Hikoya Hayatsu

Bisulfite Modification of Nucleic Acids and their Constituents

Publisher Summary This chapter discusses that nucleic acids are labile in solutions of extreme pHs and at high temperatures; the chemical modification must preferably be performed at a pH region around neutrality and at a temperature near 37°C or below. Chemical reactions that satisfy these requirements have been developed and used in nucleic acid studies. Bisulfite deaminates cytosine to give uracil. The reaction conditions are satisfactorily mild, and no deamination of adenine or guanine moieties takes place under the conditions employed. Although bisulfite does react with uracil and thymine under similar conditions, the bisulfite adducts formed are easily reverted to the parent pyrimidines. Therefore, cytosine residues in nucleic acids can be selectively transformed into uracil residues by the use of bisulfite. The most important feature of bisulfite reactions is their strict selectivity for the single-stranded regions of nucleic acids. Thus, bisulfite is a reagent that meets in excellent fashion the requirements to serve as a probe of nucleic acid functions and structures. In spite of the fact that the bisulfite reactions of nucleic acid components were discovered only several years ago, extensive studies of the reaction at the polynucleotide level have already been made and a number of applications reported. These involve (1) application for synthetic purposes, (2) studies on physical and biochemical properties of bisulfite-treated polynucleotides, (3) cross-linking of nucleic acid with protein, (4) chemical modifications of transfer RNA with the purpose of elucidating functional sites and conformations, and (5) induction of mutation in microorganisms.

The selective degradation of pyrimidines in nucleic acids by permanganate oxidation.

Abstract In our current studies on the chemical modifications of nucleic acids, we have reported on the selective reaction of semicarbazide derivatives with cytidine residues ( Hayatsu and Ukita, 1964 , Hayatsu and Ukita, 1966 ; Hayatsu et al. , 1966 ; Kikugawa et al., 1967a , Kikugawa et al., 1967b ). In this paper we describe a procedure by which pyrimidines in the single stranded region of nucleic acids are degraded selectively. Furthermore, reaction conditions are presented whereby thymidylic acid is exclusively attacked while other deoxyribonucleotides are virtually intact. This procedure consists of the permanganate oxidation of the nucleic acid under relatively mild reaction conditions of pH 6.7 and 0°C.

The mutagenic action of sodium bisulfite

Abstract This paper describes the mutagenic activity of sodium bisulfite toward phage λ. The frequency of mutation of the c gene of λ was increased about 10 times as much as that of spontaneous mutation by treatment of the phage with 3 M NaHSO3 solution of pH 5.6 at 37° for 1.5 hrs. The mutagenesis could be related to the cytosine derivative specific reaction of sodium bisulfite.

The permanganate oxidation of thymine

Abstract 1. Mild permanganate oxidation of thymine yielded cis-thymine glycol (cis-5,6-dihydroxy-5,6-dihydrothymine) and 5-hydroxy-5-methylbarbituric acid. Under certain reaction conditions, only these two compounds were the products of the reaction. 2. While cis-thymine glycol was resistant to hydrolysis at neutral pH, 5-hydroxy-5-methylbarbituric acid readily underwent hydrolysis to give methyltartronylurea. 3. In this oxidation reaction, 5-hydroxy-5-methylbarbituric acid, which is higher in the oxidation level than thymine glycol, is formed not via the thymine glycol but directly from thymine. 4. When the oxidation of thymine was carried out at an acidic pH, 5-hydroxy-5-methylbarbituric acid was predominantly produced, whereas thymine glycol was preferentially formed when the oxidation was performed at an alkaline pH. 5. The mechanism of the oxidation is discussed.

The permanganate oxidation of deoxyribonucleic acid

Abstract Mild permanganate oxidation is demonstrated to be a convenient method to selectively degrade thymine residues in DNA. The procedure consists of treatment of a denatured DNA with 5–15 mM potassium permanganate solution at pH 8.6 or in 0.02 M KOH. High selectivity of the reaction toward thymine residues can be retained until 60–70 % of total thymine residues has been oxidized. More extensive degradation accompanies oxidation of cytosine residues. Purines are not affected under the conditions employed. Only a little strand break occurs during the oxidation. The permanganate oxidation may be of value for biochemical studies of DNA.

