Species-Specific Identification of Mycobacterial 16S rRNA PCR Amplicons Using Smart Probes

Stöhr, Katharina; Häfner, Bernhard; Nolte, Oliver; Wolfrum, J.; Sauer, Markus; Herten, Dirk‐Peter

doi:10.1021/ac051447z

Cited by 58 publications

(38 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[5] In previous experiments ATTO 520-in comparison to other dyes-is efficiently quenched by guanosine, [6] due to its pronounced electron donating ability among the nucleobases. [7] Therefore, we chose dsDNA not only as rigid spacer for donor-acceptor attachment at desired distances but also as carrier of electron donating guanosine residues ( Figure 1).…”

mentioning

confidence: 99%

“…The whole set of probes is schematically drawn in Figure 1 including two more controls that were not labeled with acceptor for zero FRET efficiency (4,8). Quenching of ATTO 520 by guanosine (1-3) might either lead to an overall decrease in fluorescence intensity of donor and acceptor likewise, yielding similar FRET efficiencies as for FRET-only probes (5)(6)(7). Alternatively, ET might be competitive with FRET thus decreasing FRET efficiencies of the FRET/ET-probes (1)(2)(3) in comparison to the FRETonly probes (5)(6)(7).…”

mentioning

confidence: 99%

“…Quenching of ATTO 520 by guanosine (1-3) might either lead to an overall decrease in fluorescence intensity of donor and acceptor likewise, yielding similar FRET efficiencies as for FRET-only probes (5)(6)(7). Alternatively, ET might be competitive with FRET thus decreasing FRET efficiencies of the FRET/ET-probes (1)(2)(3) in comparison to the FRETonly probes (5)(6)(7). For all constructs the fluorescence emission spectra were recorded in bulk experiments by selectively exciting the FRET donor ( Figure 2).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Single‐Molecule Fluorescence Studies Reveal Long‐Range Electron‐Transfer Dynamics Through Double‐Stranded DNA

et al. 2009

Self Cite

View full text Add to dashboard Cite

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Single‐Molecule Fluorescence Studies Reveal Long‐Range Electron‐Transfer Dynamics Through Double‐Stranded DNA

et al. 2009

Self Cite

View full text Add to dashboard Cite

show abstract

“…[37][38][39] By combining mathematical models and in vitro experiments it has been possible to design probes with these characteristic features. The effect of loop length on the behaviour of the smart probe is typically less dramatic compared with that of the stem length and can be used to fine-tune functionality.…”

Section: Monitoring Specific Genesmentioning

confidence: 99%

“…The effect of loop length on the behaviour of the smart probe is typically less dramatic compared with that of the stem length and can be used to fine-tune functionality. [39] However, trial-and-error is still part of the optimization.…”

Section: Monitoring Specific Genesmentioning

confidence: 99%

Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

et al. 2010

Self Cite

View full text Add to dashboard Cite

The recent explosion in papers utilising single-molecule experiments pushes the envelope further for increased spatial and temporal resolution. In order to achieve this, a combination of novel fluorescent probes and spectroscopy techniques are required. Herein, we provide an overview on our contribution to developments in the field of fluorescent probes along with a palette of alternative delivery methods for introducing the probes into living cells. We discuss probe requirements arising from the use of single-molecule spectroscopy methods and the customisation of probes that depends on the target molecule, the chemical state of the molecule as well as the distance and the type of interaction between sensor and ligand. We explain how Förster resonance energy transfer (FRET) and photon-induced electron transfer (PET) can increase the probe customisation. We also discuss additional requirements that arise when performing experiments in living cells like toxicity and cell permeability. Regarding the latter, we devote a special paragraph on the different ways to introduce the desired probe into the cell and how the different properties of each probe and cell type may require different delivery methods. We offer insights based on our experience working with a variety of single-molecule methods, fluorescent probes and delivery systems. Overall, we encompass the latest developments on probe design and delivery and illustrate that the wealth of information provided by single-molecule studies goes along with increased complexity.

show abstract

Fluorophore Labeling for Single‐Molecule Fluorescence Spectroscopy (SMFS)

Sauer

Hofkens

Enderlein

2011

Handbook of Fluorescence Spectroscopy and Imaging

View full text Add to dashboard Cite

j61Nearly all proteins exhibit amine groups in terms of lysine residues and at the Nterminus. Aliphatic amines such as the e-amino group of lysine are moderately basic and reactive towards most acylating reagents. However, the concentration of the free base form of aliphatic amines below pH 8.0 is very low. Therefore, pH values of 8.5-9.5 are commonly applied in the modification of lysine residues [2]. In contrast, the a-amino group at the N-terminus of a protein can sometimes be selectively modified by reaction at a near neutral pH due to its lower pK a value of $7. Furthermore, it has to be considered that acylation reagents tend to degrade in the presence of water with increasing pH value. Therefore, a compromise between the reactivity of the amine group and the degradation of the acylation reagent in aqueous buffers has to be found for each coupling reaction. In other words, reaction time and pH value have to be carefully optimized. Aromatic amines are very weak bases and thus they are unprotonated at pH 4.0-7.0. In aqueous solution, acylating reagents are virtually unreactive with the amino group of peptide bonds and with the side-chain amides of glutamine and asparagine residues, the guanidinium group of arginine, the imidazole group of histidine and the amines found in natural nucleotides.Today N-hydroxysuccinimide (NHS) esters are most commonly used for coupling to amino groups. An NHS ester may be formed by the reaction of carboxylate with NHS in the presence of carbodiimide. To prepare stable NHS ester derivatives, the activation has to be performed in nonaqueous solvents. As exemplified in Figure 3.1, by the reaction of fluorescein-NHS with tryptophan, then the NHS or sulfo-NHS esters react with primary and secondary amines, creating stable amide and imide links, respectively. Thus, in protein molecules, NHS esters can be used to couple principally with the a-amines at the N-terminals and the e-amines of lysine side chains, depending on the pH value, that is, on the degree of deprotonation of the amines. The reaction of NHS esters with thiol or hydroxyl groups does not yield stable conjugates. NHS esters can also be prepared in situ to react immediately with amines of the target molecules in aqueous solvents. Using the water-soluble carbodiimide EDC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide] (carbodiimides are zero-length cross-linking agents used to mediate the formation of an amide or phosphoramidate linkage between a carboxylate and an amine or a phosphate and an amine, respectively), a carboxylate-containing fluorophore can be transformed into an active ester by reaction in the presence of NHS or sulfo-NHS (N-hydroxysulfosuccinimide). Sulfo-NHS esters are more water soluble than classical NHS esters, and couple rapidly with amines on target molecules with the same specificity and reactivity as NHS esters [3]. Furthermore, sulfo-NHS esters hydrolyze more slowly in water. Usually, NHS esters have a half-life of the order of hours under physiological pH conditions, but both hydrolysis and amine ...

show abstract

Species-Specific Identification of Mycobacterial 16S rRNA PCR Amplicons Using Smart Probes

Cited by 58 publications

References 26 publications

Single‐Molecule Fluorescence Studies Reveal Long‐Range Electron‐Transfer Dynamics Through Double‐Stranded DNA

Single‐Molecule Fluorescence Studies Reveal Long‐Range Electron‐Transfer Dynamics Through Double‐Stranded DNA

Fluorescent Probes and Delivery Methods for Single‐Molecule Experiments

Fluorophore Labeling for Single‐Molecule Fluorescence Spectroscopy (SMFS)

Contact Info

Product

Resources

About