group.

and 11.2 mg of sodium metaperiodate were added. The solution was stirred for 30 min and then applied to a Sephadex G-25M column (0.8 X 55 cm), eluting with water. The RNA peak eluted before periodate ion (as detected by acidic starch iodide paper). The formylated RNA showed X,, at 259 rnp, and had e300:e260 of 0.152, while the starting RNA had ~~~~~~~~~ of 0.033. The RNA fraction was concentrated to 2.7 ml and then treated with nitrous acid and analyzed as described in the above sections.

For purposes of nuclear magnetic resonance spectroscopy, the quantity of RNA used was tripled, and the other reagents were adjusted in amount accordingly. The formylated RNA was concentrated to 0.5 ml, and 10 ml of D20 were added. The solution was again evaporated to 0.5 ml, 10 ml of DzO were added, and the solution was reconcentrated to 0.5 ml. The low field portion of the nuclear magnetic resonance spectrum was taken, with 215 scans of a time-averaging computer. A broad weak peak, centered at 1 .O 7, was observed. This was not pres- ent in the spectrum of the original RNA. Its relative intensity, compared to the RNA peak centered at 2 7 (guanine H-8, adenine H-2 + H-8, cytosine and uracil H-6), was 1 to 12.6. This corresponded to the introduction of 1 formyl proton/l0 nu- cleotides.

Reaction of RNA with Kethoxal and Periodate-The reaction was run under the same conditions as the glyoxal reaction, with 1 ml of a solution containing 0.045 mole of Kethoxal (Upjohn). The reaction was followed in the ultraviolet, and the 279:254 ratio was seen to change from 0.51 to 0.67 and then level off. The reaction mixture was cooled to O”, and 22.4 mg of sodium metaperiodate were added in one portion. Stirring was con- tinued for 35 min. The RNA was purified of periodate by Sepha- dex chromatography, as described in the glyoxal periodate reac- tion. The oc-ethoxypropionyl-RNA showed X,,, at 259 rnp, and had e300:eZ260 of 0.172. The reaction of the modified RNA with nitrous acid was conducted in the same manner as with the formylated RNA.

The following variations in the reaction conditions were also run: (a) 0.090 mmole of Kethoxal at 55” and then 44.8 mg of sodium metaperiodate; (b) as in a, but at 65”; (c) as in b, but the reaction sequence conducted twice on the same RNA sample.

For the purposes of nuclear magnetic resonance spectroscopy, the reaction was scaled up 5-fold, and the procedure was con- ducted as described for the glyoxal periodate reaction. The spectrum, in D20, is shown in Fig. 2.

Isolation of N2-or-Ethoxypropionylguanosine 5’-Phosphate (V) from the Acylated RNA-The solution of ar-ethoxypropionyl-RNA (prepared as described in the above section) was concentrated to 0.5 ml. To this were added 4.8 ml of Tris-HCl buffer (0.05 M),

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:zjJr&q 200 400 600 800 1000 1200

VOLIJME(nd

FIG, 8. Separation of nucleotides from a venom phosphodiester- ase hrdrolvsate of cu.ethoxvnronionvl-RNA. The column is the

I

same described in Fig. 1. “ * -

&“, cytidine 5’-phosphate; pA, adeno- sine 5’.phosphate; pG, guanosine B-phosphate; pEG, N%-eth- oxypropionylguanosine 5’-phosphate; pU, uridine 5’-phosphate. The unlabeled peak that emerged almost immediately was non- nucleotide material.

pH (at 37”) 8.7, 0.2 ml of 0.02 M CaC12, and 0.5 mg of venom phosphodiesterase (Worthington). The reaction mixture was stirred at 37” for 24 hours and examined by thin layer chro- matography in Solvents 5 and 7. A single new spot was ob- served in each solvent, in addition to the expect,ed nucleoside 5’-phosphates. In Solvent 3, the new product (V) (RF = 0.72) ran behind uridine 5’-phosphate and ahead of adenosine 5’- phosphate as a fluorescent spot. In Solvent 7, the new product (RF 0.19) ran behind the four natural ribonucleotides.

