Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

Macíčková-Cahová, Hana; Hocek, Michal

doi:10.1093/nar/gkp845

Cited by 58 publications

(29 citation statements)

References 68 publications

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“…Two enzymes (AfeI and PspGI) even tolerate the presence of a phenyl or nitrophenyl group on dU. The cleavage patterns of the REs as a function of the size of the modification at dU were in good agreement with the results of our previous study [4] of the influence of modifications on A, in which most enzymes tolerated 7-deazaA and some even the presence of an ethynyl group at C7. This might suggest that, at least for some restriction enzymes (i.e., SacI, PspGI, RsaI, KpnI and AfeI) the specific major-groove interactions are more crucial for the recognition of G:C pairs than of A:T pairs in the target se- Figure 3.…”

Section: Discussionsupporting

confidence: 87%

“…The observed oligonucleotide fragments were longer than the expected cleavage products. In our previous paper [4] we attributed a similar observation of longer products to star activity. [22] We have now found, however, that this observation has a different explanation.…”

Section: Resultssupporting

confidence: 59%

“…As in the synthesis of 7-ethynyl-7-deazadATP, [4] the ethynylpyrimidine dNTPs (dC E TP and dU E TP) cannot be prepared directly by aqueous cross-coupling of halogenated dNTPs. [2] They were therefore prepared by phosphorylation of the corresponding nucleosides (Scheme 1 A).…”

Section: Resultsmentioning

confidence: 99%

“…Surprisingly high tolerance of several REs to some A-modifications (depending on the size of the modification) within the recognition sequence and a general tolerance to any modification next to it was observed. [4] However, data on the interaction of pyrimidine-modified DNA with REs are very scarce. Marchionni et al [5] studied cleavage of DNA from bacteriophage l containing 5-bromodeoxyuridine with EcoRI, HindIII, and SmaI REs and concluded that the modified DNA was cleaved at the same site as the unmodified DNA.…”

Section: Introductionmentioning

confidence: 99%

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

2011

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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.

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Section: Discussionsupporting

confidence: 87%

Section: Resultssupporting

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

2011

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“…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.…”

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confidence: 89%

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

et al. 2011

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

et al. 2011

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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...

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Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

Cited by 58 publications

References 68 publications

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

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

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

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

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