Beatrix Suess

Accurate reverse transcription quantitative PCR (RT-qPCR)-based quantification of circulating microRNAs (miRNAs) is limited by workflow-dependent bias arising from RNA extraction, enzymatic cDNA synthesis, amplification, matrix-derived inhibition, and sequence-dependent behavior of short RNA targets. Conventional exogenous spike-ins monitor technical process variation but are often selected without considering target-specific physicochemical properties such as GC content and predicted 3′-end structure. Here, we introduce a structure-guided framework for selecting and designing exogenous spike-in controls in small RNA quantification workflows. Native, sequence-modified, and newly engineered synthetic spike-ins were evaluated across no-extraction, pre-extraction, and post-extraction conditions, including an ethanol inhibition challenge. Secondary-structure prediction and calibration-curve-based quantification were combined with workflow-resolved coefficient of variation (CV) and ΔCq analyses. Control performance varied with the target miRNA, assay configuration, and workflow condition, with significant control, workflow, or interaction effects in selected target−assay combinations. Direct modification of predicted 3′-end structure revealed context-dependent effects on normalization precision, including a significant variant-dependent difference under the ethanol challenge, and circular dichroism (CD) spectroscopy of the hsa-miR-146a-5p variant set provided experimental support for the predicted stability differences. Finally, four engineered spike-ins spanning contrasting GC/ΔG profiles were evaluated as proof-of-concept controls for a target-informed selection framework. This study provides a measurement-focused strategy for technical internal-standard design and supports assay- and workflow-specific validation of exogenous spike-in controls in complex biofluid workflows.

Synthetic riboswitches provide protein-independent, modular control of gene expression, yet selecting aptamers that reliably couple ligand binding to regulatory switching remains challenging. Here, we identify and mechanistically characterise G12, a doxycycline-binding aptamer with remarkably high regulatory performance in yeast and human cells. We provide evidence that RNA Capture-SELEX efficiently enriches aptamers with ligand-responsive conformational switching. We compared conventional SELEX and RNA Capture-SELEX using the same starting library followed by NGS analysis and in vivo screening, which led to the identification of G12. G12 binds doxycycline with low-nanomolar affinity and strict discrimination against close derivatives, thus enabling high-dynamic-range riboswitch control of translation in yeast and splicing in human cells. Single-molecule force spectroscopy with optical tweezers revealed that doxycycline stabilises a folding intermediate independent of the closing stem P1, which primarily acts as a scaffold for correct aptamer folding. Mutational analysis and chemical probing identified tertiary contacts between loops L2 and L3 in this intermediate state. Stopped-flow fluorescence spectroscopy further supported a two-step binding mechanism consistent with efficient regulatory switching. Together, these findings deepen our understanding of regulatory aptamer selection and function and expand the synthetic biology toolbox with a high-performance doxycycline-responsive riboswitch.

RNA devices, including riboswitches, aptazymes, and RNA-based biosensors, have become essential components in synthetic and molecular biology. These systems make use of RNA’s modularity and structural diversity to build programmable tools for sensing, regulation, and cellular computation. To drive these systems, RNA aptamers usually need to undergo a conformational change upon ligand binding. However, the limited availability of such aptamers has restricted the development and application of RNA devices, particularly in mammalian systems. Traditional selection methods often prioritize high-affinity binding, resulting in a scarcity of conformation-switching aptamers. Here, we addressed this limitation by selecting a caffeine-binding aptamer with RNA Capture-SELEX and in vivo screenings. This aptamer functioned as a modular regulator of riboswitches and ribozymes in Saccharomyces cerevisiae and mammalian cells. Through new optofluidic screenings, we overcame throughput and sequence-function challenges inherent in ribozyme screening for mammalia. Additionally, a straightforward grafting method transformed the aptamer into a fluorogenic iSpinach aptasensor. This work demonstrates the successful selection of a modular and communicating aptamer for a challenging target like caffeine and establishes robust strategies for their modular integration into diverse RNA platforms. These strategies pave the way for broadening the repertoire of aptamers and expanding the potential of RNA-based synthetic biology.

