DNA-Tethered Hoechst Groove-Binding Agents:  Duplex Stabilization and Fluorescence Characteristics

Abstract

Fluorescent Hoechst 33258 analogues have been synthesized in which the terminal phenol moiety is employed as a site for the introduction of a linker to permit covalent attachment of the fluorophores to oligo(deoxynucleotides). Hybridization by the DNA−Hoechst conjugates to target sequences generates the DNA minor groove structure and triggers a binding event by the tethered Hoechst agent. The attendant fluorescence signal reports upon this hybridization event. Conjugation of the Hoechst derivatives to the DNA sequences employs a cystamine linker tethered to an internucleotide phosphorus residue. This mode of conjugation maximizes the versatility of linker placement and minimizes the associated chemistry required to introduce the linker. Two related Hoechst derivatives have been synthesized; both contain a bromoacetamido linker for conjugation to the oligonucleotides. With each Hoechst derivative, two pairs of diastereomeric (R p and S p) oligo(deoxynucleotide) conjugates were prepared to provide the tethered Hoechst groove binders with two different orientations within the dA-dT rich minor groove. T m measurements suggest that while all of the conjugates provide some increased duplex stability, the diastereomeric conjugates tentatively assigned the R p configuration exhibit nearly 20 °C increases in T m values for the double-stranded dodecameric complexes, while those tentatively assigned as the S p diastereomers exhibit only moderate 4−8 °C increases in T m values. The fluorescence characteristics of the conjugates are more variable, with one complex exhibiting a 23-fold enhancement in quantum yield effects, very similar to that observed for a free untethered Hoechst 33258 fluorophore bound to duplex DNA.

New Cyanine–Oligonucleotide Conjugates: Relationships between Chemical Structures and Properties

“…[19][20][21][22][23] Another strategy is based on modification of the signal generated by a single fluorescent or luminescent DNA binding agent linked to ODNs on hybridization with their target sequences. The minor-groove binder Hoechst 33258 [24,25] and intercalating agents such as a ruthenium complex [26][27][28] have been covalently tethered to ODNs and fluorescent or luminescent (in the case of the ruthenium complex) signal increases were observed on hybridization of these labeled probes. Pyrene-ODN conjugates have also been successfully used to discriminate perfectly matched duplexes from mismatched ones.…”

On the kinetics of distamycin binding to its target sites on duplex DNA

“…Quantitative measurement of the binding of minor groove binders has also been reported in a system where a dansyl ''semantophore'' was tethered to a base in the major groove and its fluorescence enhancement was monitored as a function of concentration of Dst (12). Direct measurement of the change in fluorescence spectra on binding to DNA has been reported for some of the more fluorescent compounds of this family, such as Hoechst 33258 and 4Ј,6-diamidino-2-phenylindole (DAPI), as well as a few others (13,14).…”

Development and Applications of Fluorescent Oligonucleotides

“…Examples of post-synthetic coupling of labels with linkers attached at internucleotidic positions via phosphoramidate [137][138][139][140], phosphothiolotriester [141,142], or phosphonate [143] linkages have been reported. In these cases, attachment of the linkers to the internucleotidic phosphate led to the formation of two stereoisomers that could be separated by chromatography.…”

“…The greatest fluorescence increase (up to 23-fold) was observed when the label was attached between two T. As above the label was tethered to an internucleotidic phosphate group leading to two isomers. The Rp isomer (directing the label towards the minor groove) gave rise to a higher fluorescence increase upon hybridization with the target sequence [138].…”