Fluorescent Oligonucleotides - Versatile Tools as Probes and Primers for DNA and RNA Analysis

Wojczewski, C.; Stolze, Karen; Engels, Joachim W.

doi:10.1055/s-1999-2914

Cited by 89 publications

(57 citation statements)

References 80 publications

(102 reference statements)

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“…F 0 and F ∞ are the fluorescence intensity in the absence of aminoglycoside or at saturation, respectively. n is the Hill coefficient or degree of cooperativity associated with the binding (1) …”

Section: Synthesismentioning

confidence: 99%

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

2007

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Fluorescent nucleobase analogs that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analog. Responsive isosteric fluorescent pyrimidine analogs are, however, rare. Appending 5-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogs with emission in the visible range. To explore the potential utility of this chromophore for studying RNA-ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial Asite, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community.

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Section: Synthesismentioning

confidence: 99%

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

2007

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“…The standard phosphoramidite DNA building block strategy enables a large variety of organic fluorophores to be routinely incorporated at specific positions within the oligonucleotide. Alternatively, postsynthetic labeling with fluorophores can be performed via amide bond formation to internal, 3′-and 5′-terminal modifiers that are commercially or synthetically available as phosphoramidites (13). In recent years, the "click" ligation strategy has become an important alternative.…”

Section: Introductionmentioning

confidence: 99%

Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

et al. 2009

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The azides 1 and 2 bearing a phenoxazinium and a coumarin fluorophore, respectively, were applied in postsynthetic "click"-type bioconjugation and coupled to oligonucleotides modified with alkyne groups using two alternative approaches: (i) as a nucleotide modification at the 2′-position of uridine and (ii) as a nucleotide substitution using (S)-(-)-3-amino-1,2-propanediol as an acyclic linker between the phosphodiester bridges. The corresponding alkynylated phosporamidites 3 and 6 were used as DNA building blocks for the preparation of alkyne-bearing DNA duplexes. The base pairs adjacent to the site of modification and the base opposite to it were varied in the DNA sequences. The modified duplexes were investigated by UV/vis absorption spectroscopy (including melting temperatures) and fluorescence spectroscopy in order to study the different optical properties of the two chromophores and to evaluate their potential for bioanalytical applications. The sequence-selective fluorescence quenching of phenoxazinium 1 differs only slightly and does not depend on the type of modification, meaning whether it has been attached to the 2′-position of uridine or as DNA base surrogate using the acyclic glycol linker. The 2′-chromophore-modified uridine still recognizes adenine as the counterbase, and the duplexes exhibit a sufficient thermal stability that is comparable to that of unmodified duplexes. Thus, the application of the 2′-modification site of uridine is preferred in comparison to glycol-assisted DNA base surrogates. Accordingly, the coumarin dye azide 2 was attached only to the 2′-position of uridine. The significant Stokes shift of ∼100 nm and the good quantum yields make the coumarin chromophore a powerful fluorescent label for nucleic acids.

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“…In particular, fluorescent nucleoside analogues that are isosteric to natural nucleobases and are sensitive to changes in their microenvironment have found wide applications in biophysics. [17][18][19][20][21][22][23] Indeed, labeling TAR with 2-aminopurine, an emissive adenosine isoster, has recently been utilized to follow up on ligand binding using steady-state emission spectroscopy. 24,25 Similarly, a TAR construct containing benzo[g]quinazoline-2,4(1H,3H)-dione, a large U analogue, displayed changes in fluorescence upon Tat binding when incorporated in the U-rich bulge.…”

Section: Introductionmentioning

confidence: 99%

Using an emissive uridine analogue for assembling fluorescent HIV-1 TAR constructs

Srivatsan¹,

Tor²

2007

Tetrahedron

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Emissive nucleoside analogues that are sensitive to their microenvironment can serve as probes for exploring RNA folding and recognition. We have previously described the synthesis of an environmentally sensitive furan-containing uridine and its triphosphate, and have demonstrated that T7 RNA polymerase recognizes this modified ribonucleoside triphosphate as a substrate in in vitro transcription reactions. Here we report the enzymatic preparation of fluorescently tagged HIV-1 TAR constructs and study their interactions with a Tat peptide. Two extreme labeling protocols are examined, where either all native uridine residues are replaced with the corresponding modified fluorescent analogue, or only key residues are site-specifically modified. For the HIV-1 Tat-TAR system, labeling all native uridine residues resulted in relatively small changes in emission upon increasing concentrations of the Tat peptide. In contrast, when the two bulge U residues were sitespecifically labeled, a reasonable fluorescence response was observed upon Tat titration. The scope and limitations of such fluorescently tagged RNA systems are discussed.

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Fluorescent Oligonucleotides - Versatile Tools as Probes and Primers for DNA and RNA Analysis

Cited by 89 publications

References 80 publications

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

Using an emissive uridine analogue for assembling fluorescent HIV-1 TAR constructs

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