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

Abstract

The sequence-specific cleavage of DNA by type II restriction endonucleases (REs) is of paramount importance in DNA manipulations, such as recombination and cloning. Hundreds of REs are commercially available and used in molecular biology. [1] Some of them are sensitive to DNA methylation, and in some bacteria REs cleave only non-methylated sequences as a defense against foreign DNA. When manipulations of large DNA sequences are required, the possibility of the selective protection of certain sequences against cleavage might be useful, especially if the recognition sequence for a particular RE is present in several copies. Base-modified nucleic acids are nowadays frequently applied in diverse areas of chemical biology, but their cleavage by REs has been only scarcely studied. DNA containing 7deazaadenine [2] (7-deazaA) or 7-deazaguanine [2e] in a recognition sequence was repeatedly reported to be resistant to some endonucleases, apparently because the N7 atom cannot form hydrogen bonds in the major groove. 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 7substituted 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 7substituted derivatives. In these studies, [4,5] permanent protection against cleavage by REs was also demonstrated. Herein, we report on the first transient and switchable protection of DNA against REs.In our previous study, [4] we noticed that several REs (RsaI, [2a] KpnI, [6] and SacI [7] ) showed high tolerance to the presence of an alkynyl group but not to a more bulky phenyl group at position 7 of 7-deazaA. Therefore, we envisaged that some silyl-substituted 7-ethynyl-7-deazaadenines might be used as transient protection against REs (analogous to the (trialkylsilyl)alkyne protection [8] for the triple click labeling of DNA by triazole formation, a method that was recently developed by the Carell group).Therefore, we tested three trialkylsilyl groups (trimethylsilyl (TMS), triethylsilyl (TES), and triisopropylsilyl (TIPS)) as protection for the acetylene at position 7 of 7-deaza-2'deoxyadenosine. [9] The synthesis and deprotection was first studied on model nucleoside monophosphates (see the Supporting Information for details). This study showed that the TMS-and TES-protected 7-ethynyl-dAMP could be easily deprotected by simple treatment with ammonia, whereas the TIPS group must be cleaved by treatment with tetra-n-butylammonium fluoride (TBAF). The next task was the preparation of (trialkylsilyl)ethynyl-modified deoxyribonucleoside triphosphates (dNTPs) as substrates for the polymerase synthesis of DNA. The polymerase construction of base-modified DNA [10] is now an established and widely used procedure. In our group, we combined this procedure with the aqueous cross-coupling of halogenated dNTPs [11] into an efficient two-step synthesis of DNA b...

Polymerase Synthesis of Photocaged DNA Resistant against Cleavage by Restriction Endonucleases

“…[1,2] The masking and modulation of sequence-specific recognition and cleavage of the DNA sequences by REs is applicable by itself in gene manipulation and cloning, [9,10] but hopefully also can be further extended to the modulation of binding of tran-scription factors in the regulation of gene expression. DNA sequences that contain naturally occuring U HM can be synthetized either directly (by polymerase incorporation of dU HM TP) or indirectly through photolysis of U NB .…”

“…[6] A series of a-branched 2-nitrobenzyl-protected 5-(hydroxymethyl)pyrimidine and 7-hydroxymethyl-7-deazapurine dNTPs have been studied as photocleavable reversible terminators for sequencing. This fact was later applied [9] in the first transient chemical protection of DNA from RE cleavage by polymerase incorporation of 7-(triethylsilylethynyl)-7-deazaadenine nucleotides to form RE-resistant protected DNA which, after desilylation by treatment with ammonia, released ethynylmodified DNA fully cleavable by several REs. This fact was later applied [9] in the first transient chemical protection of DNA from RE cleavage by polymerase incorporation of 7-(triethylsilylethynyl)-7-deazaadenine nucleotides to form RE-resistant protected DNA which, after desilylation by treatment with ammonia, released ethynylmodified DNA fully cleavable by several REs.…”

7‐Aryl‐7‐deazaadenine 2′‐Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

“…[10] Therefore, we envisaged the use of REs [11] for analyzing the products of competitive incorporations since, for any modification, we know specific REs which do not cleave the modified recognition sequence. [10] Therefore, we envisaged the use of REs [11] for analyzing the products of competitive incorporations since, for any modification, we know specific REs which do not cleave the modified recognition sequence.…”

“…They included ethynyl and phenyl derivatives, [10] redox labels (dN NO2 TP and dN NH2 TP), [12] fluorescent labels (dA BFU TP, dA ABOX TP), [13] reactive groups (dC FT TP), [14] and very bulky groups (dN STr TP). They included ethynyl and phenyl derivatives, [10] redox labels (dN NO2 TP and dN NH2 TP), [12] fluorescent labels (dA BFU TP, dA ABOX TP), [13] reactive groups (dC FT TP), [14] and very bulky groups (dN STr TP).…”

Synthesis of Nucleobase‐Functionalized Morpholino‐Modified Nucleoside Monomers Through Palladium‐Catalyzed Cross‐Coupling Reactions

“…Ideal sites for the incorporation of any group in a nucleobase without disturbing Watson-Crick base pair formation are the C-5 position in a pyrimidine base and the C-8 position in a purine base. [22] Incorporation of a propyne group in a nucleobase is known to provide better duplex stability in DNA. [17] Sonogashira, [18] Suzuki, [19] and Heck [20] coupling reactions have been used to prepare modified nucleosides.…”