Substrates for Investigation of DNA Polymerase Function: Synthesis and Properties of 4′‐<i>C</i>‐Alkylated Oligonucleotides

Detmer, Ilka; Summerer, Daniel; Marx, Andreas

doi:10.1002/ejoc.200200641

Cited by 17 publications

(25 citation statements)

References 39 publications

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“…In contrast, the heteroduplexes containing the 3Ј-methyl-modified deoxyribonucleotide, which have been shown to exhibit a sugar conformation similar to that of the 4Ј-methyl nucleosides, were significantly better substrates for human RNase H1. The observed differences in the human RNase H1 activity for the 4Ј-methylthymidine and 3Ј-methylthymidine heteroduplexes may be a function of the position of the 4Ј-methyl moiety on the furanose ring, which is predicted to contribute bulk in the minor groove, potentially interfering with enzyme binding (38). Furthermore, the loss in human RNase H1 activity observed for the 4Ј-methylthymidine heteroduplexes suggests that proper orientation of the phosphate group on the deoxyribonucleotide opposing the scissile ribonucleotide is important for human RNase H1 activity.…”

Section: Discussionmentioning

confidence: 99%

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Structural Requirements at the Catalytic Site of the Heteroduplex Substrate for Human RNase H1 Catalysis

Lima

Nichols

et al. 2004

Journal of Biological Chemistry

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Human RNase H1 cleaves RNA exclusively in an RNA/ DNA duplex; neither double-strand DNA nor doublestrand RNA is a viable substrate. Previous studies suggest that the helical geometry and sugar conformation of the DNA and RNA may play a role in the selective recognition of the heteroduplex substrate by the enzyme. We systematically evaluated the influence of sugar conformation, minor groove bulk, and conformational flexibility of the heteroduplex on enzyme efficiency. Modified nucleotides were introduced into the oligodeoxyribonucleotide at the catalytic site of the heteroduplex and consisted of southern, northern, and eastern biased sugars with and without 2-substituents, non-hydrogen bonding base modifications, abasic deoxyribonucleotides, intranucleotide hydrocarbon linkers, and a ganciclovir-modified deoxyribonucleotide. Heteroduplexes containing modifications exhibiting strong northern or southern conformational biases with and without a bulky 2-substituent were cleaved at a significantly slower rate than the unmodified substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Finally, modified heteroduplexes containing modifications predicted to mimic the sugar pucker and conformational flexibility of the deoxyribonucleotide exhibited cleavage rates comparable with those of the unmodified substrate. These data suggest that sugar conformation, minor groove width, and the relative positions of the intra-and internucleotide phosphates are the crucial determinants in the selective recognition of the heteroduplex substrate by human RNase H1 and offer immediate steps to improve the performance of DNA-like antisense oligonucleotides.RNase H hydrolyzes RNA in RNA-DNA hybrids (1). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2-7). Although RNases H constitute a family of proteins of varying molecular mass, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNases H studied to date function as endonucleases, exhibiting limited sequence specificity and requiring divalent cations (e.g. Mg 2ϩ and Mn 2ϩ ) to produce cleavage products with 5Ј-phosphate and 3Ј-hydroxyl termini (8). Recently, two human RNase H genes have been cloned and expressed (9 -11). RNase H1 is a 286-amino acid protein and is expressed ubiquitously in human cells and tissues (9). The amino acid sequence of human RNase H1 displays strong homology to RNase H1 from yeast, chicken, Escherichia coli, and mouse (9). The human RNase H2 enzyme is a 299-amino acid protein with a calculated mass of 33.4 kDa and has also been shown to be ubiquitously expressed in human cells and tissues (10).1 Human RNase H2 shares strong amino acid sequence homology with RNase H2 from Caenorhabditis elegans, yeast, and E. coli (10). Although the biological roles for the human enzymes are not fully under...

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Section: Discussionmentioning

confidence: 99%

“…phosphoramidite following the procedure described for similar compounds (37,38). Standard phosphoramidites and solid supports were used for incorporation of A, T, G, and C residues.…”

Section: Synthesis Of Modified Oligonucleotidesmentioning

confidence: 99%

Structural Requirements at the Catalytic Site of the Heteroduplex Substrate for Human RNase H1 Catalysis

