A Chromogenic and Fluorogenic Peptide Substrate for the Highly Sensitive Detection of Proteases in Biological Matrices.

The synthesis and application of a novel type of chromogenic and fluorogenic substrate for protease detection is described. The outstanding performance of the tripeptide substrates is exemplified by specific fluorescence detection of thrombin and factor Xa at only 500 fM concentration. The substrate is also applicable to the sensitive detection of the thrombin inhibitor dabigatran in human plasma and whole blood samples, highlighting its potential for a point-of-care test for instant monitoring the blood levels of this blockbuster anticoagulant drug in specific clinical situations.

Irreversible inhibition of DNA polymerase ß by small-molecule mimics of a DNA lesion.

Abasic sites are ubiquitous DNA lesions that are mutagenic and cytotoxic but are removed by the base excision repair pathway. DNA polymerase ß carries out two of the four steps during base excision repair, including a lyase reaction that removes the abasic site from DNA following incision of its 5'-phosphate. DNA polymerase ß is overexpressed in cancer cells and is a potential anticancer target. Recently, DNA oxidized abasic sites that are produced by potent antitumor agents were shown to inactivate DNA polymerase ß. A library of small molecules whose structures were inspired by the oxidized abasic sites was synthesized and screened for the ability to irreversibly inhibit DNA polymerase ß. One candidate (3a) was examined more thoroughly, and modification of its phosphate backbone led to a molecule that irreversibly inactivates DNA polymerase ß in solution (IC50 ≈ 21 µM), and inhibits the enzyme's lyase activity in cell lysates. A bisacetate analogue is converted in cell lysates to 3a. The bisacetate is more effective in cell lysates, more cytotoxic in prostate cancer cells than 3a and potentiates the cytotoxicity of methyl methanesulfonate between 2- and 5-fold. This is the first example of an irreversible inhibitor of the lyase activity of DNA polymerase ß that works synergistically with a DNA damaging agent.

Control of the photocatalytic activity of bimetallic complexes of pyropheophorbide-a by nucleic acids.

Photocatalytic activity of a photosensitizer (PS) in an oligodeoxyribonucleotide duplex 5'-PS~ODN1/ODN2~Q-3' is inhibited because of close proximity of a quencher Q. The ODN2 in this duplex is selected to be longer than the ODN1. Therefore, in the presence of a nucleic acid (analyte), which is fully complementary to the ODN2 strand, the duplex is decomposed with formation of an analyte/ODN2~Q duplex and a catalytically active, single stranded PS~ODN1. In this way the catalytic activity of the PS can be controlled by the specific nucleic acids. We applied this reaction earlier for the amplified detection of ribonucleic acids in live cells (Arian, D.; Cló, E.; Gothelf, K.; Mokhir, A. Chem.-Eur. J.2010, 16(1), 288). As a photosensitizer (PS) we used In(3+)(pyropheophorbide-a)chloride and as a quencher (Q)--Black-Hole-Quencher-3 (BHQ-3). The In(3+) complex is a highly active photocatalyst in aqueous solution. However, it can coordinate additional ligands containing thiols (e.g., proteins, peptides, and aminoacids), that modulate properties of the complex itself and of the corresponding bio- molecules. These possible interactions can lead to undesired side effects of nucleic acid controlled photocatalysts (PS~ODN1/ODN2∼Q) in live cells. In this work we explored the possibility to substitute the In(3+) complex for those ones of divalent metal ions, Zn(2+) and Pd(2+), which exhibit lower or no tendency to coordinate the fifth ligand. We found that one of the compounds tested (Pd(pyropheophorbide-a) is as potent and as stable photosensitizer as its In(3+) analogue, but does not coordinate additional ligands that makes it more suitable for cellular applications. When the Pd complex was introduced in the duplex PS~ODN1/ODN2~Q as a PS, its photocatalytic activity could be controlled by nucleic acids as efficiently as that of the corresponding In(3+) complex.

1,9-Dialkoxyanthracene as a (1)O2-sensitive linker.

