A Self-Immolative Molecular Beacon for Amplified Nucleic Acid Detection

Fluorogenic hybridization probes allow the detection of RNA and DNA sequences in homogeneous solution. Typically, one target molecule is activating the fluorescence of a single probe molecule. This limits the sensitivity of nucleic acid detection. Herein, we report a self-immolative Molecular Beacon (iMB), which escapes the one-target-one-probe dogma. The iMB probe includes a photoreductively cleavable N-alkylpicolinium (NAP) linkage within the loop region. A fluorophore at the 5'-end serves, on the one hand, as a reporter group and, on the other hand, as a photosensitizer of a NAP-linker cleavage reaction. In the absence of a target, the iMB adopts a hairpin shape. Quencher proups prevent photo-induced cleavage. The iMB opens upon hybridization with target, and both fluorescent emission as well as photo-inductive cleavage of the NAP-linker can occur. In contrast to previous chemical amplification probes, iMBs are unimolecular. Cleavage leads to products that have lower target affinity than the probes before reaction. Aided by catalysis, the method allowed the detection of 5 pM RNA target within 100 min. <br>

A Self-Immolative Molecular Beacon for Amplified Nucleic Acid Detection

Fluorogenic hybridization probes allow the detection of RNA and DNA sequences in homogeneous solution. Typically, one target molecule is activating the fluorescence of a single probe molecule. This limits the sensitivity of nucleic acid detection. Herein, we report a self-immolative Molecular Beacon (iMB), which escapes the one-target-one-probe dogma. The iMB probe includes a photoreductively cleavable N-alkylpicolinium (NAP) linkage within the loop region. A fluorophore at the 5'-end serves, on the one hand, as a reporter group and, on the other hand, as a photosensitizer of a NAP-linker cleavage reaction. In the absence of a target, the iMB adopts a hairpin shape. Quencher proups prevent photo-induced cleavage. The iMB opens upon hybridization with target, and both fluorescent emission as well as photo-inductive cleavage of the NAP-linker can occur. In contrast to previous chemical amplification probes, iMBs are unimolecular. Cleavage leads to products that have lower target affinity than the probes before reaction. Aided by catalysis, the method allowed the detection of 5 pM RNA target within 100 min. <br>

Synthesis of Polyanionic C5-Modified 2′-Deoxyuridine and 2′-Deoxycytidine-5′-Triphosphates and Their Properties as Substrates for DNA Polymerases

Modified 2′-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

Application of the Inverse-Electron-Demand Diels-Alder Reaction for Metabolic Glycoengineering

The inverse electron-demand Diels-Alder (IEDDA or DAinv) reaction is an emerging bioorthogonal ligation reaction that finds application in all areas of chemistry and chemical biology. In this review we highlight its application in metabolic glycoengineering (MGE). MGE is a versatile tool to introduce unnatural sugar derivatives that are modified with a chemical reporter group into cellular glycans. The IEDDA reaction can then be used to modify the chemical reporter group allowing, for instance, the visualization or isolation of glycoconjugates. During the last years, many different sugar derivatives as well as reporter groups have been published. These probes are summarized, and their chemical and biological properties are discussed. Furthermore, we discuss examples of MGE and subsequent IEDDA reaction that highlight its suitability for application within living systems.

Recent advances in glycosidase probes used in Escherichia coli detection

Pathogenic Escherichia coli poses a serious threat to global public health and are especially dangerous with the increase of antibiotic resistance. β-Glucuronidase (GUS) and some other glycosidases can serve as useful biomarkers or indicators for detection of E. coli. The probes made up of a glycosyl residue (recognition group), a label or reporter group, and a linkage that is generally a direct glycosidic bond, are powerful analytical tools. Upon hydrolysis of the glycosidic linkages by the corresponding glycosidases, these probes irreversibly release detectable labels or reporter molecules. A variety of such glycosidase probes have been developed and applied for detection of E. coli or for the development of various corresponding detection methods. This paper provides an overview of recent advances in this field, covering the development and applications of chromogenic, fluorogenic, luminogenic, and electrochemical glycosidase substrates. The challenges and opportunities in the probe development for detection of E. coli are also discussed.

Fragment-Based Identification of Ligands for Bromodomain-Containing Factor 3 of Trypanosoma Cruzi

The <i>Trypanosoma cruzi</i> (<i>T. cruzi</i>) parasite is the cause of Chagas disease, a neglected disease endemic in South America. The life cycle of the <i>T. cruzi</i> parasite is complex and includes transitions between distinct life stages. This change in phenotype (without a change in genotype) could be controlled by epigenetic regulation, and might involve the bromodomain-containing factors 1-5 (<i>Tc</i>BDF1-5). However, little is known about the function of the <i>Tc</i>BDF1-5. Here we describe a fragment-based approach to identify ligands for <i>T. cruzi</i> bromodomain-containing factor 3 (<i>Tc</i>BDF3). We expressed a soluble construct of <i>Tc</i>BDF3 in <i>E. coli</i>, and used this to develop a range of biophysical assays for this protein. Fragment screening identified twelve compounds that bind to the <i>Tc</i>BDF3 bromodomain. Based on this screen, we developed functional ligands containing a fluorescence or ¹⁹F reporter group, and a photo-crosslinking probe for <i>Tc</i>BDF3. These tools compounds will be invaluable in future studies on the function of <i>Tc</i>BDF3 and will provide insight into the biology of <i>T. cruzi</i>.

Fragment-Based Identification of Ligands for Bromodomain-Containing Factor 3 of Trypanosoma Cruzi

The <i>Trypanosoma cruzi</i> (<i>T. cruzi</i>) parasite is the cause of Chagas disease, a neglected disease endemic in South America. The life cycle of the <i>T. cruzi</i> parasite is complex and includes transitions between distinct life stages. This change in phenotype (without a change in genotype) could be controlled by epigenetic regulation, and might involve the bromodomain-containing factors 1-5 (<i>Tc</i>BDF1-5). However, little is known about the function of the <i>Tc</i>BDF1-5. Here we describe a fragment-based approach to identify ligands for <i>T. cruzi</i> bromodomain-containing factor 3 (<i>Tc</i>BDF3). We expressed a soluble construct of <i>Tc</i>BDF3 in <i>E. coli</i>, and used this to develop a range of biophysical assays for this protein. Fragment screening identified twelve compounds that bind to the <i>Tc</i>BDF3 bromodomain. Based on this screen, we developed functional ligands containing a fluorescence or ¹⁹F reporter group, and a photo-crosslinking probe for <i>Tc</i>BDF3. These tools compounds will be invaluable in future studies on the function of <i>Tc</i>BDF3 and will provide insight into the biology of <i>T. cruzi</i>.