Unlocked nucleic acids with a pyrene-modified uracil: synthesis, hybridization studies, fluorescent properties and i-motif stability.

The synthesis of two new phosphoramidite building blocks for the incorporation of 5-(pyren-1-yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene-modified nucleobase component were found to destabilize an i-motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene-modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene-modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady-state fluorescence emission studies of oligonucleotides containing pyrene-modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.

Electrochemistry of single metalloprotein and DNA-based molecules at Au(111) electrode surfaces.

We have briefly overviewed recent efforts in the electrochemistry of single transition metal complex, redox metalloprotein, and redox-marked oligonucleotide (ON) molecules. We have particularly studied self-assembled molecular monolayers (SAMs) of several 5'-C6-SH single- (ss) and double-strand (ds) ONs immobilized on Au(111) electrode surfaces via Au-S bond formation, using a combination of nucleic acid chemistry, electrochemistry and electrochemically controlled scanning tunnelling microscopy (in situ STM). Ds ONs stabilized by multiply charged cations and locked nucleic acid (LNA) monomers have been primary targets, with a view on stabilizing the ds-ONs and improving voltammetric signals of intercalating electrochemical redox probes. Voltammetric signals of the intercalator anthraquinone monosulfonate (AQMS) at ds-DNA/Au(111) surfaces diluted by mercaptohexanol are significantly sharpened and more robust in the presence than in the absence of [Co(NH3)6](3+). AQMS also displays robust Faradaic voltammetric signals specific to the ds form on binding to similar LNA/Au(111) surfaces, but this signal only evolves after successive voltammetric scanning into negative potential ranges. Triply charged spermidine (Spd) invokes itself a strong voltammetric signal, which is specific to the ds form and fully matched sequences. This signal is of non-Faradaic, capacitive origin but appears in the same potential range as the Faradaic AQMS signal. In situ STM shows that molecular scale structures of the size of Spd-stabilized ds-ONs are densely packed over the Au(111) surface in potential ranges around the capacitive peak potential.

A quencher-free molecular beacon design based on pyrene excimer fluorescence using pyrene-labeled UNA (unlocked nucleic acid).

A quencher-free molecular beacon capable of generating pyrene excimer fluorescence has been constructed using strategically positioned pyrene-UNA monomers. Hybridization of a fully complementary RNA target was accompanied by a pyrene excimer emission increase of more than 900%, and detection of RNA in living cells was demonstrated.

Electrochemistry and in situ scanning tunnelling microscopy of pure and redox-marked DNA- and UNA-based oligonucleotides on Au(111)-electrode surfaces.

We have studied adsorption and electrochemical electron transfer of several 13- and 15-base DNA and UNA (unlocked nucleic acids) oligonucleotides (ONs) linked to Au(111)-electrode surfaces via a 5'-C6-SH group using cyclic voltammetry (CV) and scanning tunnelling microscopy in aqueous buffer under electrochemical potential control (in situ STM). 2,2',6',2''-Terpyridine (terpy) onto which the transition metal ions Fe(2+/3+), Os(2+/3+) and Ru(2+/3+) could be coordinated after UNA monolayer formation was attached to UNA via a flexible linker. The metal centres offer CV probes and in situ STM contrast markers, and the flexible UNA/linker a potential binder for intercalation. CV of pure and mercaptohexanol diluted ON monolayers displayed reductive desorption signals but also, presumably capacitive, signals at higher potentials. Distinct voltammetric signals arise on metal binding. Those from Ru-binding are by far the strongest and in accord with multiple site Ru-attachment. In situ STM disclosed molecular scale features in varying coverage on addition of the metal ions. The Ru-derivatives showed a bias voltage dependent broad maximum in the tunnelling current-overpotential correlation which could be correlated with theoretical frames for condensed matter conductivity of redox molecules. Together the data suggest that Ru-units are bound to both terpy and the UNA-DNA backbone.

Polycation induced potential dependent structural transitions of oligonucleotide monolayers on Au(111)-surfaces.