Larger amounts of V were separated by anion exchange chro- matography, with the method described under “Ion Exchange Chromatographic Analysis of RNA.” The elution pattern obtained is given in Fig. 3. The tubes containing V were col- lected and lyophilized to 1.5 ml. Compound V was separated from contaminating guanosine 5’-phosphate by preparative thin layer chromatography in Solvent 5. It was eluted with 0.05 N

sodium acetate buffer, pH 5.5. In the ultraviolet, it showed: PI-I 1, maximum at 261 rnp (E = lO,ZOO), minimum at 226 rnp, em: EZGO = 0.68, e250: eZcO = 0.87; pH 7, maximum at 260 rnp, minimum at 232 rnp, E~~,J:~x,~ = 0.66, Q~~:~x,o = 0.82. The extinction coefficient at 261 rnp, pH 1, was estimated on the basis of the quantitative conversion of this compound to guanosine 5’-phosphate in NH&H solution (see below).

Upon addition of 1 drop of NH,OH to a solution of V, its .ultraviolet spectrum changed rapidly. After 14 min, it had changed to that of guanosine 5’-phosphate, as measured at pH 1, 7, and Il. Upon thin layer chromatography in Solvent 5, one spot with the RF (0.36) of guanosine 5’-phosphate was observed.

Conversion of W-wEthoxypropionylguamsine 5’-Phosphate (V) to N2-cY-Ethoxypropionylguanine (VII)-A periodate cleavage and aniline-catalyzed elimination sequence were used (12). To approximately 0.5 mg of V were added 2 ml of 0.15 M sodium acetate buffer, pH 5.0. The solution was cooled to 0” and 0.5 mg of sodium metaperiodate was added. The reaction mixture was stirred for 1 hour at 0” and then allowed to warm up to 20”. Ethylene glycol (0.2 mg) was added and stirring was continued for 15 min. Aniline (1.9 mg, distilled from zinc), dissolved in 0.5 ml of absolute ethanol, was then added. The reaction mix- ture was stirred for 3 hours at 25” and extracted with ether. The aqueous layer was concentrated to 1 drop and examined by thin layer chromatography in Solvents 6 and 8. A single spot corresponding to N%-ethoxypropionylguanine (VII) (RF 0.45 in Solvent 6, 0.65 in Solvent 8) was observed. The spot, on

elution into water, showed ultraviolet spectra at 111-I 1 and 7 identical with those of VII (3).

RESULTS

Previous studies have indicated that glyoxal, at concentrations of less than about 0.8 M, reacts specifically with guanine deriva- tives (1, 4, 7). As the specificity of our acylation sequence (consisting of reaction with a glyoxal, followed by periodate cleavage of the resulting adduct) had not been tested, this was now studied. Bases and deoxynucleosides were used as model compounds. Modification of deoxyguanosine by glyoxal and periodate led to its quantitative conversion to a single new sub- stance, presumably N2-formyldeoxyguanosine (II, R = H, R’ = P-n-2-deo,xyribofuranosyl). As expected, cytosine and deoxycytidine were not affected by these conditions. Sur- prisingly, however, when adenine (IIIa) was heated with glyoxal and periodate was added to the reaction, a new substance was formed. Its analysis and nuclear magnetic resonance spectrum showed it to be a formyladenine, and its ultraviolet spectrum resembled that of NC-butyryladenine (13). It was assigned the structure, Ne-formyladenine (IVa). In Scheme II, for IIIa and IVa, R = 1-T; for IIIb and IVb, R = 2-deoxyfl-n-ribofuranosyl.

9 H-C-NH

I) CHOCIHO

2) IO,--

k k

Itt m SCHEME II

The yields of this product were very erratic and varied from 0 to 90% from run to run. The procedure listed under “Experi- mental Procedure” was finally found to give reproducible yields of about 13%. Application of this procedure to deoxyadenosine (IIIb) led to its partial conversion to a new substance, con- sidered to be Na-formyldeoxyadenosine (IVb) . The instability of this substance prevented it.s large scale isolation. It decom-

posed to Ne-formyladenine upon attempted chromatography, and gave deoxyadenosine upon treatment with ammonia.

