Niels Bomholt

Significantly enhanced DNA thermal stability resulting from porphyrin H-aggregate formation in the minor groove of the duplex

(Figure Presented) Too groovy: The covalent attachment of up to four porphyrins to complementary strands led to the formation of DNA porphyrin zippers with significantly increased DNA duplex stability. This is a result of H-aggregate formation in the minor groove. To the best of our knowledge this is the first report showing such a significant thermal duplex stabilization.

Triplex Formation by Pyrene-Labelled Probes for Nucleic Acid Detection in Fluorescence Assays

Triplex‐forming homopyrimidine oligonucleotides containing insertions of a 2′–5′ uridine linkage featuring a pyrene moiety at the 3′‐position exhibit strong fluorescence enhancement upon binding to double‐stranded DNA through Hoogsteen base pairing. It is shown that perfect matching of the new modification to the base pair in the duplex is a prerequisite for strong fluorescence, thus offering the potential to detect single mutations in purine stretches of duplex DNA. The increase in the fluorescence signal was dependent on the thermal stability of the parallel triplex, so a reduction in the pH from 6.0 to 5.0 resulted in an increase in thermal stability from 25.0 to 55.0 °C and in an increase in the fluorescence quantum yield (ΦF) from 0.061 to 0.179, while the probe alone was fluorescently silent (ΦF=0.001–0.004). To achieve higher triplex stability, five nucleobases in a 14‐mer sequence were substituted with α‐L‐LNA monomers, which provided a triplex with a Tm of 49.5 °C and a ΦF of 0.158 at pH 6.0. Under similar conditions, a Watson–Crick‐type duplex formed with the latter probe showed lower fluorescence intensity (ΦF=0.081) than for the triplex.

High physiological thermal triplex stability optimization of twisted intercalating nucleic acids (TINA)

The structure of the monomer (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol () in twisted intercalating nucleic acids (TINA) was optimized for stabilizing interactions between the intercalator and surrounding nucleobases when used as a triplex forming oligonucleotide (TFO). Enhancement of pi-pi interactions with nucleobases of the TFO was achieved by increasing the aromatic surface using the (R)-1-O-[4-(1-pyrenylethynyl)naphthylmethyl]glycerol monomer (). Bulge insertion of in the middle of a Hoogsteen-type triplex increased the triplex thermal stability, DeltaT(m) = +2.0 degrees C compared with at pH 7.2. Syntheses and thermal denaturation studies of triplexes and duplexes are described for three novel TINA monomers. The influence of pi-pi interactions, link length and the positioning of the ether in the linker in the TINA derivatives are described.

Synthesis of locked pyranosyl nucleic acid (LpNA)

A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation.

Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole intercalator to a triplex oligonucleotide and to a three-way junction

A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.