Detektion von Nukleinsäuren durch postsynthetisch modifizierte Fluoreszenzsonden auf Basis photostabiler Cyaninfarbstoffe

Die Arbeit beschreibt die Synthese von 25 neuen Cyaninfarbstoffen, wobei eine große Photostabilität angestrebt wurde. Durch chemische Variation wurde die Halbwertszeit stark erhöht und zugleich eine gesteigerte Fluoreszenz erhalten. Ausgewählte Fluorophore wurden anschließend postsynthetisch an DNA gebunden und zu Energietransferpaaren kombiniert. Geeignete Paare dienten zudem in Nukleinsäuresonden der Detektion von miRNA, Verfolgung von Strangaustauschprozessen und Duplexinvasion in DNA

Development of a Wavelength-Shifting Fluorescent Module for the Adenosine Aptamer Using Photostable Cyanine Dyes

DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide–alkyne cycloaddition by two structurally different approaches: 1) as nucleotide modifications at the 2′-position of uridines and 2) as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications

Exploring the photochemical reactivity of multifunctional photocaged dienes in continuous flow

Flow reactors become more and more automated by enabling on-line reaction monitoring and adjusting the process parameters. On-line monitoring of chemical processes is a valuable tool to steer processes, leading to precise engineering of macromolecular materials. Detailed information about specific product patterns, end-group functionality or material composition can be obtained by coupling a flow reactor to a mass spectrometer (e. g. ESI-MS). In this work, we study the deprotection of maleimides and its subsequent photoenol functionalization to synthesize complex macromolecules using a photo flow reactor coupled to an ESI-MS. Using a trapping agent (TA), furan is efficiently removed from the maleimide Diels−Alder adduct within just minutes at 175 °C and quantitatively converted into an unreactive species that does not interfere with further reactions of the maleimide. The photoenol reaction was likewise shown to be highly effective to proceed in microreactors, reaching quantitative conversion of trifunctional molecules in as little as 2 min.</p

Resolving Chemical Modifications to a Single Amino Acid within a Peptide Using a Biological Nanopore

While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427-1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores.BN/Chirlmin Joo LabBN/Christophe Danelon LabChemE/Advanced Soft MatterBN/Cees Dekker La