Attempts to obtain evidence for the formation of an initial adduct (before oxidation) between adenine and glyoxal were fruitless. It is probable that this adduct was very labile, and decomposed under the dilution conditions used in ultraviolet spectroscopy or thin layer chromatography. Furthermore, if the pH of the reaction was kept above 5, no iV6-formyladenine was formed, so that the initial adduct presumably formed only in acidic solution. Thus, by working at pH 6 or above, the specificity of the acylation procedure for guanine or deoxy- guanosine was reestablished.

One goal of our research has been t,o develop a blocking group capable of protecting t,he amino group of a guanine within a nucleic acid from attack by nit)rous acid. The effectiveness of the formyl group for this purpose was studied, with the deox2r- nucleosides as a model system. The kinetics of deamination by 1 N HNOz (pH 5, 32”) was followed, with the general methods already reported for ribonucleosides (8). The reactions were

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followed for the first 4 hours only, as after that time the situation was complicated by N-glycosyl cleavage of deoxyxanthosine and, to a lesser extent, deoxyinosine. This has also been observed by other workers (14). The pseudo-first order rate constants (mi&) for deamination of the natural deoxyribonucleosides were: deoxyguanosine, 3.1, deoxyadenosine, 5.0, and deoxycyti- dine, 0.76. The relative reactivity for the three nucleosides was thus 4.1:6.6:1. It is of interest to note that in the ribonucleo- side series, under roughly comparable conditions (0.57 M NaN02, pH 5, 37”), the order of reactivity of guanosine, adenosine, and cytidine was 6.0:2.7: 1 (8). Thus, the removal of a sugar hydroxyl group has had a significant effect on the relative reac- tivity of the bases. When Nz-formyldeoxyguanosine was allowed to react with HNOt under the conditions used for the other deoxynucleosides, it was converted to xanthine with a k (min-l) of 1.5. Thus it was only about 50% effective as a blocking group. This was undoubtedly due to its slow hydrolysis to deoxyguanosine under the reaction conditions.

It was desired to test the formylation procedure at the nucleic acid level. As the lability of the formyl group precluded any attempt to isolate formylated bases or nucleosides from an RNA hydrolysate, a problem was raised as to how the specificity and extent of reaction could be shown. Generally, radioactive labels and ultraviolet spectroscopy have been used for this purpose. The former method, although exceedingly sensitive, gives little information about the nature of the transformation taking place, and requires the availability of a suitably labeled reagent. The latter method requires either that the absorption of the bases of the nucleic acid be significantly altered by the reaction or that the reagent itself absorb beyond 300 rnp. We therefore used this opportunity to test alternative methods for charac- terizing a chemically modified nucleic acid: nuclear magnetic resonance spectroscopy, and alterations in chemical reactivity.

The formylation procedure was applied to yeast RNA. Ex- cess periodate and other salts were removed by Sephadex chro- matography. The 300:260 absorbance ratio in the ultraviolet of the RNA was increased from 0.033 to 0.15 by this procedure. Other workers have synthesized oligodeoxynucleotides contain- ing Nz-acetylguanine and reported values of about 0.18 to 0.27 for their products (15, 16). The low field region of the nuclear magnetic resonance spect.rum (in DzO) of the formylated RNA was obtained with the aid of a time-averaging computer. A broad weak peak was observed near 1.0 7, in addition to the peaks observed in the nuclear magnetic resonance spectrum of the untreated RNA.

The formylated RNA was then exposed for a set period of time to deamination by nitrous acid. The RNA was then de- salted by Sephadex chromatography and hydrolyzed in alkali and the nucleotide composition was analyzed. We first at- tempted to use an ion exchange column described by Carbon (17) for this purpose, but in our hands this did not give a satisfactory separation of the components. A modified scheme was therefore devised, which is described under “Experimental Procedure.” The composition was used to compute the extent of deamination of each amino base in the RNA, as well as the amount of each nucleotide and its deamination product recovered. These were compared to control runs in which the RNA was analyzed without deamination and in which the RNA was deaminated without prior acylation. The results of these studies are sum- marized in Table I. Since the percentage of deamination was

found to vary by up to 7% in duplicate runs, multiple runs were used and the values were averaged.

