Olalla Vázquez

Bis-4-aminobenzamidines: versatile, fluorogenic A/T-selective dsDNA binders.

N(1),N(3)-Bis(4-amidinophenyl)propane-1,3-diamine (BAPPA, R = H) is a bisaminobenzamidine fluorogenic derivative that displays a large increase in its emission fluorescence when bound to the minor groove of specific A/T DNA sites (K(d) for an AATT site approximately 79 +/- 6 nM). Moreover, the structural characteristics of BAPPA allow the easy introduction of functional groups that protrude out of the DNA surface.

Straightforward access to bisbenzamidine DNA binders and their use as versatile adaptors for DNA-promoted processes

Bisbenzamidines are an important family of minor groove DNA-binding agents. We present a one-step synthesis of aromatic aza-bisbenzamidines that allows straightforward and versatile access to a large number of these molecules. One of them, the azide-aza-bisbenzamidine 13, can be readily modified via click-chemistry with a variety of functionalities that can, therefore, be delivered to the vicinity of an A/T-rich DNA minor groove. This strategy, therefore, provides a simple means for triggering site selective, DNA-promoted biochemical and physicochemical processes.

Templated native chemical ligation: peptide chemistry beyond protein synthesis.

Native chemical ligation (NCL) is a powerful method for the convergent synthesis of proteins and peptides. In its original format, NCL between a peptide containing a C‐terminal thioester and another peptide offering an N‐terminal cysteine has been used to enable protein synthesis of unprotected peptide fragments. However, the applications of NCL extend beyond the scope of protein synthesis. For instance, NCL can be put under the control of template molecules. In such a scenario, NCL enables the design of conditional reaction systems in which, peptide bond formation occurs only when a specific third party molecule is present. In this review, we will show how templates can be used to control the reactivity and chemoselectivity of NCL reactions. We highlight peptide and nucleic‐acid‐templated NCL reactions and discuss potential applications in nucleic acid diagnosis, origin‐of‐life studies and gene‐expression‐specific therapies. Copyright

Efficient DNA Binding and Nuclear Uptake by Distamycin Derivatives Conjugated to Octa-arginine Sequences

Efficient targeting of DNA by designed molecules requires not only careful fine‐tuning of their DNA‐recognition properties, but also appropriate cell internalization of the compounds so that they can reach the cell nucleus in a short period of time. Previous observations in our group on the relatively high affinity displayed by conjugates between distamycin derivatives and bZIP basic regions for A‐rich DNA sites, led us to investigate whether the covalent attachment of a positively charged cell‐penetrating peptide to a distamycin‐like tripyrrole might yield high affinity DNA binders with improved cell internalization properties. Our work has led to the discovery of synthetic tripyrrole–octa‐arginine conjugates that are capable of targeting specific DNA sites that contain A‐rich tracts with low nanomolar affinity; they simultaneously exhibit excellent membrane and nuclear translocation properties in living HeLa cells.

Sequence-selective DNA recognition with peptide-bisbenzamidine conjugates.

Transcription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza-bisbenzamidines, which are readily accessible molecules that interact with A/T-rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza-benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine-GCN4 binding sites (TCAT⋅AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity (KD≈12 nM) and specificity to a composite AATTT⋅GAGA sequence containing both the bisbenzamidine and the TF consensus binding sites.

Cytotoxic peptide–PNA conjugates obtained by RNA-programmed peptidyl transfer with turnover

We describe a RNA-programmed peptidyl transfer reaction, which triggers the formation of a cytotoxic, cell permeant 14-mer peptide–PNA conjugate from inactive fragments. Turnover in the RNA template is required to evoke the bioactivity. HeLa cells enabled the read-out of the reaction, which proceeded rapidly when it was performed on matched RNA templates.

A Far‐Red Fluorescent DNA Binder for Interaction Studies of Live Multidrug‐Resistant Pathogens and Host Cells

Transgene expression of green fluorescent protein (GFP) has facilitated the spatiotemporal investigation of host-pathogen interactions; however, introduction of the GFP gene remains challenging in drug-resistant bacteria. Herein, we report a novel far-red fluorescent nucleic acid stain, 6-TramTO-3, which efficiently labels bacteria through a DNA binding mode without affecting growth and viability. Exemplarily, we stained Klebsiella pneumoniae, a major threat to hospitalized patients, and deciphered divergent interaction strategies of antibiotic-resistant and antibiotic-sensitive Klebsiella strains with immune cells. 6-TramTO-3 constitutes an off-the-shelf reagent for real-time analysis of bacterial infection, including strains for which the use of genetically encoded reporters is not feasible. Eventually, our approach may aid the development of strategies to combat a major worldwide health threat: multidrug-resistant bacteria.

Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains.

We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac‐R7‐GG‐KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine‐containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol‐rich domains in a GPMV model, thus documenting the potential of our approach for membrane‐partitioning studies.