Synthesis of a Photostable Energy‐Transfer Pair for “DNA Traffic Lights”

Abstract

A new cyano‐substituted thiazole red derivative as a red emitter and a novel green fluorescent donor dye of the cyanine styryl type were synthesized in good yields. Characterization of their optical properties revealed excellent photostabilities and large apparent Stokes' shifts. Both dyes can be incorporated into oligonucleotides through postsynthetic “click”‐type chemistry and combined in a diagonal arrangement in double‐stranded DNA. As a result, both dyes combine to an energy‐transfer pair in DNA that shows remarkable optical properties such as significant emission wavelength shift from green to red upon hybridization owing to high energy‐transfer efficiency between the two dyes and remarkable emission red/green color contrast ratio. The combination of these dyes as an energy‐transfer pair according to our concept of “DNA traffic lights” has high potential for applications in molecular imaging.

Strand displacement and duplex invasion into double-stranded DNA by pyrrolidinyl peptide nucleic acids

“…In order to readout the strand displacement dynamics with acpcPNA not only by conventional fluorescence intensity changes at a single emission wavelength, we followed our concept of wavelength-shifting nucleic acid probes. 44 In order to apply such fluorescence color changes to directly report the dynamics of strand displacements in vitro we synthesized the 14mers PNA1 and PNA2 that carry the blue emitting dye 1 in the middle of the sequence (Fig. 25,43 Moreover, we showed that this concept works also for pairs of fluorescent dyes attached to the 2′-positions in two different DNA strands and yields even better emission color contrasts as readout.…”

“…25,43 Moreover, we showed that this concept works also for pairs of fluorescent dyes attached to the 2′-positions in two different DNA strands and yields even better emission color contrasts as readout. 44,45 The dye 1-labeled PNA1 and PNA2 were separately annealed with the unmodified 7mer oligonucleotide counterstrand DNA1 to obtain the starting duplexes for strand displacement experiments. 2).…”

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

“…, A fluorescence color contrast (red/green) of up to 20:1 was achieved together with a large wavelength shift of 140 nm between excitation and emission. Moreover, dyes with significantly enhanced photostability can be combined as 2′‐modifications in a diagonal arrangement similar to the previous base substitutions in DNA and yield derivatives with remarkable wavelength‐shifting fluorescent properties …”

“…The photostabilities of both dyes in the presence of double‐stranded DNA (random sequence, see Supporting Information) under irradiation with a xenon lamp (75 W, 305 nm cut‐off filter) were compared with the fluorescence half‐lives ( t 1/2 ) (Figure ). Compared with thiazole orange ( t 1/2 =32 min) and thiazole red ( t 1/2 =7 min), both the green‐emitting dye ( 1 ) and the red‐emitting dye ( 2 ) showed significantly improved photostability with t 1/2 values of 293 min and 317 min, respectively.…”

“…The single‐stranded oligonucleotides DNA1 – DNA22 were synthesized according to our published protocols using the copper(I)‐catalyzed cycloaddition between the propargyl groups as part of the oligonucleotides and azide‐modified dyes. The synthesis of the azide of dye 1 has previously been published; the preparation of the azide of dye 2 is described in the Supporting Information. Based on the right‐handed helicity of the double‐stranded DNA, variations of the diagonal orientation of the two dyes in the optical module was achieved by combining one of the oligonucleotides, DNA1 – DNA4 and DNA9 – DNA12 , with one of the complementary counterparts, DNA5 – DNA8 and DNA13 – DNA16 .…”