The results differed from those expected on the basis of the study with the deoxynucleosides. A reduction in the extent of deamination of guanine was indeed obtained, but also of cyto- sine, and, to a lesser extent, adenine. Therefore, the specificity exhibited at the nucleoside level was not operative at the nucleic acid level. The amount of adenine and inosine nucleotides recovered from the nitrous acid-treated RNA was equal to the adenosine 2’(3’)-phosphate recovered from native RNA. How- ever, the recovery of cytosine and uracil nucleotides was only about 90% of that expected. This suggested destruction of one of these nucleotides by nitrous acid. A similar situation pre- vailed with guanine and xanthine nucleotide pairs. In one run, the nitrous acid reaction time was doubled, and the recoveries of both nucleotide pairs fell to 80%.

Another reagent was examined to see whether it would provide the desired specific acylation of guanine in RNA. It had been shown that treatment of guanine with Kethoxal (oL-keto-fi- ethoxybutyraldehyde), followed by periodate cleavage of the resulting adduct, yielded N*-a-ethoxypropionylguanine (VII) (3). This substance had proved fully resistant to attack by nitrous acid. Yeast RNA was therefore treated with Kethoxal and then with periodate. The product, freed of excess salts, exhibited a 300:260 absorbance ratio in the ultraviolet of 0.17. The nuclear magnetic resonance spectrum of the product (in D20) was obtained with the aid of a time-averaging computer, and is shown in Fig. 2. The presence of t,he modifying group in the RNA is confirmed by the peak at about 9 7, which repre- sents the 6 protons of the two methyl groups. A peak at 6.7 7 (partly overlapping a peak from the RNA) can be assigned to the ethoxy CH2 protons of the modifying group. The size of the methyl peak was obt.ained by integration, and this was compared to the size of the RNA peak near 2 7. The latt’er is composed of absorbances from the guanine H-8, adenine H-2 and H-8, and uracil and cytosine H-6 protons (18). The relative sizes of the methyl and aromatic peaks were estimated as 1.2 to 1. From the composition of the RNA, it was calculated that this corresponded to the introduction of 25 acyl groups/100 nucleo- tides of the RNA. If the acyl groups were only on guanine, this corresponded to about 88% acylation of guanine. As a further control, a known quantity of sodium acetate was added to the solution. The observed ratio of the size of the acetate peak to the size of the RNA peaks was in accord with the known concentrations of these substances.

The deamination of the cr-ethoxypropion>I-RNA was investi- gated by the procedure used for the formylated RNA. The results are summarized in Table I. As can readily be seen, the extent of deamination of cytosine and adenine was the same, within experimental error, as in the unprotected RNA, while the deamination of guanine was reduced by two-thirds. Fur- thermore, the recovery of guanine and xanthine nucleotides was excellent, indicating that the side reaction leading to the destruc- tion of one of them was largely eliminated. This confirmed the specificity of the acylation for guanine. Some efforts were made to eliminate the residual amount of deamination of guanine. Greater amounts of Kethoxal were used, at a higher temperature. In one run, the acylated RNA was subjected to a second treat- ment with Kethoxal and periodate. This was to no avail, as the results were the same as they were in the runs with the origi- nal conditions. In a control experiment, the Kethoxal was

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2638 Specific Acylation of Guanine Residues of RNA Vol. 245, No. 10

TABLE I Analysis of chemically modified RNA

Nucleotideb isolated

RNA used’=

CP I UP I AP 1 GP 1 ‘IP / XP

tmwles x 103

Unmodified RNAd.. . . . . . 2.3 3.1 2.8 3.2 RNA + HN02, 5 his’.. . . . . . . . . . 0.81 4.0 0.73 0.65 2.2 2.2 RNA + HN02, 10 hrs . . . 0.47 3.9 0.39 0.50 2.6 2.0 Formyl-RNA + HN02, 5 hrsd.. . 1.1 3.5 1.0 1.6 1.8 1.6 a-Ethoxypropionyl-RNA + HN02,

