Targeting of mixed sequence double-stranded DNA using pyrene-functionalized 2′-amino-α-l-LNA

Hrdlicka, Patrick J.; Kumar, T. Santhosh; Wengel, Jesper

doi:10.1039/b506986f

Cited by 50 publications

(93 citation statements)

References 26 publications

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“…Moreover, attachment of perylene to 2′-amino-α-L-LNA results in significantly higher binding affinity than previously introduced diastereoisomeric 2′-amino-LNA 35 . On the other hand, similar stabilization effect to modification T* was previously reported for pyrene 2′-amino-α-L-LNA analogue which was proposed to intercalate into the double helix 36 . This was also confirmed by red-shifted pyrene absorbance upon hybridization with complementary DNA.…”

Section: Synthesis Of Modified Phosphoramidite Reagentsupporting

confidence: 55%

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Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Astakhova

Kumar

Wengel

2011

Collect. Czech. Chem. Commun.

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Dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition of his outstanding contributions to the area of nucleic acid chemistry.Herein, a novel fluorescent nucleotide analogue, perylene-2′-amino-α-L-LNA, has been prepared and studied within synthetic oligonucleotides of different sequences. The phosphoramidite reagent was synthesized in 85% overall yield starting from 2′-amino-α-L-LNA nucleoside. Incorporation efficiency of the resulting perylene-2′-amino-α-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards complementary DNA and RNA targets, batochromically shifted excitation/emission wavelengths with respect to the often applied polyaromatic hydrocarbon pyrene, high fluorescent quantum yields and very low target detection limits (5-10 nM). Fluorescence of single stranded LNA/DNA mixmer oligonucleotide having two incorporations of monomers T* was quenched (quantum yield Φ F = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Φ F = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues sequences containing monomers T* at 5′-and 3′-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose that a covalent link between two T* monomers in the double-labeled probe provides a remarkable degree of rigidity in the double helix which enforces positioning of the bulky perylene moieties in the nonpolar groove resulting in reduced fluorescence quenching.

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Section: Synthesis Of Modified Phosphoramidite Reagentsupporting

confidence: 55%

“…Reported T m values and absorbance maxima are averages of at least two measurements. T* = 2′-N-(perylen-3-yl)methyl-2′-amino-α-L-LNA monomer; SSP = single stranded probe since this type of orientation has been also previously proposed for Nfunctionalized 2′-amino-α-L-LNA 36 .…”

Section: Synthesis Of Modified Phosphoramidite Reagentmentioning

confidence: 96%

Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Astakhova

Kumar

Wengel

2011

Collect. Czech. Chem. Commun.

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“…We originally used 2′- N -(pyren-1-yl)methyl-2′-amino-α-L-LNA (Locked Nucleic Acid) nucleotides as the key activating components of Invader probes, 15 but recently discovered that they can be replaced by 2′- O -(pyren-1-yl)methyl-ribonucleotides (Figure 1). 16 The resulting probes display similar dsDNA-recognition efficiency and are much easier to synthesize.…”

Section: Introductionmentioning

confidence: 99%

Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

et al. 2014

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Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-alkylated uridine monomers X–Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔTm/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.

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“…Among these, double stranded 2 -N-(pyren-1-yl)methyl-2 -amino-α-L-LNA have been shown to target double-stranded DNA. [6] Herein, we present the synthesis and biophysical studies of N2 -functionalized 2 -amino-α-L-LNA.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Biophysical Studies of N2′-Functionalized 2′-Amino-α-L-LNA

Kumar

Madsen

Wengel

et al. 2007

Nucleosides, Nucleotides and Nucleic Acids

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Targeting of mixed sequence double-stranded DNA using pyrene-functionalized 2′-amino-α-l-LNA

Cited by 50 publications

References 26 publications

Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

Synthesis and Biophysical Studies of N2′-Functionalized 2′-Amino-α-L-LNA

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