Frédéric Ducongé

The chemical instability of mRNA complicates its future clinical application, including issues surrounding its storage, handling, and potential for delivery to patients. Herein, we describe a chemical formulation, inspired by the dynamic multidimensional structure of a virus, with the potential to address many mRNA delivery limitations by enhancing stability, effective cytosolic delivery, and enabling the ability to target mRNA expression to a specific cell receptor. It consists of a liposomal core, stabilized by a metal organic framework, encapsulated within our nucleic acid nanocapsule formulation, ultimately imparting an enzymatic trigger to gate mRNA release and expression in cells, much as a virus is degraded by proteolytic enzymes under acidic conditions in the endosome to release its genetic cargo. We refer to this formulation as a liposomal metal organic framework-nucleic acid nanocapsule, wherein we show how a multilayered delivery carrier can use both pH and enzyme-specific triggers to gate efficient mRNA delivery to the cytosol of cells in vitro, as well as in vivo. Our system can achieve sustained expression from mRNA when stored at room temperature (∼14 days), mRNA structural integrity after >100 days at -20 °C, and therapeutic efficacy in an avian influenza in vitro model. In vivo, we show the expression of therapeutically relevant proteins and changes in the distribution of fluorescent proteins within a tumor microenvironment, observing more homogeneous expression using aptamers. This work provides an approach to mRNA delivery, which extends its shelf life while providing a route to receptor-specific delivery.

Decoration of aptamers with chemical modifications at the level of nucleobases grants access to alternative binding modes, which often result in improved binding properties. Most functional groups involved in such endeavours mimic the side chains of amino acids or are based on sp²-dominated moieties. While this approach has met undeniable success, trends in modern drug discovery seem to favor sp³-rich compounds over aromatic derivatives. Here, we report the use of a nucleotide modified with the three-dimensional, highly flexible cyclooctatetraene carboxylate (COTc). This nucleotide was engaged in an SELEX experiment against the biomarker PvLDH. Tightly binding aptamers were identified, which displayed dissociation constants in the low nM range, representing a significant improvement compared to previously identified cubamers. These modified aptamers clearly underscore the usefulness of COTc as a bioisostere replacement of aromatic moieties not only in small compounds but also in functional nucleic acids.

Decoration of aptamers with chemical modifications at the level of nucleobases grants access to alternative binding modes which often result in improved binding properties. Most functional groups involved in such endeavours mimic the side chains of amino acids or are based on sp2-dominated moieties. While this approach has met undeniable success, trends in modern drug discovery seem to favor sp3-rich compounds over aromatic derivatives. Here, we report the use of a nucleotide modified with the three dimensional, highly flexible cyclooctatetraene (COT). This nucleotide was engaged in a SELEX experiment against the biomarker PvLDH. Tightly binding aptamers, coined COTmers, were identified which displayed dissociation constants in the low nM range, representing a significant improvement compared to previously identified cubamers. COTmers clearly underscore the usefulness of COT as a bioisostere replacement of aromatic moieties not only in small compounds but also in functional nucleic acids.

Synucleinopathies, including dementia with Lewy bodies (DLB), Parkinson's disease (PD), and multiple system atrophy (MSA), are characterized by the presence of α-synuclein (α-syn) aggregates in the central nervous system. Recent evidence suggests that the heterogeneity of synucleinopathies may be partly explained by the fact that patients may have different α-syn fibrillar polymorphs with structural differences. In this study, we identify nuclease resistant 2′fluoro-pyrimidine RNA aptamers that can differentially bind to structurally distinct α-syn fibrillar polymorphs. Moreover, we introduce a method, AptaFOOT-Seq, designed to rapidly assess the affinity of a mixture of these aptamers for different α-SYN fibrillar polymorphs using next-generation sequencing. Our findings reveal that the binding behavior of aptamers can be very different when they are tested separately or in the presence of other aptamers. In this case, competition and cooperation can occur, providing a higher level of information, which can be exploited to obtain specific ‘footprints’ for different α-Syn fibrillar polymorphs. Notably, these footprints can distinguish polymorphs obtained from patients with PD, DLB or MSA. This result suggests that aptaFOOT-Seq could be used for the detection of misfolded or abnormal protein conformations to improve the diagnosis of synucleinopathies.

Dolichols are isoprenoid end-products of the mevalonate and 2C-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a cis-prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques.

Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G₂-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G₂-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.

Ovarian cancer is the deadliest gynecological cancer. With non-specific symptoms of the disease and the lack of effective diagnostic methods, late diagnosis remains the crucial hurdle of the poor prognosis. Therefore, development of novel diagnostic approaches are needed. The purpose of this study is to develop DNA-based aptamers as potential diagnostic probes to detect ovarian cancer biomarker Human epididymis protein 4 (HE4) in urine. HE4 is a protein overexpressed in ovarian cancer, but not in healthy or benign conditions. With high stability and diagnostic value for detection of ovarian cancer, urine HE4 appears as an attractive non-invasive biomarker. The high-affinity anti-HE4 DNA aptamers were selected through 10 cycles of High Fidelity Systematic Evolution of Ligands by EXponential enrichment (Hi-Fi SELEX), a method for aptamer selection based on digital droplet PCR. The anti-HE4 aptamers were identified using DNA sequencing and bioinformatics analysis. The candidate aptamer probes were characterized in urine for binding to HE4 protein using thermofluorimetry. Two anti-HE4 aptamers, AHE1 and AHE3, displayed binding to HE4 protein in urine, with a constant of dissociation in the nanomolar range, with K_d (AHE1) = 87 ± 9 nM and K_d (AHE3) aptamer of 127 ± 28 nM. Therefore, these aptamers could be promising tools for application in diagnostics and future development of urine tests or biosensors for ovarian cancer.

Although SELEX can identify high-affinity aptamers, Doped-SELEX is often performed post-selection for the identification of better variants. Starting from a partially randomized (doped) library derived from an already identified aptamer, this method can screen rapidly several thousand substitutions in order to identify those that can improve the binding of the aptamers. It can also highlight the positions that do not tolerate substitutions, which suggest they are crucial for the interaction of the aptamer with its target. High-throughput sequencing (HTS), also named next-generation sequencing (NGS), can dramatically improve this method by studying millions of sequences. This high number of sequences ensures a statistically robust analysis of variants even for those with a low frequency in the library. It can reduce the number of selection rounds and provide a more in-depth analysis of the positions that are crucial for the aptamer affinity. In this chapter, we provide a protocol to simultaneously study and improve an aptamer using Doped-SELEX and HTS analysis, including the design of the doped library, the selection, HTS, and analysis. This protocol could be useful to improve the affinity of an aptamer and to reduce its size as well as to improve ribozyme. Key words Aptamer Doped-SELEX High-throughput sequencing Next-generation sequencing

Micelle-forming amphiphilic drug conjugates were synthesized starting from a biologically active epipodophyllotoxin derivative which was covalently inserted in between a hydrophilic targeting spermine unit, and a hydrophobic stearyl chain. The amphiphilic drug conjugates were further assembled into the corresponding micelles and evaluated in vitro for the active targeting of tumor cells overexpressing the polyamine transport system.