The Structural Basis for Optimal Performance of Oligothiophene‐Based Fluorescent Amyloid Ligands: Conformational Flexibility is Essential for Spectral Assignment of a Diversity of Protein Aggregates

Abstract

Protein misfolding diseases are characterized by deposition of protein aggregates, and optical ligands for molecular characterization of these disease-associated structures are important for understanding their potential role in the pathogenesis of the disease. Luminescent conjugated oligothiophenes (LCOs) have proven useful for optical identification of a broader subset of disease-associated protein aggregates than conventional ligands, such as thioflavin T and Congo red. Herein, the molecular requirements for achieving LCOs able to detect nonthioflavinophilic Aβ aggregates or non-congophilic prion aggregates, as well as spectrally discriminate Aβ and tau aggregates, were investigated. An anionic pentameric LCO was subjected to chemical engineering by: 1) replacing thiophene units with selenophene or phenylene moieties, or 2) alternating the anionic substituents along the thiophene backbone. In addition, two asymmetric tetrameric ligands were generated. Overall, the results from this study identified conformational freedom and extended conjugation of the conjugated backbone as crucial determinants for obtaining superior thiophene-based optical ligands for sensitive detection and spectral assignment of disease-associated protein aggregates.

Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands

“…1D) were included in the study, since the length of the thiophene backbone as well as the position of the anionic groups along the backbone, have been identified as important structural determinants for thiophene-based ligands efficient for spectral separation of protein aggregates. 20,22,30 All 4 dyes successfully stained PrP aggregates associated with each of the distinct strains and the deposits were easily identifiable due to the bright fluorescence from the thiophene-based ligands (Fig. 1).…”

“…In contrast to p-FTAA and h-FTAA, the emission spectra from p-KTAA bound to PrP deposits lacked the characteristic double-peaks previously observed from a variety of LCOs bound to protein deposits. 20,22,30 Overall, all the dyes exhibited altered emission properties associated with pronounced planarization of the backbone and/or stacking between adjacent thiophene chains when bound to mSS deposits.…”

“…In addition, LCOs bound to amyloid deposits exhibit unique excitation spectra that reflect the conformational constraints of the dyes upon interaction with the deposits and excitation profiles can be utilized to assess conformational alterations of the dyes. 20,22,30 Herein, we utilized the more sensitive methods of lifetime measurements and excitation spectroscopy, as well as emission spectroscopy from LCPs and LCOs bound to PrP deposits in brain tissue sections from mice inoculated with 2 distinct prion strains. The obtained results clearly demonstrate how multimodal fluorescence microscopy can be utilized for enhanced optical separation of PrP deposits associated with distinct prion strains.…”

Poly-and oligothiophenes : Optical probes for multimodal fluorescent assessment of biological processes

“…Substitution of a thiophene moiety with a selenophene or a phenylene units [5] alters the conformational freedom and thereby affect the conjugation length. Thus, these chemical modifications of the conjugated backbone can efficiently be utilized to tune the fluorescent properties of the probes.…”

“…Chemical modifications embracing substitution in the thiophene backbone has also been evaluated for probable enhancement in spectral discrimination of Aβ plaques and neurofibrillary tangles [5]. By replacing one thiophene unit with a selenophene or a phenyl ring the spectral separation was reduced.…”