To increase the apparent Stokes' shift of perylene, pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to achieve the efficient fluorescence resonance energy transfer (FRET) from pyrene to perylene. Multiple donors were introduced in the vicinity of acceptors through D-threoninol and natural base pairs were inserted between the dyes. Accordingly, donors and acceptors could be accumulated inside the DNA without forming an undesired excimer/exciplex. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 460 nm was observed from perylene when excited at 345 nm at which pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high apparent FRET efficiency (Phi>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R(0)) of the donor pyrene. Next, this FRET system was used to enlarge the Stokes' shift of the DNA probe, which can discriminate a one-base deletion mutant from wild type with a model system by incorporation of multiple donors into DNA. Two perylene moieties were tethered to the DNA on both sides of the intervening base, and two pyrenes were further inserted in the vicinity of the perylenes as an antenna. Hybridization of this FRET probe with a fully matched DNA allowed monomer emission of perylene when the pyrenes were excited. In contrast, excimer emission was generated by hybridization with a one-base deletion mutant. Thus, the apparent Stokes' shift was enhanced without loss of efficiency in the detection of the deletion mutant.

Off means off: An in‐stem molecular beacon in which D‐threoninol units tether perylene and anthraquinone in the stem region effectively detected target sequences and was able to discriminate a one‐base‐deletion mutant from the wild‐type (full‐match) sequence without background emission (see picture).

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Aus heißt auch aus: Ein neuartiger molekularer Beacon wurde entwickelt, der in der Stamm‐Region Pseudobasenpaare aus dem Fluorophor (Perylen) und dem Fluoreszenzlöscher (Anthrachinon) enthält, wobei D‐Threoninol‐Einheiten als Verbindungsstück dienen (in‐stem molecular beacon, ISMB). Der ISMB war in der Lage, eine Einzeldeletionsmutante von der Wildtypsequenz zu unterscheiden, ohne dass Hintergrundemission auftrat (siehe Bild).

Pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to increase the apparent Stokes' shift of perylene. Multiple donors were introduced in the vicinity of acceptors via D-threoninol and natural base pairs were inserted between donors and acceptors in order to suppress undesired interactions between them. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 495 nm was observed from perylene when excited at 345 nm where pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high FRET efficiency (Phi>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R(0)) of the donor pyrene.