Chemical modification of tRNAyeastTyr with bisulfite. A new method to modify isopentenyladenosine residue

Abstract When tRNAyeastTyr was treated with bisulfite at pH 7, the uracil (U) residue next to the 5′-end of the anticodon and the isopentenyladenine (iA) residue adjacent to the 3′-end of it were chemically modified. On treatment of this transformed tRNA with weak alkali, the modified U residue regenerated U, while the modified iA remained unaltered. The modification of both the U and iA residues did not affect the amino acid accepting activity of the tyrosine tRNA, while the specific modification of the iA residue resulted in the decrease of the ability of the tyrosyl tRNA to bind to the messenger-ribosome complex.

Phosphorus-Proton Spin-Spin Coupling and Conformation of a Dinucleoside Phosphate

The phosphorus-31 nuclear magnetic resonance spectrum of β-adenosine-3-β-adenosine-5-phosphoric acid in its aqueous solution (pH = 9.2) was studied. The signal consisted of eight peaks caused by the spin-spin coupling of the phosphorus nucleus with three protons, two on the 5 carbon, and one on the 3 carbon. The coupling constants were 3.4, 6.5, and 8.1 hertz; from these values the dihedral angles of the three P-O-C-H systems were estimated.

The modification of nucleosides and nucleotides

Abstract 1. Semicarbazide has been found to replace the C-4 amino group of cytidine and deoxycytidine with a semicarbazido residue. The reaction occurred at a maximum rate at pH 4.2 and followed pseudo-first-order kinetics. 2. The reaction was found to be absolutely specific for cytosine nucleosides or nucleotides and no reaction between semicarbazide and adenosine, guanosine, uridine, or their corresponding nucleotides was observed in the pH range 4.0–9.5 and 37°. 3. The product of the reaction between semicarbazide and cytidine was identified as 4-deamino-4-semicarbazido cytidine. This latter compound was synthesized from 2′,3′,5′-tri- O -benzoyl-4-thiouridine by reaction with semicarbazide followed by debenzoylation. 4. 4-Deamino-4-semicarbazido cytidine 2′,3′-cyclic phosphate was more slowly hydrolyzed than uridine 2′,3′-cyclic phosphate by bovine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2′-transferase (cyclizing), EC 2.7.7.16). 5. An application of the reaction to the selective modification of cytidine or deoxycytidine residues in RNA and DNA is proposed.

Inhibition of nitrosamine formation in vitro by sorbic acid.

Abstract Sorbic acid ( trans,trans -2,4-hexadienoic acid), a food preservative, was found to react rapidly with nitrite in acidic media. One of the products of the reaction was isolated and identified as an oxime nitrite of sorbic acid. Sorbic acid inhibited the in vitro formation of N -nitrosodimethylamine from dimethylamine and nitrite. The extent of the inhibition by sorbic acid was approximately the same as that by ascorbic acid under comparable conditions. Against the formation of N -nitrosomorpholine, the inhibitory action of sorbic acid was weaker than that of ascorbic acid, and unlike the latter acid, sorbic acid had no inhibitory effect on the nitrosation of N -methylaniline.

The modification of nucleosides and nucleotides: IV. The reaction of semicarbazide with nucleic acids

Abstract 1. Cytidine residues of yeast RNA have been specifically converted into semicarbazide derivatives without altering any other bases. In this reaction the C-4 amino groups of cytidine residues were converted into a semicarbazido group. 2. tRNA modified with semicarbazide was completely hydrolysed to nucleoside 5′-phosphate with snake venom phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1). The base composition of the tRNA was estimated after separation of the nucleotides by ion-exchange column chromatography. A quantitative estimation of the modified cytidine residues in the tRNA was achieved by this method. 3. The results of sedimentation analysis and gel filtration of the tRNA modified with semicarbazide indicated that no splitting of the internucleotide bonds of the RNA occurred during the reaction. 4. The temperature-absorbance relationship plotted for the modified tRNA indicated that the secondary structure in the tRNA decreased with increasing extent of modification. 5. The rate of this modification reaction is low when cytosine groups are highly hydrogen bonded (double-stranded DNA). 6. A simple method for estimating the extent and rate of the modification reaction of cytidine residues in nucleic acids is proposed. In this procedure the specific light absorption of the 4-deamino-4-semicarbazidocytidine residue in the range 300–340 mμ at pH 13 is followed.