An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA

Hirao, Ichiro; Kimoto, Michiko; Mitsui, Tsuneo; Fujiwara, Tsuyoshi; Kawai, Rie; Sato, Akira; Harada, Yuka; Yokoyama, Shigeyuki

doi:10.1038/nmeth915

Cited by 229 publications

(329 citation statements)

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“…Most of these investigations were driven by the search for additional base pairs to be used for the extension of the genetic alphabet, [1][2][3][4][5][6][7][8] as tools in biotechnology, [9][10][11] for probing recognition, fidelity, and nucleotide processing by DNA polymerases, [12][13][14][15] or for designing novel genetic systems. [16,17] Of special interest amongst these artificial constructs are aromatic base replacements that interact with each other, specifically without the formation of hydrogen bonds, merely on the basis of edge-on or face-on hydrophobic or stacking interactions.…”

Section: Introductionmentioning

confidence: 99%

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

Grigorenko

Leumann

2008

Chemistry A European J

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Three 2′‐phenanthrenyl‐C‐deoxyribonucleosides with donor (phenNH2), acceptor (phenNO2), or no (phenH) substitution on the phenanthrenyl core were synthesized and incorporated into oligodeoxyribonucleotides. Duplexes containing either one or three consecutive phenR residues, which were located opposite each other, were formed. Within these residues, the phenR residues are expected to recognize each other through interstrand stacking interactions, in much the same way as described previously for biphenyl DNA. The thermal, thermodynamic, and fluorescence properties of such duplexes were determined by UV melting analysis and fluorescence spectroscopy. Depending on the nature of the substituent, the thermal stability of single‐modified duplexes can vary between −2.7 to +11.3 °C in Tm and that of triple‐modified duplexes from +7.8 to +11.1 °C. Van′t Hoff analysis suggested that the observed higher thermodynamic stability in phenH‐ and phenNO2‐containing duplexes is of enthalpic origin. A single phenH or phenNO2 residue in a bulge position also stabilizes a corresponding duplex. If a phenNO2 residue is placed in a bulge position next to a base mismatch this can lead, in a sequence‐dependent manner, to duplex destabilization. The phenNO2 residue was found to be a highly efficient (10–100‐fold) quencher of phenH and phenNH2 fluorescence if placed in the opposite position to the fluorophores. When phenH and phenNH2 residues were placed opposite each other, efficient quenching of phenH and enhancement of phenNH2 fluorescence was found, which is an indicator for electron‐ or energy‐transfer processes between the aromatic units.

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Section: Introductionmentioning

confidence: 99%

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

Grigorenko

Leumann

2008

Chemistry A European J

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“…Similarly, Hirao and coworkers have shown that the unnatural nucleotide 2-amino-6-(2-thienyl)purine can direct the sitespecific incorporation of 2-oxopyridine into RNA (54). More recently, the same group has expanded the scope of the hydrophobic base pairs developed by Schweitzer and Kool (55) and Romesberg et al (56) for the site-specific incorporation of nucleotide analogs in RNA (57). In addition, they have demonstrated site-specific incorporation of cross-linking analogs, biotinylated nucleotides, and fluorescent analogs into RNA (58)(59)(60).…”

