Development and validation of AI/ML derived splice-switching oligonucleotides.

Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFß pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.

An In Vitro Selection Platform to Identify Multiple Aptamers against Multiple Cell-Surface Markers Using Ligand-Guided Selection.

Aptamer ligand discovery against multiple molecules expressed on whole cells is an essential component in molecular tool development. However, owing to their intrinsic structural characteristics, cell-surface receptors have proven to be challenging targets in ligand discovery. Several variants to systematic evolution of ligands by exponential enrichment (SELEX) have been introduced to address the ″target problem″ for aptamer screening. To this end, we introduced a variant of SELEX, termed ligand-guided selection (LIGS), to identify highly specific aptamers against complex cell-surface markers in their native state. So far, the application of LIGS has been aimed at identifying aptamers against the most dominant receptors on the cell surface. Here, we report that LIGS can be expanded to identify two receptors on the same cell surface, paving the way to generate a multiplexed ligand discovery platform based on SELEX-targeting membrane receptors in their native functional state. Using CD19 and CD20 expressed on Toledo cells as a model system, multiple aptamer families were evolved against Toledo cells. We then utilized two monoclonal antibodies (mAbs) against CD20 and CD19 to selectively partition specific aptamers against CD19 and CD20. Following biochemical characterization, we introduce two specific aptamers against CD19 and two specific aptamers against CD20 with high affinity. Multi-target LIGS, as reported here, demonstrates a successful combinatorial approach for nucleic acid library screening to generate multiple artificial nucleic acid ligands against multiple receptors expressed on a single cell.

Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3ε.

Here we are reporting, for the first time, a ligand-guided selection (LIGS) experiment using an artificially expanded genetic information system (AEGIS) to successfully identify an AEGIS-DNA aptamer against T cell receptor-CD3ε expressed on Jurkat.E6 cells. Thus, we have effectively combined the enhanced diversity of an AEGIS DNA library with LIGS to develop a superior screening platform to discover superior aptamers. Libraries of DNA molecules from highly diversified building blocks will provide better ligands due to more functional diversity and better-controlled folding. Thus, a DNA library with AEGIS components (dZ and dP) was used in LIGS experiments against TCR-CD3ε in its native state using two clinically relevant monoclonal antibodies to identify an aptamer termed JZPO-10, with nanomolar affinity. Multiple specificity assays using knockout cells, and competition experiments using monoclonal antibodies utilized in LIGS, show unprecedented specificity of JZPO-10, suggesting that the combination of LIGS with AEGIS-DNA libraries will provide a superior screening platform to discover artificial ligands against critical cellular targets.

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines.

Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called "Ligand-Guided-Selection" (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.

Ligand-guided selection of aptamers against T-cell Receptor-cluster of differentiation 3 (TCR-CD3) expressed on Jurkat.E6 cells.

We recently introduced a screening technology termed ligand-guided selection, (LIGS), to selectively identify target-specific aptamers from an evolved cell-SELEX library. Cell-SELEX utilizes a large combinatorial single-stranded oligonucleotide library and progressively selects DNA ligands against whole cells with variable DNA-binding affinities and specificities by repeated rounds of partition and amplification. LIGS exploits the partition step and introduces a secondary, pre-existing high-affinity monoclonal antibody (mAb) ligand to outcompete and elute specific aptamers towards the binding target of the antibody, not the cell. Here, using anti-CD3ε mAb against the cluster of differentiation 3 (CD3ε), as the guiding ligand against one of the domains of the T-cell Receptor (TCR) complex expressed on Jurkat.E6 cells, we discovered three specific aptamers against TCR complex expressed on an immortalized line of human T lymphocyte cells. In sum, we demonstrate that specific aptamers can be identified utilizing an antibody against a single domain of a multidomain protein complex in their endogenous state with neither post- nor pre-SELEX protein manipulation.

Ligand Guided Selection (LIGS) of Artificial Nucleic Acid Ligands against Cell Surface Targets.

With the success of RNA-based therapeutic drugs, the demand has increased for sophisticated nucleic-acid-based targeting agents. Nucleic acid aptamers (NAAs), in this regard, represent a suitable class of molecules with synthetic versatility. Aptamers are composed of single-stranded RNA/DNA/XNA molecules, which can be identified using a method called systematic evolution of ligands by exponential enrichment (SELEX) against any molecule. This Spotlight summarizes the recent introduction of ligand guided selection (LIGS), which will permit the identification of a wide range of functional aptamers against complex targets such as cell surface receptors while maintaining their native functional state. Aptamers identified from LIGS will allow researchers to develop aptamers in biomedicine as low-cost, stable therapeutic agents and diagnostic molecules or biochemical devices.

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands.

