Figure 1.
The principle of non-conjugated small molecular FRET.
Left: Mixing non-conjugated FRET partner molecules with the Aβ aggregates/fibrils, which consist of numbers of β-sheet ladders, has a high probability of incorporating the partner into the Aβ species within 10 nm proximity to generate a viable FRET signal. Right: Chemical structures of curcumin, CRANAD-2 and CRANAD-5.
Figure 2.
Validation of FRET phenomenon in solution with Aβ40 aggregates.
(A) Spectra overlay showing FRET phenomenon in a solution of CRANAD-5 and CRANAD-2 with Aβ40 aggregates (a) (red); spectrum of control solution of CRANAD-5 with Aβ40 aggregates (b) (blue); spectrum of control solution of CRANAD-2 with Aβ40 aggregates (c) (black, overlapped with the green line); spectrum of control solution of CRANAD-5 and CRANAD-2 without Aβ40 aggregates (d) (green, overlapped with the black line). (B) Excitation spectra overlay of solution (a) (red), (b) (blue) and (c) (black) with the emission at 700 nm. Note two excitation peaks for solution (a). (C) Spectra overlay of solutions of curcumin and CRANAD-2 with Aβ40 aggregates: FRET (red), spectrum of control solution of curcumin with Aβ40 aggregates (blue), spectrum of control solution of CRANAD-2 with Aβ40 aggregates (black), and spectrum of control solution of curcumin and CRANAD-2 without Aβ40 aggregates (green).
Figure 3.
Differentiation of Aβ monomers from higher molecular weight Aβ species, which include aggregates/fibrils, and dimers.
(A) Spectra overlay of Aβ42 dimers (red) and Aβ42 monomers (black). (B) Spectra overlay of Aβ40 aggregates (red) and Aβ40 monomers (black).
Figure 4.
Testing with commercial available Aβ40 peptide.
(A) Standard curve of FRET with Aβ40 aggregates. (B) Fluorescence spectra of Aβ40 sample from rPeptide with CRANAD-5/-2 FRET pair.
Figure 5.
Flow chart and principle for differentiating various Aβ species based on non-conjugated FRET technique.
(A-1) Differentiating Aβ monomers from high molecular weight species; (A-2, A-3) differentiating large size species (A-2) from small oligomers (A-3).