Section: Enzymatic Synthesismentioning

confidence: 96%

Combined Approaches to Site-Specific Modification of RNA

2008

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Both natural and unnatural modifications in RNA are of interest to biologists and chemists. More than 100 different analogs of the four standard RNA nucleosides have been identified in nature. Unnatural modifications are useful for structure and mechanistic studies of RNA. This Review highlights chemical, enzymatic, and combined (semisynthesis) approaches to generate sitespecifically modified RNAs. The availability of these methods for site-specific modifications of RNAs of all sizes is important in order to study the relationships between RNA chemical composition, structure, and function.RNAs undergo specific post-transcriptional modification by a wide variety of enzymes and ribonucleoprotein complexes (1,2). More than 100 different modifications of the four standard RNA nucleosides, adenosine, cytidine, guanosine, and uridine, have been identified (3). Examples from each of the four major categories of base isomerization, base modification, sugar modification, and hypermodification are shown in Figure 1, panel a. Similarly, a large number of unnatural modifications have been synthesized and used for structure and mechanistic studies of RNA (4-6). A few examples are shown in Figure 1, panel b (7-10). Natural modified nucleotides are often found in the functionally important regions of RNA, such as the peptidyl transferase center of ribosomal RNA (rRNA), the anticodon loop of transfer RNAs, or the branch site of spliceosomal . Several modifications, such as pseudouridine (14), have been known for almost 50 years. Their locations in natural RNAs can, in some cases, be highly conserved; yet, our understanding of their biological roles is still incomplete (15). Through a combination of biological, chemical, and biophysical approaches, much can be learned regarding the roles of modified nucleotides. Similarly, the incorporation of unnatural modifications may be useful in order to study the biological roles and functions of RNA (5).In a number of cases, the enzymes or small nucleolar RNAs (snoRNAs) that are responsible for site-specific RNA modification have been identified (2). Knock-out studies of the individual modifying enzymes or snoRNAs then allow the function of the modification to be deduced. In cases where the enzymes are not known, or the modification is not a natural one, alternative approaches must be considered. Over the past several decades, several chemical methods have been developed to study the effects of modifications in small RNA model systems. These studies have led to newer, more powerful approaches that allow site-specific modification of large RNAs, reconstitution into biological systems, and studies of RNA structure and function. Chemical SynthesisChemical synthesis of RNA allows for site-selective incorporation of modified nucleotides. Five decades ago, Michelson and coworkers synthesized a thymidylate dinucleotide by the *Corresponding author csc@chem.wayne.edu. Figure 2): i) coupling at the 5′ site with a protected phosphoramidite, ii) capping of the unreacted 5′-hydroxyl groups, ...

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“…Hirao and colleagues at RIKEN and TagCyx previously reported that the hydrophobic unnatural nucleotide 7-(2-thienyl) imidazo [4,5-b]pyridine (Ds) and its base pair partner, a diol-modified 2-nitro-4-propynylpyrrole, could be incorporated into DNA and were compatible with both SELEX and PCR amplification. [2][3][4] …”

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confidence: 99%

“…[2][3][4] Now, Hirao's group has assessed the functional consequences of using an expanded genetic alphabet consisting of four natural nucleotides plus Ds to generate libraries of DNA aptamers. In vitro selection experiments against human VEGF-165 and interferon-g (IFNG; IFN-g) identified DNA aptamers from these libraries that bound to the proteins with subpicomolar and subnanomolar K d values.…”

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confidence: 99%

Expanding the aptamer alphabet

Lou¹

2013

Science-Business eXchange

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Researchers at the RIKEN Center for Life Science Technologies and TagCyx Biotechnologies have used an expanded genetic alphabet containing unnatural nucleotides to generate DNA aptamers that have 100-fold higher binding affinity for a target protein than counterparts created using only natural nucleotides.1 The group plans to develop the aptamers for diagnostic applications before exploring therapeutic applications.Nucleic acid aptamers consist of single strands of DNA or RNA and are usually generated via in vitro approaches such as SELEX (systematic evolution of ligands by exponential enrichment), in which aptamers from a library undergo iterative rounds of selection to enrich for aptamers with the highest affinity for a particular target.However, generating molecules with the desired level of functionality usually is less efficient for nucleic acid aptamers than for peptides and proteins. The reason is that a four-nucleotide genetic alphabet limits chemical and structural diversity, according to Ichiro Hirao, leader of the synthetic molecular biology team at the RIKEN Center for Life Science Technologies and founder, president and CEO of TagCyx.Conventional peptides and proteins are generated using a 20-aminoacid alphabet.For more than a decade, Hirao and colleagues in Japan have been developing base pairs of unnatural nucleotides that could be used to expand the standard genetic alphabet and yield oligonucleotides with new properties and functionality. These nucleotide pairs are designed to be compatible with DNA/RNA replication and transcription but only form base pairs with one another and not with any of the four natural nucleotides. Hirao and colleagues at RIKEN and TagCyx previously reported that the hydrophobic unnatural nucleotide 7-(2-thienyl) imidazo [4,5-b]pyridine (Ds) and its base pair partner, a diol-modified 2-nitro-4-propynylpyrrole, could be incorporated into DNA and were compatible with both SELEX and PCR amplification. [2][3][4]

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An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA

Cited by 229 publications

References 29 publications

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

Combined Approaches to Site-Specific Modification of RNA

Expanding the aptamer alphabet

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