We developed a (1)O(2)-sensitive linker based on a 9,10-dialkoxyanthracene structure. Its cleavage in the presence of (1)O(2) is quick and high-yielding. A phosphoramidite containing this fragment was prepared and coupled to a variety of molecular fragments, including nucleosides, fluorescent dyes, and a cholesteryl derivative. On the basis of this building block we prepared a fluorogenic probe for monitoring (1)O(2) in live mammalian cells and visible-light-activated "caged" oligodeoxyribonucleotides. In particular, the fluorogenic (1)O(2) probe is a conjugate of 4,7,4',7'-tetrachlorofluorescein and N,N,N',N'-tetramethylrhodamine coupled to each other via the (1)O(2)-sensitive linker. Fluorescence of the dyes in this probe is quenched. In the presence of (1)O(2), the linker is cleaved with formation of 9,10-anthraquinone and two strongly fluorescent dyes: 4,7,4',7'-tetrachlorofluorescein and N,N,N',N'-tetramethylrhodamine derivatives. We observed that the fluorescence of the probe correlates with the amount of (1)O(2) present in solution. The red-light-activated "caged" oligodeoxyribonucleotides are stable duplexes, which consist of an unmodified strand and a blocker strand. The (1)O(2)-sensitive linker is introduced in the interior of the blocker strand. Upon exposure of the duplex to red light in the presence of In(3+)(pyropheophorbide-a) chloride, the linker is cleaved with formation of the unstable duplex structure. This product decomposes spontaneously, releasing the unmodified strand, which can bind to the complementary target nucleic acid. This uncaging reaction is high-yielding. In contrast, previously reported visible-light-activated reagents are uncaged inefficiently due to competing reactions of sulfoxide and disulfide formation.

Single molecule atomic force microscopy studies of photosensitized singlet oxygen behavior on a DNA origami template.

DNA origami, the folding of a long single-stranded DNA sequence (scaffold strand) by hundreds of short synthetic oligonucleotides (staple strands) into parallel aligned helices, is a highly efficient method to form advanced self-assembled DNA-architectures. Since molecules and various materials can be conjugated to each of the short staple strands, the origami method offers a unique possibility of arranging molecules and materials in well-defined positions on a structured surface. Here we combine the action of light with AFM and DNA nanostructures to study the production of singlet oxygen from a single photosensitizer molecule conjugated to a selected DNA origami staple strand on an origami structure. We demonstrate a distance-dependent oxidation of organic moieties incorporated in specific positions on DNA origami by singlet oxygen produced from a single photosensitizer located at the center of each origami.

A nucleic acid dependent chemical photocatalysis in live human cells.

Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment of cancer. We report here the first example of a chemical reaction that generates a cytotoxic drug from a nontoxic prodrug in the presence of a specific endogeneous ribonucleic acid in live mammalian cells. In this case, the prodrug is triplet oxygen and the drug is singlet oxygen. The key component of this reaction is an inert molecule (InP-2'-OMe-RNA/Q-2'-OMe-RNA; P: photosensitizer; Q: quencher), which becomes an active photosensitizer (InP-2'-OMe-RNA) in the presence of single-stranded nucleic acid targets. Upon irradiation with red light, the photosensitizer produces over 6000 equivalents of toxic singlet oxygen per nucleic acid target. This reaction is highly sequence specific. To detect the generation of singlet oxygen in live cells, we prepared a membrane-permeable and water-soluble fluorescent scavenger, a derivative of 2,5-diphenylisobenzofurane. The scavenger decomposes upon reaction with singlet oxygen and this is manifested in a decrease in the fluorescence intensity. This effect can be conveniently monitored by flow cytometry.

A simple method for monitoring protein-DNA interactions.

A simple, efficient and cheap method is reported for monitoring interactions between single stranded desoxyribonucleic acids and proteins, using fluorescence spectroscopy and complexes of 5'-dye-DNA conjugates with bovine serum albumin as probes. In the presence of a single stranded DNA-binding protein the complexes with bovine serum albumin are disrupted, which results in a reduction of fluorescence intensity.