Rapid fluorescence imaging of miRNAs in human cells using templated Staudinger reaction

Górska, Katarzyna; Keklikoglou, Ioanna; Tschulena, Ulrich; Winssinger, Nicolas

doi:10.1039/c1sc00216c

Cited by 75 publications

(82 citation statements)

References 37 publications

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“…The use of the (bis-azido)-rhodamine GPNA probe 90 increased the signal-to-noise ratio and the sensitivity (Scheme 11e). 78 Fluorescence measurements showed 20% conversion into product oligonucleotide 92 at 2 nM DNA template (0.02 equiv. ), with virtually no signal in the absence of the target.…”

Section: Nucleic Acid-templated Nitrogen Release Reactions Employing mentioning

confidence: 99%

Amplification by nucleic acid-templated reactions

2014

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Nucleic acid-templated reactions enable the design of conditional reaction systems, in which bond formation occurs only when a particular DNA or RNA molecule is present. Such reaction systems are currently being explored for applications in DNA/RNA diagnosis, drug screening and as a means to design gene expression specific therapy. However, biological nucleic acid templates usually have low abundance. Therefore, either the targeted nucleic acid template has to be multiplied by means of an amplification step or the template itself has to act as a catalyst which amplifies product formation. This critical review highlights the recent advancements in nucleic acid-templated reactions that proceed with turnover in template and thereby provide a means of amplification. Improvements in reaction engineering and the development of new chemistries have pushed the limits from 10(1) to 10(2)-10(3) turnovers. This includes reaction systems that lead to the ligation of oligonucleotides or to the interconversion of appended functional groups beyond ligation as well as templated chemistries that enable the activation of catalysts for subsequent triggering of reactions between non-nucleotidic substrates. The present limitations and future opportunities are discussed.

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Section: Nucleic Acid-templated Nitrogen Release Reactions Employing mentioning

confidence: 99%

Amplification by nucleic acid-templated reactions

2014

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“…Though the data clearly indicate that product cyclization speeds up ligation reactions when substoichiometric template is used, the observed efficiency still is inferior to the efficiencies reported for templated reactions without ligation, such as acyl transfer, [25,26,[58][59][60]62] Wittig transfer, [61] transition metal catalysis, [64,65] or nitrogen release reactions. [24,44,45,47,49,50,52,56,57] We performed melting experiments to assess the template affinities of the molecules involved in the templated ligation-cyclization reaction (Figure 4). The melting temperature (T M ) of the PNA-DNA duplex comprised of cyclic PNA 9 and template T was 11 8C lower (T M = 46 8C) than that of the corresponding linear (cyclization-deficient) duplex 22·T (T M = 57 8C).…”

Section: Cycligation At Substoichiometric Template Loadsmentioning

confidence: 99%

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Roloff¹,

Seitz²

2013

ChemBioChem

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Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA-triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid-programmed ligation reactions. A DNA-triggered ligation-cyclization sequence ("cycligation") of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA-thioesters and isocysteinyl-PNA-cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product-template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur-halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2-3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25-0.01 equiv). We believe that cyclization of products from DNA-templated reactions could ultimately afford systems that completely overcome product inhibition.

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“…More recent studies are based on the application of templated chemistry where a reaction is triggered between two oligonucleotide probes hybridizing on adjacent sites on the nucleic acid target 5 . Attractive are templated chemical reactions that de-quench a fluorophore via removal of a covalently attached quencher, 6 - 9 chemically convert a profluorophore into a fluorophore, 10 - 22 or de novo generate a fluorescent dye 23 , 24 . An intriguing asset of templated reactions is their potential to occur in a catalytic manner, leading to an amplification of the fluorescence signal and, consequently, increased sensitivity 25 .…”

Section: Introductionmentioning

confidence: 99%

Nucleic acid sensing by an orthogonal reporter system based on homo-DNA

Stoop

Désiron²,

Leumann

2013

Artificial DNA: PNA & XNA

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We have developed an assay for single strand DNA or RNA detection which is based on the homo-DNA templated Staudinger reduction of the profluorophore rhodamine-azide. The assay is based on a three component system, consisting of a homo-DNA/DNA hybrid probe, a set of homo-DNA reporter strands and the target DNA or RNA. We present two different formats of the assay (Omega probe and linear probe) in which the linear probe was found to perform best with catalytic turnover of the reporter strands (TON: 8) and a match/mismatch discrimination of up to 19. The advantage of this system is that the reporting (homo-DNA) and sensing (DNA) domain are decoupled from each other since the two pairing systems are bioorthogonal. This allows independent optimization of either domain which may lead to higher selectivity in in vivo imaging.

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Rapid fluorescence imaging of miRNAs in human cells using templated Staudinger reaction

Cited by 75 publications

References 37 publications

Amplification by nucleic acid-templated reactions

Amplification by nucleic acid-templated reactions

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Nucleic acid sensing by an orthogonal reporter system based on homo-DNA

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