Lima

Nichols

et al. 2004

Journal of Biological Chemistry

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“…Desilylation and 5'-O-tritylation of 2 a-c yielded 3 a-c, which were subsequently coupled to a succinylated long-chain alkyl amine-modified controlled pore glass (LCAA-CPG) support. [33] The solid supports 4 a-c were used in standard automated DNA synthesis to yield oligonucleotides 5-7 that carry the respective, modified thymidine moiety at their 3' termini. [33] We also synthesized oligonucleotides bearing 4'-C-hydroxymethylene moieties at their 3' termini by following a protocol published by Wengel and co-workers.…”

Section: Resultsmentioning

confidence: 99%

“…[33] The solid supports 4 a-c were used in standard automated DNA synthesis to yield oligonucleotides 5-7 that carry the respective, modified thymidine moiety at their 3' termini. [33] We also synthesized oligonucleotides bearing 4'-C-hydroxymethylene moieties at their 3' termini by following a protocol published by Wengel and co-workers. [34] Attempts to synthesize the 4'-Cethoxymethylene analogue from 1 failed; therefore, we developed the synthesis by the de novo construction of the nucleoside.…”

Section: Resultsmentioning

confidence: 99%

Tuning Single Nucleotide Discrimination in Polymerase Chain Reactions (PCRs): Synthesis of Primer Probes Bearing Polar 4′‐C‐Modifications and Their Application in Allele‐Specific PCR

Gaster

Marx

2005

Chemistry A European J

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There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.

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“…The hydrophobic 4 0 -C-modifications were introduced by conversion of the alcohol 56 into iodide 57 by treatment with iodine, triphenylphosphine, and imidazole (Scheme 4.10) [59,66]. Reduction by hydrogenation with palladium on carbon in the presence of triethylamine and subsequent cleavage of the silylethers with tetra-n-butylammonium fluoride (TBAF) yielded 4 0 -C-methyl thymidine 58a.…”

Section: Design and Synthesismentioning

confidence: 99%

2′‐Deoxyribose‐Modified Nucleoside Triphosphates and their Recognition by DNA Polymerases

Jung

Marx

2008

Modified Nucleosides

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The entire blueprint of life is stored in the DNA of every living species. Generally, DNA is built from the four nucleotide building blocks that contain one of the four nucleobases, a phosphate that bridges the nucleotides via phosphodiester bonds, and a 2 0 -deoxyribose unit. The interconnected building blocks result in DNA strands that pair to form the well-known double helix via Watson-Crick pairing [1]. DNA is central to the evolution of life, and the transmission of genetic information from the parental DNA strand to the offspring is crucial for the survival of any living species [2][3][4][5][6][7][8]. This process must be sufficiently error-free (i.e., transmission according to the WatsonCrick pairing) to ensure the integrity of the genome, yet also sufficiently error-prone in order to spur evolution and foster the preservation of a species. Clearly, this process must be well balanced, and along these lines several intriguing aspects related to DNA have long inspired and challenged scientists of many disciplines. The mechanisms of the above-depicted information transfer from the ancestor to the offspring have been a matter of interest since the first proposal of the DNA structure, and even until now they are not fully understood. Following another aspect of DNA function, scientists have investigated the causative of the present-day nucleic acids, namely DNA and RNA [9,10]. Insights into the chemical etiology of DNA and RNA are crucial for our understanding of the evolution, for example, of DNA as a generic genetic material.In nature, all DNA synthesis is catalyzed by DNA polymerases [11]. These enzymes catalyze proceeding DNA synthesis in a template-directed manner by recognizing the template to correctly insert the complementary nucleotide. DNA polymerase selectivity is crucial for the survival of any living species. These enzymes are presented with a pool of four structurally similar dNTPs, and of course NTPs, from which the sole correct (i.e., Watson-Crick base-paired dNTP) substrate must be selected for incorporation into the growing DNA strand. The mechanisms by which these remarkable enzymes achieve this tremendous task have been a matter of interest and intensive Modified Nucleosides: in Biochemistry, Biotechnology and Medicine. Edited by Piet Herdewijn

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Substrates for Investigation of DNA Polymerase Function: Synthesis and Properties of 4′‐C‐Alkylated Oligonucleotides

Cited by 17 publications

References 39 publications

Structural Requirements at the Catalytic Site of the Heteroduplex Substrate for Human RNase H1 Catalysis

Structural Requirements at the Catalytic Site of the Heteroduplex Substrate for Human RNase H1 Catalysis

Tuning Single Nucleotide Discrimination in Polymerase Chain Reactions (PCRs): Synthesis of Primer Probes Bearing Polar 4′‐C‐Modifications and Their Application in Allele‐Specific PCR

2′‐Deoxyribose‐Modified Nucleoside Triphosphates and their Recognition by DNA Polymerases

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