Cleavage of Functionalized DNA Containing 5‐Modified Pyrimidines by Type II Restriction Endonucleases

Abstract

A series of six pyrimidine-modified dNTPs--5-ethynyl-, 5-phenyl-, and 5-(3-nitrophenyl)deoxycitidine and -deoxyuridine triphosphates--were prepared and incorporated by primer extension with Vent (exo-)polymerase to specific DNA sequences within or next to the recognition sequences of selected restriction endonucleases. The cleavage of these pyrimidine-modified DNA sequences by 13 restriction enzymes was then studied. Whereas the presence of any modified C within the target sequence completely prevented any restriction cleavage, most enzymes tolerated the presence of 5-ethynylU and two of them even the presence of 5-phenyl- and 5-(3-nitrophenyl)U. Modifications outside the recognition sequence were tolerated except in the case of phenyl derivatives with the PvuII enzyme. 5-EthynylC was used for protection of the recognition sequence from cleavage in the presence of the second unmodified copy of the same sequence that was cleaved.

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA–Protein Cross‐Linking

“…Nucleophilic substitution with sodium azide [34] followed by reactionw ith 2-chloroethanesulfonyl chloride affordedt he desired compound 3 in acceptabley ield (34 %o vert wo steps). [36,37] Therefore, the study required the preparation of 2'-deoxycytidine triphosphates that contained an alkyne moiety at position 5( dC E TP and dC O TP)a ss ubstrates for the polymerase synthesis of alkyne-modified DNA. [36,37] Therefore, the study required the preparation of 2'-deoxycytidine triphosphates that contained an alkyne moiety at position 5( dC E TP and dC O TP)a ss ubstrates for the polymerase synthesis of alkyne-modified DNA.…”

Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7‐Substituted 7‐Deazaguanine Nucleobases

“…On the other hand, some less bulky modificationsa re often tolerated at position7 of 7-deazaadenine and at position 5o fu racil. [14][15][16] This might indicatet hat, at least for some REs, the major-grooves pecific recognition of the G:Cp airs might be more important than that of A:Tp airs. However,b ecause even the enzymes that tolerate modifications on Aa nd T do not cleave the sequences with A:Tt oT :A mutation, they still must have other means for the recognitiono fA:T pairs that are not affected by small majorgroove substituents.…”

“…The residue was co-evaporated several times with water.T he product was purified by HPLC chromatography [aq. TEAB ( (100) (16,dG Ac TP):N ucleoside 14 (53 mg, 0.146 mmol) was suspended in trimethyl phosphate (426 mL) in an argonpurged vial, and the suspension was cooled to 0 8C. Then, non-redistilled POCl 3 (20 mL, 0.215 mmol) was added.…”

Transient and Switchable (Triethylsilyl)ethynyl Protection of DNA against Cleavage by Restriction Endonucleases

“…The incorporation of 7-deazaA was used [3] as a means of permanently protecting DNA against cleavage by certain REs. Recently, we reported a systematic study of the cleavage of DNA containing 7-substituted 7-deazaadenines [4] and 5-substituted pyrimidines; [5] we found a surprising tolerance of several REs to the presence of not only 7-deazaadenine but also some 7-substituted derivatives. In these studies, [4,5] permanent protection against cleavage by REs was also demonstrated.…”