To discover DNA ligands against a predetermined receptor protein complex, we introduce a comprehensive version of ligand-guided selection (LIGS). LIGS is, itself, a variant of systematic evolution of ligands by exponential enrichment (SELEX). Herein, we have optimized LIGS to identify higher affinity aptamers with high specificity. In addition, we demonstrate the expandability of LIGS by performing specific aptamer elution at 25°C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries obtained through Illumina high-throughput (HT) DNA sequencing were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data in this report introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape.

The role of G-quadruplex structures of LIGS-generated aptamers R1.2 and R1.3 in IgM specific recognition.

Exploiting a variant of SELEX called "Ligand-Guided Selection" (LI-GS), we recently identified two novel truncated G-rich aptamers, called R1.2 and R1.3, specific for membrane-bound IgM (mIgM), the hallmark of B cells. Herein, the conformational behaviour of these aptamers has been analysed by multiple biophysical methods. In order to investigate their functional secondary structures, these studies have been carried out in pseudo-physiological buffers mimicking different cellular environments. Both aptamers proved to be highly polymorphic, folding into stable, unimolecular G-quadruplex structures in K+-rich buffers. In turn, in buffered solutions containing Na+/Mg2+ ions, R1.2 and R1.3 formed mainly duplex structures. Remarkably, these aptamers were able to effectively bind mIgM on B-cell lymphoma exclusively in the presence of potassium ions. These findings demonstrate the key role of G-quadruplex folding in the molecular recognition and efficient binding of R1.2 and R1.3 to mIgM expressed in lymphoma and leukemia cells, providing a precious rational basis for the design of effective aptamer-based biosensors potentially useful for the detection of cancer-relevant biomarkers.

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells.

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.

Systematic optimization and modification of a DNA aptamer with 2'-O-methyl RNA analogues.

Nucleic acid aptamers (NAAs) are short synthetic DNA or RNA molecules that specifically fold into distinct three-dimensional structures able to specifically recognize a target. While NAAs show unprecedented promise in a variety of applications, including sensing, therapeutics and diagnostics, one major limitation involves the lack of stability towards omnipresent nucleases. Therefore, we herein report a systematic truncation and incorporation of 2'-O-methyl bases to a DNA aptamer, which results in increased stability without affecting affinity. One of the newly designed analogues is stable up to 24 hours, demonstrating that 2'-O-methyl RNA is an attractive modification to DNA aptamers, especially when therapeutic applications are intended.

Ligand-Guided Selection of Target-Specific Aptamers: A Screening Technology for Identifying Specific Aptamers Against Cell-Surface Proteins.

We report on a new strategy for identifying highly specific aptamers against a predetermined epitope of a target. Termed "ligand-guided selection" (LIGS), this method uniquely exploits the selection step, the core of SELEX (Systematic Evolution Exponential enrichment). LIGS uses a naturally occurring stronger and highly specific bivalent binder, an antibody (Ab) interacting with its cognate antigen to outcompete specific aptamers from a partially enriched SELEX pool, as a strategy. We demonstrate the hypothesis of LIGS by utilizing an Ab binding to membrane-bound Immunoglobulin M (mIgM) to selectively elute aptamers that are specific for mIgM from a SELEX pool that is partially enriched toward mIgM expressing Ramos cells. The selected aptamers show specificity toward Ramos cells. We identified three aptamer candidates utilizing LIGS that could be outcompeted by mIgM Ab, demonstrating that LIGS can be successfully applied to select aptamers from a partially evolved cell-SELEX library, against predetermined receptor proteins using a cognate ligand. This proof-of-concept study introduces a new biochemical-screening platform that exploits the binding of a secondary stronger molecular entity to its target as a partition step, to identify highly specific artificial nucleic acid ligands.

Cited3 activates Mef2c to control muscle cell differentiation and survival.

Vertebrate muscle development occurs through sequential differentiation of cells residing in somitic mesoderm - a process that is largely governed by transcriptional regulators. Our recent spatiotemporal microarray study in zebrafish has identified functionally uncharacterized transcriptional regulators that are expressed at the initial stages of myogenesis. cited3 is one such novel gene encoding a transcriptional coactivator, which is expressed in the precursors of oxidative slow-twitch myofibers. Our experiments placed cited3 into a gene regulatory network, where it acts downstream of Hedgehog signaling and myoD/myf5 but upstream of mef2c. Knockdown of expression of cited3 by antisense morpholino oligonucleotides impaired muscle cell differentiation and growth, caused muscle cell death and eventually led to total immotility. Transplantation experiments demonstrated that Cited3 cell-autonomously activates the expression of mef2c in slow myofibers, while it non-cell-autonomously regulates expression of structural genes in fast myofibers. Restoring expression of cited3 or mef2c rescued all the cited3 loss-of-function phenotypes. Protein truncation experiments revealed the functional necessity of C-terminally conserved domain of Cited3, which is known to mediate interactions of Cited-family proteins with histone acetylases. Our findings demonstrate that Cited3 is a critical transcriptional coactivator functioning during muscle differentiation and its absence leads to defects in terminal differentiation and survival of muscle cells.