Cellular analysis and PNA encoded libraries

A peptide nucleic acid (PNA) encoded 1296 member peptide library was synthesised and incubated with a variety of cell types. Library members entering cells were extracted, hybridised onto DNA microarrays and the peptide identity was determined via deconvolution. Global consensus analysis highlighted the tetrapepide, Glu-Llp- Glu-Glu (Llp is 6-hexamine-N-aminoacetic acid), a surprise in view of the basic residues typically observed in cell penetrating peptides. When evaluated, Glu-Llp- Glu-Glu revealed cellular uptake comparable to a known basic peptide (tetraLlp). In depth delineation via clustering analysis allowed assessment of differential cellular uptake, with the identified peptides showing clear cellular specificity. This was verified by peptide synthesis and cellular uptake analysis by flow-cytometry, and in all cases an endocytic uptake mechanism was confirmed. This approach establishes a strategy for the identification of short peptides as tools for selective delivery into specific cell types. The incubation of a 10,000 member PNA-encoded peptide library with D54 and HEK293T transfected with CCR6 cells followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new ligands for integrins and G-protein coupled receptors and offers a novel approach to ligand discovery allowing the comparative analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between various cell types. In addition, this work included the development of a novel method for the indirect amplification of a PNA library by amplification of a complementary DNA library hybridised to the PNA. The generation of 10,000 defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification of immobilised DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3875; 10,000 defined oligonucleotides) were used to validate the process, with sequences verified by selective hybridisation to a complementary DNA microarray with DNA sequencing demonstrating error rates of ca ≈ 0.2%. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA primers, while the DNA-arrays can be used as “factories” allowing specific DNA oligonucleotide pools to be generated with or without masking. This study also demonstrated a significant variance observed between the sequence frequencies found via Solexa sequencing compared to microarray analysis

Live-imaging rate-of-kill compound profiling for Chagas disease drug discovery with a new automated high-content assay

Chagas disease, caused by the protozoan intracellular parasite Trypanosoma cruzi, is a highly neglected tropical disease, causing significant morbidity and mortality in central and south America. Current treatments are inadequate, and recent clinical trials of drugs inhibiting CYP51 have failed, exposing a lack of understanding of how to translate laboratory findings to the clinic. Following these failures many new model systems have been developed, both in vitro and in vivo, that provide improved understanding of the causes for clinical trial failures. Amongst these are in vitro rate-of-kill (RoK) assays that reveal how fast compounds kill intracellular parasites. Such assays have shown clear distinctions between the compounds that failed in clinical trials and the standard of care. However, the published RoK assays have some key drawbacks, including low time-resolution and inability to track the same cell population over time. Here, we present a new, live-imaging RoK assay for intracellular T. cruzi that overcomes these issues. We show that the assay is highly reproducible and report high time-resolution RoK data for key clinical compounds as well as new chemical entities. The data generated by this assay allow fast acting compounds to be prioritised for progression, the fate of individual parasites to be tracked, shifts of mode-of-action within series to be monitored, better PKPD modelling and selection of suitable partners for combination therapy

Oligonucleotide sequences not seen by Solexa sequencing and their background-corrected average microarray intensities.

<p>Oligonucleotide sequences not seen by Solexa sequencing and their background-corrected average microarray intensities.</p

General sequences of microarray supported oligonucleotides and primer sequences.

<p>H = A, C, or T.</p

DNA gel electrophoresis.

<p>(<b>a</b>) PCR products from the 1, 10, 3,875, 10,000 oligonucleotide microarrays. (<b>b</b>) Products from 5 repeats of PCR from the 10,000 oligonucleotide array. (<b>c</b>) dsDNA-10,000 and dsDNA-3875 (left) and their EcoICRI digestion (right). (<b>d</b>) PCR amplification with two primers producing dsDNA-10,000-FAM and dsDNA-3,875-FAM and dsDNA-10-FAM (left) and asymmetric PCR with a single primer producing ssDNA-10,000-FAM and ssDNA-3,875-FAM (right).</p

The background corrected average intensities plotted versus the number of replicates.

<p>(<b>a</b>) The dsDNA-10-FAM library. (<b>b</b>) The ssDNA-3,875-FAM library. (<b>c</b>) The ssDNA-10,000-FAM library. Error bars indicate ± s.d.</p

Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution

Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA–fluorophore complexes would extend the utility of this technology, the identification of novel RNA–fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA–fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in <i>Escherichia coli</i>. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (<i>Z</i>)-4-(3,5-difluoro-4-hydroxy­benz­ylidene)-1,2-dimethyl-1<i>H</i>-imid­azol-5­(4<i>H</i>)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells