Amplification of 4‘-ThioDNA in the Presence of 4‘-Thio-dTTP and 4‘-Thio-dCTP, and 4‘-ThioDNA-Directed Transcription in Vitro and in Mammalian Cells

Inoue, Naonori; Shionoya, Aki; Minakawa, Noriaki; Kawakami, Akiko; Ogawa, Naoki; Matsuda, Akira

doi:10.1021/ja075953c

Cited by 34 publications

(32 citation statements)

References 11 publications

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“…Using this DNA polymerase, we prepared a modified DNA library involving C5-modified thymidine and successfully screened modified DNA aptamers bound to sialyllactose, R-isomer of thalidomide derivative, and so on by SELEX (23–25). Recently, Inoue et al (69) reported that double-stranded 4′-thioDNAs were directly amplified by PCR using KOD Dash and triphosphates of 4′-thio-nucleoside. Thus, KOD Dash DNA polymerase could accept a broad range of nucleotide modifications and might be best suited for enzymatic preparation of functional modified DNA.…”

Section: Resultsmentioning

confidence: 99%

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2′,4′-bridged nucleosides

Kuwahara

Obika

Nagashima

et al. 2008

Nucleic Acids Research

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In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2′,4′-bridged nucleotides and three types of 2′,4′-bridged nucleoside-5′-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2′-O,4′-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2′,4′-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2′,4′-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2′,4′-bridged nucleotides into extending strands using 2′,4′-bridged nucleoside-5′-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.

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Section: Resultsmentioning

confidence: 99%

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2′,4′-bridged nucleosides

Kuwahara

Obika

Nagashima

et al. 2008

Nucleic Acids Research

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“…2′-Deoxy-4′-thiocytidine 5′-triphosphate (dSCTP) was prepared according to our previous reports. [13] Oligonucleotides were purchased from FASMAC (Kanagawa, Japan). Rhodamine-labeled anti-GFP siRNA (21-mer, 5′-gcugacccugaaguucauctt-3′, 5′-gaugaacuucagggucagctt-3′) was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

Hasan

Tarashima

Fujikawa

et al. 2016

Science and Technology of Advanced Materials

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An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

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“…These polymerases could accept NTP analogs of 2′-thio (–SH) [17], 2′-amino (–NH 2 ) [19], 2′-azido (–N 3 ) [29], 2′-hydroxymethyl (–CH 2 OH) [33], and 4′-thio (–S–) [39] in addition to 2′-F and 2′-OMe as substrates. Furthermore, NTP analogs with base modification (e.g., C5-modified uridines and cytidines) and with phosphate-modified nucleosides (e.g., 5′-( α -thio)triphosphates and 5′-( α -borano)triphosphates) [21, 30], which are available for modified RNA polymerization, have also been reported.…”

Section: Enzymatic Modified Rna/dna Polymerizationmentioning

confidence: 99%

Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries

Kasahara

Kuwahara

2012

Journal of Nucleic Acids

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Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.

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Amplification of 4‘-ThioDNA in the Presence of 4‘-Thio-dTTP and 4‘-Thio-dCTP, and 4‘-ThioDNA-Directed Transcription in Vitro and in Mammalian Cells

Cited by 34 publications

References 11 publications

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2′,4′-bridged nucleosides

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2′,4′-bridged nucleosides

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries

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