We have studied self-assembled molecular monolayers (SAMs) of several 3'-C3-SH conjugated single-strand (ss) and double-strand (ds) 20-base oligonucleotides (ONs) immobilized on single-crystal, atomically planar Au(111)-electrode surfaces in the presence of the triply positively charged base spermidine (Spd). This cation binds strongly to the polyanionic ON backbone and stabilizes the ds-form relative to the ss-form. A combination of chemical ON synthesis, melting temperature measurements, cyclic voltammetry (CV), and in situ scanning tunneling microscopy (STM) in aqueous biological buffer under electrochemical potential control was used. Spd binding was found to increase notably the ds-ON melting temperature. CV displays capacitive features associated with ss- and ds-ON. A robust capacitive peak around -0.35 V versus saturated calomel electrode (SCE), specific to ds-ON and highly sensitive to base pair mismatches, was consistently observed. The peak is likely to be caused by surface structural reorganization around the peak potential and located close to reported peak potentials of several DNA intercalating or covalently tethered redox molecules reported as probes for long-range electron transfer.

Pyrene-modified unlocked nucleic acids: synthesis, thermodynamic studies, and fluorescent properties.

Two pyrene-modified UNA monomers were synthesized and incorporated into 21-mer DNA oligonucleotides. Melting temperatures and thermodynamic properties of the modified duplexes were measured, and the fluorescence properties of single strands and duplexes containing one or more pyrene-UNA modifications were studied. It was found that incorporation of pyrene-UNA monomers increased duplex stability relative to UNA monomers, and thermodynamic studies revealed significant mismatch discriminative capabilities of the pyrene-UNA modified oligonucleotides. Furthermore, the steady-state fluorescence emission intensities of pyrene-UNA modified oligonucleotides were increased upon hybridization to DNA, which to the best of our knowledge is unprecedented for an acyclic pyrene modification in DNA. Interestingly, pyrene excimer emission was observed for single-stranded oligonucleotides containing three pyrene-UNA modifications, whereas this excimer emission disappeared after hybridization to DNA. In view of both the pyrene monomer and the excimer fluorescence emission, the triply modified oligonucleotides show intriguing properties relating to the development of new DNA/RNA detection tools.

Synthesis of an unlocked nucleic acid terpyridine monomer and binding of divalent metal ion in nucleic acid duplexes.

Herein we present the synthesis and thermal stability studies of modified oligonucleotides containing an unlocked nucleic acid (UNA) terpyridine monomer. Incorporation of this monomer into both strands of a DNA duplex allowed reversible thermal stability modulation upon addition or withdrawal of divalent metal ions. A likely explanation of this phenomenon is interstrand complexation between two terpyridine units and a metal ion. This system could be useful in the development of nanoscale devices based on DNA hybridization.

Selection of LNA-containing DNA aptamers against recombinant human CD73.

LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.

Structure-Activity Study of Dihydrocinnamic Acids and Discovery of the Potent FFA1 (GPR40) Agonist TUG-469.

The free fatty acid 1 receptor (FFA1 or GPR40), which is highly expressed on pancreatic ß-cells and amplifies glucose-stimulated insulin secretion, has emerged as an attractive target for the treatment of type 2 diabetes. Several FFA1 agonists containing the para-substituted dihydrocinnamic acid moiety are known. We here present a structure-activity relationship study of this compound family suggesting that the central methyleneoxy linker is preferable for the smaller compounds, whereas the central methyleneamine linker gives higher potency to the larger compounds. The study resulted in the discovery of the potent and selective full FFA1 agonist TUG-469 (29).

Discovery of potent and selective agonists for the free fatty acid receptor 1 (FFA(1)/GPR40), a potential target for the treatment of type II diabetes.

A series of 4-phenethynyldihydrocinnamic acid agonists of the free fatty acid receptor 1 (FFA(1)) has been discovered and explored. The preferred compound 20 (TUG-424, EC(50) = 32 nM) significantly increased glucose-stimulated insulin secretion at 100 nM and may serve to explore the role of FFA(1) in metabolic diseases such as diabetes or obesity.