RNA aptamers targeting bacterial repressors show significant promise in synthetic biology. We determined the crystal structure of the SELEX-derived aptamer Dap1 in complex with its target, the GntR/HutC repressor family member DasR. In the complex, two monomers of Dap1 individually sequester the two DNA-binding domains (DBDs) of the dimeric repressor. The DBDs of DasR interact with Dap1 in a highly similar fashion to what is observed in GntR–dsDNA complexes. This also holds true at the level of single amino acids, and identical arginine residues play crucial roles in both RNA and DNA recognition. The structure shows how DasR binding to Dap1 competes with DasR binding to its operator dre sites and, hence, how Dap1 is able to induce DasR-controlled gene transcription in vivo. A structure-informed Dap1 aptamer pool randomized at only four positions allowed for the identification of the novel aptamer Dap1m1 that binds and de-represses the DasR homologous protein NagR instead of DasR. This shows that the complex structure can serve as a blueprint for the design of novel aptamer–protein interaction pairs, opening up new venues for the generation of aptamers directed against other GntR/HutC family members and possibly also against other repressor classes.

The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations that place no excessive metabolic burden on the host cell. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5′ UTR of a messenger RNA, these devices can regulate translation initiation upon specific binding of one or both ligands without the need for additional auxiliary factors. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we improved the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near-digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment-driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes.

Synthetic riboswitches offer a versatile and protein-independent solution for conditional gene regulation. They consist of a regulatory domain and an aptamer domain that binds a specific ligand, with their performance largely depending on the ability of the aptamer to control the regulatory domain. Expanding the range of synthetic riboswitches therefore requires the discovery and characterization of new regulatory aptamers. In the present study, we identified a doxycycline-binding aptamer with outstanding regulatory properties in both yeast and human cells which are based on a unique structural dynamic upon ligand binding. Single-molecule force spectroscopy revealed that doxycycline binding strongly stabilizes an intermediate aptamer conformation offering mechanistic insights into its function. The identification of the aptamer through a combination of parallel SELEX and subsequent in vivo screening in yeast, also provided valuable insights into selection dynamics and the first proof for the effectiveness of RNA Capture-SELEX in aptamer selection. Together, the presented data deepen our understanding of regulatory aptamer selection and functionality while adding a high-performing doxycycline-responsive aptamer to the synthetic biology toolbox.

Synthetic riboswitches have undergone great development in the past decade, evolving into valuable regulatory tools. Operating entirely at the RNA level and independently of auxiliary proteins, they offer a promising alternative to protein-based systems such as TetON/OFF or CRISPR-Cas. As compact, modular RNA elements they unite sensing and regulatory functions within a single molecule, giving them the advantages of high modularity, portability and low metabolic burden. Here, we explore the unique features of synthetic riboswitches, highlight key applications, assess current bottlenecks and limitations and put them in context with emerging solutions, to emphasise the potential of synthetic riboswitches.

Recently, a novel tobramycin-responsive riboswitch was developed by a combination of Capture-SELEX and in vivo screening. This riboswitch regulates translation initiation in eukaryotes with a high dynamic range and remarkable ligand affinity and selectivity. Its secondary structure differs from all previously described aminoglycoside-binding RNA motifs, suggesting a novel mode of ligand recognition. To provide a structural basis for the remarkable regulatory efficiency and ligand selectivity of this riboswitch, we investigated its structure in complex with its cognate ligand tobramycin by high-resolution solution nuclear magnetic resonance spectroscopy. The structure of the complex reveals a novel structural organization for an aminoglycoside binding motif with a unique pattern of intermolecular hydrogen bonds and electrostatic interactions between the RNA and functional groups of all three rings of the ligand. In contrast to other aminoglycoside binding motifs, ligand binding of the tobramycin riboswitch is coupled with the formation of an extensive network of noncanonical RNA–RNA interactions, rationalizing the high ligand affinity of this small hairpin RNA. Comparison with the free form of the RNA shows that the latter is much less compact, lacking many RNA–RNA interactions, in particular in the bulge regions, thereby immediately providing a rationale for the exceptional switching efficiency of this synthetic riboswitch.

The application of synthetic riboswitches or aptamer-based biosensors for the monitoring of engineered metabolic pathways greatly depends on a high degree of target molecule specificity. Since metabolic pathways include close derivatives that often differ only in single moieties, the binding specificity of aptamers utilized for these systems has to be high. In the present study, we selected an RNA aptamer that is highly specific in its binding to homoeriodictyol while discriminating its close derivatives eriodictyol and naringenin. This high degree in specificity was achieved through three consecutive SELEX approaches while the selection parameters were adjusted and refined from one to the next. The adjustments along the process, with the selection outcome and next-generation sequencing analysis of the selection rounds, led to valuable insights into the stringency necessary to facilitate target specificity in aptamers obtained from SELEX. From the third selection, we obtained a highly binding specific aptamer and examined its structure and binding properties. Overall, our results connect the importance of selection stringency with SELEX outcome and aptamer specificity while providing a highly selective, homoeriodictyol-binding RNA aptamer.