5 hrsg............................ 0.90 3.9 0.71 2.3 2.0 0.85

0 For the exact experimental conditions, see “Experimental Procedure.”

Deamination Recovery

Cpc AP GP I I

%

CP+UPI Ap+Ip(Gp+Xp

61 74 77 74 87 80 44 64 50

100”

91 80 85

% 100’ 100 100 100

100” 88 78

100

56 74 27 89 96 98

b The initials Cp, Up, Ap, Gp, Ip, and Xp refer to the 2’(3’)-phosphates of cytidine, uridine, adenosine, guanosine, inosine, and xanthosine, respectively.

c This was computed in the following way. The fraction Cp/(Cp + Up) was computed before and after deamination and the dif- ference was obtained. This was divided by the fraction Cp/(Cp + Up) before deamination and multiplied by 100.

d Average of three runs. 6 By definition. f Average of two runs. 9 Average of four runs.

replaced by an equimolar amount of 2-ethoxyethanol, and the same procedure was followed. The extent of deamination observed was identical with that observed with unmodified RNA.

The greater stability of W-ar-ethoxypropionylguanine, as compared to Wformylguanine, permitted the isolation of the former as its ribonucleotide from the RNA. For this purpose, the RNA was hydrolyzed by venom phosphodiesterase, rather than alkali. The acylated RNA resisted hydrolysis at a pH (7.4) which sufficed for hydrolysis of the untreated RNA. At a more optimal pH (8.4), and with a higher concentration of enzyme, degradation of the modified RNA to 5’-ribonucleotides was successful. Model studies, however, showed that the acyl group of N2-a-ethoxypropionylguanine was about half-hydro- lyzed under these conditions, so that a nucleotide analysis of the RNA hydrolysate could not be used to determine the extent of acylation of the guanine in the RNA. Analysis of the hydrol- ysate by thin layer or ion exchange chromatography did show the presence of only one new product, which was identified as N2-a-ethoxypropionylguanosine 5’-phosphate (V). The elution

0

PH 5 CH,CH,O

SCHEME III

pattern of the ion exchange column used to separate this product is shown in Fig. 3. The acyl nucleotide, V, overlapped guano- sine 5’-phosphate and was separated from the latter by prepara- tive thin layer chromatography. Its ultraviolet spectra at pH 1 and 7 resembled those of N2-a-ethoxypropionylguanine (3). Treatment of V with ammonia converted it to guanosine 5’- phosphate (VI). Finally, application of a sequence consisting of periodate oxidation, followed by aniline-catalyzed elimination (12) to V, converted it to NQ-ethoxypropionylguanine (VII).

DISCUSSION

Treatment of yeast RNA with Kethoxal, followed by periodate, leads to the specific attachment of a-ethoxypropionyl groups onto the guanine amino groups of the RNA. This was estab- lished by the marked reduction in the amount of deamination by nitrous acid of the guanines in the RNA, by nuclear magnetic resonance spectroscopy of the modified RNA, and by the iso- lation of the acylated guanine nucleotide from the modified RNA. No other products were observed in the hydrolysate of the acylated RNA, nor was any significant alteration in the amount of deamination by nitrous acid of the other amino bases detected.

