Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Wawel około roku 1250

Wiek XIII w dziejach rezydencji książęcej na Wawelu obfituje w wydarzenia natury politycznej, wiążące się z walkami o tron wielkoksiążęcy w Krakowie, kolejnymi najazdami tatarskimi i wreszcie opanowaniem Małopolski przez Wacława II Czeskiego w roku 12911. Jednakże w sferze materialnej okres ten był do niedawna niemal nieuchwytny dla archeologów i badaczy architektury, oprócz niezbyt licznych znalezisk przedmiotów ruchomych, które - z większym lub mniejszym prawdopodobieństwem - można byłoby odnosić do XIII stulecia.Thirteenth century in the history of the prince's residence at Wawel rich in events of a political nature, involving the struggles of the ducal throne in Krakow, successive invasions of Tatar and finally mastery of Malopolska by Wenceslaus II of Bohemia in the year 12911