Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes

Marras, Salvatore A. E.; Kramer, Fred Russell; Tyagi, Sanjay

doi:10.1093/nar/gnf121

Cited by 587 publications

(433 citation statements)

References 26 publications

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“…Moreover, the fitted value of K close , 59 ± 30, corresponds to a free energy of −10.6 (±1.4) kJ/mol for the formation of the two-mismatch-containing stem. This, in turn, agrees to within experimental uncertainty with the −12.2 (±1.6) kJ/mol predicted by adding the −4.6 kJ/mol stability of the stem as predicted by the "DINAMelt Mfold" secondary structure prediction algorithm (32,33) to the −7.6 (±1.6) kJ/mol prior literature estimates of the stabilization produced by the fluorophore-quencher pair we have used (34,35). Encouraged by these successful test case design efforts, we next adapted our simple strategy to engineering cooperativity into two structurally more complex receptors.…”

Section: Resultssupporting

confidence: 83%

“…Specifically, Mfold (32,33) predicts that the parent aptamer forms a stem loop structure with folding free energy that is unstable by 0.75 kJ/mol (per monomeric aptamer) in the absence of doxorubicin. When added to the favorable association energy of the fluorophore-quencher pair (34,35), this yields a closing free energy of −6.1 kJ/mol and a K close of 11.2 for the tandem repeat. Inserting the latter value into Eq.…”

Section: Resultsmentioning

confidence: 99%

“…Specifically, the folding free energy of the fluorophore-and-quenchermodified parent aptamer is −7.5 kJ/mol. Given the known stabilizing effects of the fluorophore-quencher pair (34,35), we thus estimate that the folding free energy of the dye-free parent aptamer is +0.1 kJ/mol. The folding free energy of a dimer of aptamers, one of which is dye-labeled, should thus be −7.4 kJ/mol, which in turn corresponds to a K close of 19.…”

Section: Resultsmentioning

confidence: 99%

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Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Simon

Vallée‐Bélisle

Ricci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hilltype" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsicdisorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. ultrasensitivity | intrinsically disordered proteins | biosensors | synthetic biology | ribozymes

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Simon

Vallée‐Bélisle

Ricci

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…2A). Spacing at a distance of seven bases and short linkers avoided contact quenching (24). A BLAST search was performed with the sequence of the 16-mer to minimize specific interactions with genomic DNA or messenger RNA.…”

Section: Resultsmentioning

confidence: 99%

Hybridization kinetics is different inside cells

Schoen

Krammer

Braun

2009

Proc. Natl. Acad. Sci. U.S.A.

114

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“…494 nm [37] Supposedly similar as free chromophore max ϭ wavelength of maximum absorption; em ϭ wavelength of maximum fluorescence emission; ex ϭ excitation wavelength; ⌽ ϭ fluorescence quantum yield;…”

Section: Minor Groovementioning

confidence: 99%

Electron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores

Gabelica

Rosu

Pauw

et al. 2007

J. Am. Soc. Mass Spectrom.

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Double stranded DNA multiply charged anions coupled to chromophores were subjected to UV-Vis photoactivation in a quadrupole ion trap mass spectrometer. The chromophores included noncovalently bound minor groove binders (activated in the near UV), noncovalently bound intercalators (activated with visible light), and covalently linked fluorophores and quenchers (activated at their maximum absorption wavelength). We found that the activation of only chromophores having long fluorescence lifetimes did result in efficient electron photodetachment from the DNA complexes. In the case of ethidium-dsDNA complex excited at 500 nm, photodetachment is a multiphoton process. The MS 3 fragmentation of radicals produced by photodetachment at ϭ 260 nm (DNA excitation) and by photodetachment at Ͼ 300 nm (chromophore excitation) were compared. The radicals keep no memory of the way they were produced. A weakly bound noncovalent ligand (m-amsacrine) allowed probing experimentally that a fraction of the electronic internal energy was converted into vibrational internal energy. This fragmentation channel was used to demonstrate that excitation of the quencher DABSYL resulted in internal conversion, unlike the fluorophore 6-FAM. Altogether, photodetachment of the DNA complexes upon chromophore excitation can be interpreted by the following mechanism: (1) ligands with sufficiently long excited-state lifetime undergo resonant two-photon excitation to reach the level of the DNA excited states, then (2) the excited-state must be coupled to the DNA excited states for photodetachment to occur. Our experiments also pave the way towards photodissociation probes of biomolecule conformation in the gas-phase by Since then, some major advances must be mentioned in electron activation methods [3][4][5] and in infrared photodissociation [6].We recently started exploring the gas-phase reaction pathways of multiply charged DNA single strands and double strands upon UV irradiation around 260 nm [7,8]. To our surprise, we found out that, instead of fragmentation, electron detachment was the major reaction pathway with strands containing guanines. Electron photodetachment itself is not useful for DNA structure analysis, but subsequent collisional activation of the oligonucleotide radicals produced by electron photodetachment gives fragmentation into w, d, a· and z· ions with good sequence coverage [7]. This technique combining electron photodetachment and collision-induced dissociation was coined electron photodetachment dissociation (EPD). It has now been shown to apply to peptides and proteins as well [9].Apart from the sequencing applications of EPD, numerous questions remain about the electron photodetachment mechanism, and how it compares with electron detachment dissociation [3][4][5] and thermal electron detachment [10,11]. We will briefly summarize our current understanding of the photodetachment mechanism [8]. The electron binding energy in multiply charged DNA anions depends on the balance between electron binding energy of the different DNA...

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Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes

Cited by 587 publications

References 26 publications

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Hybridization kinetics is different inside cells

Electron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores

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