Some ambiguity remains as to the extent of acylation of the guanines of the RNA. The nuclear magnetic resonance suggests that almost complete acylation (88%) had taken place, and this was supported by the failure to get further reaction under forcing conditions. However, deamination of the guanines of the RNA by nitrous acid took place to an extent that suggested that only about two-thirds of them were protected. Two possible explanations for this apparent discrepancy can be suggested. Yeast RNA is heterogeneous, and it may be that a portion of the guanines was protected from acylation by strong secondary structure. The nuclear magnetic resonance result could then be ascribed to inaccuracy in integration, or other undetermined error. However, Litt and Hancock (5) reported that the gua- nines of tRNA were able to react completely with Kethoxal, despite hydrogen bonding. An alternative explanation is that slow loss of the acyl groups occurred under the conditions of nitrous acid treatment, leading to some dea,mination. Although

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Issue of May 25, 1970 R. Shapiro, B. I. Cohen, and D. C. Clagett 2639

Nz-a-ethoxypropionylguanine (VII) was stable under these con- ditions, the same stability need not apply at the nucleic acid level.

This procedure for the acylation of guanines in RNA should provide a useful tool for studies of the structure and function of nucleic acids. One obvious application is to label single stranded regions of an RNA. Litt and Hancock (5) have demonstrated that the initial reaction of Kethoxal with tRNA can be controlled so that only the guanines in single stranded regions react. Perio- date treatment would then convert the initial labile adducts to more stable acyl groups. The guanine acylation method is complementary to a reported procedure for the acetylation of cytosine residues of RNA (19, 20). It should be noted that the cytosine acylations were also found to stop after two-thirds of the bases were acylated.

Our results indicate that treatment of RNA with glyoxal, followed by periodate cleavage, results in nonspecific formylation of all three amino bases. This conclusion is based on the nitrous acid deamination study of the formylated RNA. Unless some very unusual acylation mechanism is operating, the nonspecific- ity must reside in the fact that glyoxal reacts with adenine and cytosine, as well as guanine, within RNA. This result is strik- ingly different from that observed at the nucleoside level, at which specificity for guanine exists under the same reaction conditions. It suggests that one should proceed with caution in extrapolating reactivities observed at the nucleoside level to nucleic acids. This study casts doubt upon claims, based upon extrapolations of this type, of the use of glyoxal for the specific modification of the guanines of RNA and DNA (4,7).

An interesting feature of this work is the use of nuclear mag- netic resonance spectroscopy to characterize a chemically modi- fied nucleic acid. This technique is routinely used in organic chemistry and seems especially applicable to RNA because of the presence of considerable areas of the spectrum free of inter- ference from the RNA protons. This would be particularly useful when it is necessary to distinguish between alternative chemical reactions, which give rise to differing absorptions in the nuclear magnetic resonance spectrum. The most obvious draw- back is the relative insensitivity (as compared to ultraviolet or fluorescence spectroscopy) of the nuclear magnetic resonance instrumentation routinely available in most laboratories. This would make the technique useful only when a considerable amount of modification of the RNA has taken place.

A new observation was made in the studies of the nitrous acid deamination of yeast RNA. Care was taken to determine quantitatively the recovery of nucleotides, as compared to suit-

able controls. It was found that not all of the guanine lost can be accounted for as xanthine. This has also been reported by other workers in studies on the deamination of tobacco mosaic virus RNA by nitrous acid (21). In the present case, however, some destruction of either cytosine or uracil was also observed. This has not been reported before, and may be responsible for some of the many anomalies reported in nucleic acid-nitrous acid reactions. For a more extensive discussion of this, see Shapiro and Pohl (8).

Ackncnuledgments-We wish to thank Dr. Edward Reich for valuable advice and encouragement and Dr. Paul W. O’Connell of the Upjohn Company for a gift of Kethoxal.

REFERENCES

1. STAEHELIN, M., Biochim. Biophys. Ada, 31, 448 (1959). 2. SHAPIRO, R., AND HACHMANN, J., Biochemistry, 6, 2799 (1966). 3. SHAPIRO, R., COHEN, B. I., SHIUEY, S.-J., AND MAURER, H.,

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(1969). 17. CARBON, J. A., Biochim. Biophys. Acta, 96, 550 (1965). 18. SMITH. I. C. P., YAMANE, T., AND SCHULMAN, R. G., Science,

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Robert Shapiro, Bertram I. Cohen and Donald C. ClagettSpecific Acylation of the Guanine Residues of Ribonucleic Acid

1970, 245:2633-2639.J. Biol. Chem. 

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