Mechanism of Degradation of Purine Nucleosides by Formamide. Implications for Chemical DNA Sequencing Procedures † Raffaele Saladino,* ,‡ Enrico Mincione, ‡ Claudia Crestini, ‡ Rodolfo Negri, § Ernesto Di Mauro, §,⊥ and Giovanna Costanzo ⊥ Contribution from the Dipartimento ABAC, UniVersita ` degli Studi della Tuscia, Viterbo 01100, Italy, Centro di Studio per gli Acidi Nucleici, CNR, Roma, Italy, and Fondazione “Istituto Pasteur - Fondazione Cenci Bolognetti”, c/o Dipartimento di Genetica e Biologia Molecolare, UniVersita ` “La Sapienza”, Roma, Italy ReceiVed October 20, 1995 X Abstract: We describe the reaction of formamide with 2′-deoxyadenosine and 2′-deoxyguanosine to give imidazole ring opening by nucleophilic addition on the electrophilic C(8)-position of the purine ring. This information allows improvement of the one-lane chemical DNA sequencing procedure based on the base-selective reaction of formamide with DNA. The reactivity with formamide of several 7-deazapurine analogues (7-deaza-2′-deoxyinosine, 7-deaza- 2′-deoxyguanosine, and 7-deaza-2′-deoxyadenosine) incorporated into polynucleotides is also described. The wide spectrum of different sensitivities to formamide displayed by these purine analogues provides the single-lane DNA chemical sequencing procedures with the possibility of wide-ranging signal intensity modulation and thus increased specificity. Introduction Methods for accurately establishing the order of the four bases along given DNA fragments are based on two different principles. One method uses chemical reagents that react with specific bases to break DNA preferentially at given nucleotides, 1 the other is based on the analysis of the products of DNA polymerization selectively interrupted with chain terminating deoxyribonucleotides. 2 The ideal DNA sequencing method should yield unambiguous and complete information in a single electrophoretic lane and should be simple, rapid, economical and accurate. Compression of DNA sequencing procedures is crucial for development and improvement of automated analytical systems. Methods for partial sequence data compression have been reported, 3,4 and a single-lane sequencing procedure is currently available, based upon the dideoxy Sanger methodology, analyzed in automated sequences. This method (four fluorochromes/four bases) cannot a priori be compressed further. Chemical DNA sequencing analysis offers the potential of complete compression: if one could obtain unambiguous and complete sequence information in a single lane, then four different DNAs, each labeled with a different fluorochrome, could be analyzed in the same electro- phoretic lane (four fluorochromes/four DNAs). The methods developed so far which aim to simplify the sequencing procedures by introducing chemical alternatives to the classical Maxam and Gilbert protocols 1 have been reviewed. 5-7 The DNA sequencing procedure based on the selective degradation of nucleic acids by formamide provides complete sequence information in a single electrophoretic lane, by measuring intensity of the signal corresponding to each cleaved sequence position. 8-11 This procedure depends on the degrada- tion of the purine and pyrimidine bases by formamide at high temperature (>100 °C) followed by scission of the glycosidic linkages through -elimination reactions. In the presence of a weak base such as formamide, only the 3′--elimination occurs; 8 efficient -elimination at 5′ (leading to unbiased sequencing of 5′-labeled DNAs) requires the use of piperidine 9,10 as a second reaction step. Irrespective of the specific protocol used for the phosphodiester bond breakage, the selective (i.e., base-specific) part of the reaction is carried out by formamide. 8-11 In any sequencing protocol, the best analytical condition is one in which the difference in reactivity between the four bases is homogeneous and large. The order of sensitivity observed in the formamide reaction is G > A > C . T, where the reactivity ratio Gs/As ranges from 1 to 1.5 depending on the * Corresponding author: Raffaele Saladino, Dipartimento ABAC Uni- versita ` degli Studi della Tuscia, Via Camillo De Lellis, Viterbo 01100, Italy. Tel. + 39.761.357230. Fax. + 39.761.357242. ‡ Universita ` degli Studi della Tuscia. § Centro di Studio per gli Acidi Nucleici. ⊥ Universita ` “La Sapienza”. † Abbreviations: G, guanosine; A, adenosine; I, inosine; C, cytidine; T, thymidine; ATP, adenosine triphosphate; CTP, cytidine triphosphate; dATP, 2′-deoxyadenosine triphosphate; dGTP, 2′-deoxyguanosine triphosphate; dITP, 2′-deoxyinosine triphosphate; deazaA, 7-deaza-2′-deoxyadenosine; deazadATP, , 7-deaza-2′-deoxyadenosine triphospate; deazadGTP, 7-deaza- 2′-deoxyguanosine triphosphate; deazadITP, 7-deaza-2′-deoxyinosine tri- phosphate; PCR, polymerase chain reaction; PE, primer extension; TLC, thin layer chromatography; EDTA, ethylenediaminetetraacetic acid; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide. X Abstract published in AdVance ACS Abstracts, June 1, 1996. (1) Maxam, A. M.; Gilbert, W. Proc. Natl. Acad. Sci. US.A. 1977, 74, 560-563. (2) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463-5467. 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