Arrays of oligonucleotide probes (DNA chips) immobilized on glass or silicon surfaces have emerged as powerful new tools for the analysis of DNA and RNA. [1] These specific DNA sensors operate through hybridization reactions which are based on the mutual recognition of two complementary nucleic acid strands that establish hydrogen bonds between their nucleic bases. Hybridization is most commonly assayed by fluorescence, and hence requires that fluorophore labels are covalently attached to the DNA target fragments to be analyzed.This technology, however, is still fraught with a number of drawbacks and requires new developments. For example, it is still difficult to assess the quality of the oligonucleotides attached to the surface. The homogeneity and the reproducibility of the procedures for preparing the DNA-functionalized surfaces, and consequently the DNA surface density, are difficult to control. Furthermore the polymerase chain reaction (PCR) step, which serves to amplify the DNA targets, and the subsequent chemical step, for the attachment of the fluorophore to the target DNA, are responsible for significant modifications in the relative proportions of the different populations of the nucleic acids to be analyzed.We propose that some of these problems might be simply solved if the detection label (here a fluorophore) is incorporated on the array capture DNA strand and not on the target to be detected. This strategy might have many advantages. First it greatly reduces the number of manipulations of the targets. Second, it provides a way to control the quality of the DNA array (by using standard fluorescence scanners) in the absence of the targets. Such an array requires that: 1) the fluorescence is not quenched by interaction of the fluorophore with the surface; 2) the label on the probe does not affect its affinity for its complementary oligonucleotide; and 3) the fluorescence is significantly changed as a specific consequence of hybridization of the functionalized DNA[1] a)

The synthesis of oligonucleotides from dimers to a hexamer by a H‐phosphonate approach in solution using solid‐supported acyl chloride is reported herein. This strategy avoids the use of chromatography. The work‐ups have been simplified and involve filtration, aqueous extraction and precipitation. The synthesis was scaled up to 1 mmol for the dimers and 340 μmol for the hexamer. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A new solution-phase phosphoramidite approach is reported for oligonucleotide synthesis employing recyclable solid-supported reagents. It uses polyvinyl pyridinium tosylate as the activator of a nucleoside-3'-O-phosphoramidite in the coupling step with a 5'-OH nucleoside or dinucleotide. The resulting phosphite triester was either sulfurized or oxidized using polystyrene-bound trimethylammonium tetrathionate or periodiate. This method avoids complicated purification steps, as excess reagents are easily removed by filtration. [reaction: see text]

Sensitive detection of DNA on the basis of hybridization to a complementary DNA probe and complex-specific signal (for example, fluorescence) detection, may be improved by molecular amplification methods.[1] DNA target amplification is one of the most widely used methods and is mainly based on the polymerase chain reaction (PCR). Recently, signalamplification techniques based on catalytic reactions, which might be useful for PCR-independent detection of label-free DNA sequences, have also been investigated. [2][3][4][5] These methods allow each probe-hybridization event to be converted into many signal events because the catalyst (a chemical or an enzyme) turns over many copies of the sensing-reaction substrate. As a consequence, high sensitivity can be attained. For example, the insertion of ferrocene moieties or redox-active intercalators allows hybrids to catalyze electrochemical reactions that can be monitored either amperometrically or chemically. [2,3] Herein we propose an original and simple strategy that involves the cofactor of an enzyme as the catalytic species. In this system, the DNA probe is an oligonucleotide covalently attached to the cofactor, and the enzyme is selected on the basis of its ability to catalyze the cofactor-dependent conversion of a fluorogenic substrate into an optically silent product (Figure 1). If the enzyme is functional only with a single-stranded cofactoroligonucleotide conjugate, and not when the latter is hybridized to its complementary strand, enzymatic conversion of the substrate, monitored by fluorescence spectroscopy, can serve as a tool to differentiate whether the probe is hybridized or not (Figure 1). Since the enzyme turns over many copies of the fluorogenic substrate, the difference in the fluorescence signals obtained with the free and the hybridized probes can be greatly amplified enzymatically.This new concept is illustrated herein with an enzyme that catalyzes the oxidation of reduced pyridine nucleotides, either nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), by molecular oxygen in the presence of a riboflavin, either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), as a cofactor. Such an enzyme is called an NAD(P)H:flavin oxidoreductase or flavin reductase.[6] We selected the flavin reductase from Escherichia coli, named Fre, which is soluble, monomeric, and very easy to purify in large amounts. [7,8] Structural, mechanistic, and substrate-specificity studies in our laboratory [9][10][11][12] have shown that Fre contains an active site which accomodates both the flavin and the reduced pyridine nucleotide and that the reaction proceeds in two steps (Scheme 1): first, a hydride transfer from NAD(P)H to the oxidized flavin and then an oxidation of the reduced flavin by molecular oxygen, thereby regenerating the cofactor for a new cycle. With small amounts of flavin it is thus possible to oxidize large excesses of NAD(P)H, a process that can be easily monitored spectrophotometrically since NAD(P)H ...