N

TAGCCG-Gly

19-times increased fluorescence upon binding of 11A: 5'-CGGCTATTACGGC-3' 5-times increased fluorescence upon binding of 11C: 5-CGGCTCTTACGGC-3 Fig. 4.2.7. Homogeneous DNA detection with FIT-probes.

diationless fluorescence resonance energy transfer (FRET) to an acceptor group. Single-labeled probes, termed ResonSense probes, bind to target DNA (Figure 4.2.8a) [24]. A DNA-intercalator such as SYBR Gold can bind to the double-stranded segment serving as fluorescence donor that sensitizes the fluorescence of the probe-bound acceptor dye. More commonly, both fluorescence donor and a fluorescence acceptor group are appended to probe molecules. In the adjacent probe format two probes are designed to bind adjacent to one another on the target strand, which brings a 5'-label in close proximity to a 3'-label (Figure 4.2.8b) [25, 26]. As a result the fluorescence of the donor dye becomes quenched whereas acceptor fluorescence is enhanced. An alternative approach utilizes double-stranded probes that dissociate on competitive hybridization with the target DNA [27]. This leads to disruption of the energy transfer between the 5 '-label and the 3 '-label, thereby increasing the fluorescence of the donor dye. The molecular beacons (Box 18) are an intramolecular version of this approach (Figure 4.2.8c) [28]. Molecular beacons are designed to form a hairpin structure with a target unrelated double-stranded stem sequence that holds two reporter groups in close proximity. Accordingly, the fluorescence is quenched as a result of FRET and static and dynamic (collisional) quenching. When the single-stranded loop segment anneals to the target sequence, structural reorganization increases the donor-quencher distance within the formed duplexes and fluorescence can occur. PCR-primers equipped with a molecular beacon structure are known as Scorpion probes [29, 30]. Molecular beacons incorporated in the DNA-template strand enable real-time monitoring of the DNA polymerase reaction [31]. It has been discovered that hybridization of linear dual-labeled oligonucleotides devoid of secondary structure-forming segments also leads to fluorescence enhancement [32]. This has been attributed to hybridization-induced stiffening that reduces quenching processes that operate in the flexible single-stranded probes. For example, oligonucleotide probes in which

Fig. 4.2.8. Homogeneous DNA detection by and fluorescence-labeled probe molecules;

employing fluorescence resonance energy (b) adjacent probes and (c) in Molecular transfer between two labels. Dual label inter- Beacons; (d) in TaqMan probes, and upon (e)

action between (a) dyes that bind to dsDNA template-directed dye-terminator incorporation.

Fig. 4.2.8. Homogeneous DNA detection by and fluorescence-labeled probe molecules;

employing fluorescence resonance energy (b) adjacent probes and (c) in Molecular transfer between two labels. Dual label inter- Beacons; (d) in TaqMan probes, and upon (e)

action between (a) dyes that bind to dsDNA template-directed dye-terminator incorporation.

terminal FAM donors and TAMRA acceptors were spaced by 20 to 27 bases afforded fluorescence increases of a factor of seven when hybridized to complementary nucleic acids [33].

In our work we explored the utility of dual-labeled PNA probes [34-36]. It was observed that thermal denaturation of single-stranded PNA, in contrast with DNA, resulted in a phase transition and considerable hyperchromicity, which indicated that base stacking might be a favorable process even in unhybridized PNA [37]. We reckoned that a suitably appended fluorescence donor could be located in close proximity to the fluorescence quencher because of a possible inter- or intramolecular association of PNA single-strands (Figure 4.2.9). As a result collisional quenching and fluorescence resonance energy transfer (FRET) would reduce the fluorescence of the single-strand. It was expected that quenching should be more

Fig. 4.2.9. Linear PNA-beacons: in unhybrid-ized peptide nucleic acids (PNA) the averaged distance between appropriately appended fluorescence donor and fluorescence quencher groups is smaller than in the duplex structure.

Fig. 4.2.9. Linear PNA-beacons: in unhybrid-ized peptide nucleic acids (PNA) the averaged distance between appropriately appended fluorescence donor and fluorescence quencher groups is smaller than in the duplex structure.

The fluorescence is quenched by collisional quenching and fluorescence resonance energy-transfer (FRET). When the probe sequence anneals to the target sequence fluorescence occurs.

The fluorescence is quenched by collisional quenching and fluorescence resonance energy-transfer (FRET). When the probe sequence anneals to the target sequence fluorescence occurs.

efficient in dual-labeled PNA-probes than in dual-labeled DNA-probes. Hybridization to a complementary nucleic acid would, however, induce a structural reorganization that would lead to an increase of the averaged donor-quencher-distance. Thus, in the duplex, fluorescence would occur.

It was unclear how the hybridization-induced change of the distance between the donor and the quencher group could be maximized. We developed a highly flexible and automatable strategy which enabled us to perform all reactions, including the labeling steps, on the solid phase (Scheme 4.2.2). The use of the HYCRON-linker enabled the solid phase synthesis of unprotected PNA-resins such as 14 (for more information on linkers see Chapter 6.1). The resin-bound PNA 14 was subjected to a set of orthogonal ligation reactions. A Pd(0)-catalyzed allyl transfer accomplished the final detachment furnishing dual-labeled PNA conjugates such as 16. The hybridization experiments revealed that duplex formation was accompanied by fluorescence enhancements reaching factors of 6. Use of mixed-sequence PNA oligomers that contain terminally appended fluorescent labels suggested that the fluorescence behavior of linear dual-labeled PNA oligomers is a general phenomenon that is independent of the sequence context [35]. Recent work compared the performance of Molecular Beacons, linear dual-labeled DNA, and linear duallabeled PNA [38]. Briefly, dual-labeled PNA was found to hybridize rapidly and to provide a fluorescence response that was independent of the salt concentration.

Dual-labeled oligonucleotide probes known as TaqMan probes are amongst the most widely used type of probe in real-time PCR analysis (Figure 4.2.8d) [33, 39]. This is because of the 5'-3' exonuclease activity of Taq-polymerase which provides an effective means of separating the donor from the acceptor fluorophore. During PCR the dual-labeled probe binds to the template. When the Taq-polymerase extends the primer it eventually reaches the 5 '-end of the hybridized probe, which is then degraded by the exonuclease activity. This leads to an irreversible donor fluorescence increase that accumulates with each round of PCR-amplification. Irreversible cleavage of triple-stranded regions forms the basis of the Invader assay [29]. The opposite, namely the template-directed coupling of a fluorescence-labeled dye-terminator or oligonucleotide segment, forms the basis of the homogeneous variants of the Primer Extension [40, 41] (Figure 4.2.8e) and Oligonucleotide Ligation assays [42], respectively.

(CH2)4NH2 Trt (CH2)4NH-Boc

Ac-Cys-t tattattattatta t-Gly-HYCRON -<--Ac-Cys-t taRttaRttaRttaRtta t-Gly-HYCRON

Ac-Cys-t tattattattatta t-Gly-HYCRON —^^ -Ac-Cys-t tattattattatta t-Gly-COOH

15 16

R=Bhoc

R=Bhoc

0 0

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