Riboswitch Mechanisms for Regulation of P1 Helix Stability.

Riboswitches are highly structured RNA regulators of gene expression. Although found in all three domains of life, they are particularly abundant and widespread in bacteria, including many human pathogens, thus making them an attractive target for antimicrobial development. Moreover, the functional versatility of riboswitches to recognize a myriad of ligands, including ions, amino acids, and diverse small-molecule metabolites, has enabled the generation of synthetic aptamers that have been used as molecular probes, sensors, and regulatory RNA devices. Generally speaking, a riboswitch consists of a ligand-sensing aptamer domain and an expression platform, whose genetic control is achieved through the formation of mutually exclusive secondary structures in a ligand-dependent manner. For most riboswitches, this involves formation of the aptamer's P1 helix and the regulation of its stability, whose competing structure turns gene expression ON/OFF at the level of transcription or translation. Structural knowledge of the conformational changes involving the P1 regulatory helix, therefore, is essential in understanding the structural basis for ligand-induced conformational switching. This review provides a summary of riboswitch cases for which ligand-free and ligand-bound structures have been determined. Comparative analyses of these structures illustrate the uniqueness of these riboswitches, not only in ligand sensing but also in the various structural mechanisms used to achieve the same end of regulating switch helix stability. In all cases, the ligand stabilizes the P1 helix primarily through coaxial stacking interactions that promote helical continuity.

1-L Transcription in Prion Diseases.

Understanding the pathogenesis and mechanisms of prion diseases can significantly expand our knowledge in the field of neurodegenerative diseases. Prion biology is increasingly recognized as being relevant to the pathophysiology of Alzheimer's disease and Parkinson's disease, both of which affect millions of people each year. This bioinformatics study used a theoretical protein-RNA recognition code (1-L transcription) to reveal the post-transcriptional regulation of the prion protein (PrPC). The principle for this method is directly elucidated on PrPC, in which an octa-repeat can be 1-L transcribed into a GGA triplet repeat RNA aptamer known to reduce the misfolding of normal PrPC into abnormal PrPSc. The identified genes/proteins are associated with mitochondria, cancer, COVID-19 and ER-stress, and approximately half are directly or indirectly associated with prion diseases. For example, the octa-repeat supports CD44, and regions of the brain with astrocytic prion accumulation also display high levels of CD44.

Solution-based biophysical characterization of conformation change in structure-switching aptamers.

Structure-switching aptamers have become ubiquitous in several applications, notably in analytical devices such as biosensors, due to their ease of supporting strong signaling. Aside from their ability to bind specifically with their respective target, this class of aptamers also undergoes a conformational rearrangement upon target recognition. While several well-studied and early-developed aptamers (e.g., cocaine, ATP, and thrombin) have been found to have this structure-switching property, the vast majority do not. As a result, it is common to try to engineer aptamers into switches. This proves challenging in part because of the difficulty in obtaining structural and functional information about aptamers. In response, we review various readily available biophysical characterization tools that are capable of assessing structure switching of aptamers. In doing so, we delve into the fundamentals of these different techniques and detail how they have been utilized in characterizing structure-switching aptamers. While each of these biophysical techniques alone has utility, their real power to demonstrate the occurrence of structural change with ligand binding is when multiple techniques are used. We hope that through a deeper understanding of these techniques, researchers will be better able to acquire biophysical information about their aptamer-ligand systems and accelerate the translation of aptamers into biosensors.

Mechanism of Dual-Site Recognition in a Classic DNA Aptamer.

Nucleic acid aptamers possess unique advantages in specific recognition. However, the lack of in-depth investigation into their dynamic recognition mechanisms has restricted their rational design and potential applications in fields such as biosensing and targeted therapy. We herein utilized enhanced sampling molecular dynamics to address affinities of adenosine monophosphate (AMP) to the dual binding sites in the DNA aptamer, focusing on the dynamic recognition mechanism and pathways. The present results indicate that in addition to the widely known intermolecular interactions, inequivalence of chemical environments of the two binding sites leads to slightly higher stability of AMP binding to the site proximal to the aptamer terminus. In the presence of two AMPs captured by the two sites, each binding free energy is enhanced. In particular, an additional hydrogen bond of AMP to A10 is introduced in the dual-site binding complex, which increases the binding energy from -4.25 ± 0.47 to -9.48 ± 0.33 kcal mol-1 in the site close to the loop. For the dual-site recognition process, the free energy landscape and minimum free energy pathway calculations elucidate the crucial role of electrostatic interactions between the AMP phosphate groups and Na+ ions in positively cooperative binding mechanisms.

Plug-and-play protein biosensors using aptamer-regulated in vitro transcription.

Molecular biosensors that accurately measure protein concentrations without external equipment are critical for solving numerous problems in diagnostics and therapeutics. Modularly transducing the binding of protein antibodies, protein switches or aptamers into a useful output remains challenging. Here, we develop a biosensing platform based on aptamer-regulated transcription in which aptamers integrated into transcription templates serve as inputs to molecular circuits that can be programmed to a produce a variety of responses. We modularly design molecular biosensors using this platform by swapping aptamer domains for specific proteins and downstream domains that encode different RNA transcripts. By coupling aptamer-regulated transcription with diverse transduction circuits, we rapidly construct analog protein biosensors and digital protein biosensors with detection ranges that can be tuned over two orders of magnitude and can exceed the binding affinity of the aptamer. Aptamer-regulated transcription is a straightforward and inexpensive approach for constructing programmable protein biosensors that could have diverse applications in research and biotechnology.

Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

A novel aptasensor based endogenous enzyme-powered DNA walker for ATP imaging in specific living cells.

Highly sensitive and specific imaging of ATP in living cells remains a challenge. Here, a novel aptasensor based endogenous enzyme-powered DNA walker for imaging ATP was proposed. The strategy leverages the highly expressed APE1 in tumor cells as the driving force of the DNA walker, achieving high sensitivity and superior imaging contrast. The method can detect ATP as low as 3.43 µM within 1 h. The approach can also effectively monitor intracellular ATP expression fluctuations and successfully differentiate between normal and cancer cells with high contrast.

Development of an Ultrasensitive Competitive Double-Enzyme Cascade Fluorescence Signal Amplification Method for Factor XIa Inhibitor Screening.

Coagulation factor XIa (FXIa) is associated with a low risk of bleeding and has been identified as an effective and safe target for the development of novel anticoagulant drugs. In this study, we established an ultrasensitive competitive dual-enzyme cascade signal amplification method for the quantitative analysis and screening of FXIa inhibitors. Due to the specific recognition of FXIa's active site by the aptamer AptE40, the AptE40-QDs-EK recognition probe modified with enterokinase (EK) and the aptamer AptE40, was attached to the MNPs-FXIa capture probe. When FXIa inhibitor was present, it competed with AptE40 for binding to FXIa, resulting in the detachment of AptE40-QDs-EK from MNPs-FXIa. After magnetic separation, the enterokinase of AptE40-QDs-EK in the supernatant hydrolyzed N-terminal hexapeptide of trypsinogen, leading to the production of a large amount of trypsin as part of the first-stage signal cascade amplification. Next, trypsin could hydrolyze the hexameric arginine peptide (RRRRRR, R6), leading to the dissociation of RQDs from the R6-RQDs signal probe; this resulted in a dramatic increase in the fluorescence intensity of the supernatant as the second-stage signal cascade was amplified. The feasibility of the method was investigated using the FXIa inhibitor aptamer FELIAP as a positive model drug. Furthermore, the method was applied to screen the FXIa inhibitors in Eupolyphaga sinensis Walker. Two fractions with more active anticoagulated ingredients were successfully identified and validated via the conventional method, and the results were consistent. The established method provides a key technique for the sensitive detection, high-throughput analysis, and screening of the FXIa inhibitors.

Unveiling Putative Excited State and Transmission of Binding Information in the Fluoride Riboswitch.

Riboswitches regulate downstream gene expression by binding to specific small molecules or ions with multiple mechanisms to transfer the binding information. In the case of the fluoride riboswitch, the transcription termination signal is conveyed through a transient excited state (ES). In this work, we performed conventional molecular dynamics (MD) simulations, totaling 180 µs, to obtain the ES structure and investigate the mechanism underlying information transmission in Mg2+/F- binding within the fluoride riboswitch aptamer. The Mg2+/F- binding pocket exhibits various conformations in its apo form. A series of ES structures were extracted from the MD trajectories of the apo form. The dynamics of the Mg2+/F- binding pocket influenced key pair A40-U48 in ES structures. The pathway connecting the binding pocket to the pair involves interactions between the phosphate groups of U7 and G8 and the nucleobases of G8-C47-U48. Our work presents a structural ensemble of the ES and elucidates a pathway for transferring Mg2+/F- binding information, thereby facilitating the understanding of how the holo-like apo state achieves transcriptional repression.

Unlocking precision in aptamer engineering: a case study of the thrombin binding aptamer illustrates why modification size, quantity, and position matter.

The thrombin binding aptamer (TBA) is a prototypical platform used to understand the impact of chemically-modified nucleotides on aptamer stability and target affinity. To provide structural insight into the experimentally-observed effects of modification size, location, and number on aptamer performance, long time-scale molecular dynamics (MD) simulations were performed on multiple binding orientations of TBA-thrombin complexes that contain a large, flexible tryptophan thymine derivative (T-W) or a truncated analogue (T-K). Depending on modification position, T-W alters aptamer-target binding orientations, fine-tunes aptamer-target interactions, strengthens networks of nucleic acid-protein contacts, and/or induces target conformational changes to enhance binding. The proximity and 5'-to-3' directionality of nucleic acid structural motifs also play integral roles in the behavior of the modifications. Modification size can differentially influence target binding by promoting more than one aptamer-target binding pose. Multiple modifications can synergistically strengthen aptamer-target binding by generating novel nucleic acid-protein structural motifs that are unobtainable for single modifications. By studying a diverse set of modified aptamers, our work uncovers design principles that must be considered in the future development of aptamers containing chemically-modified nucleotides for applications in medicine and biotechnology, highlighting the value of computational studies in nucleic acids research.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

Optimization of Exon-Skipping Riboswitches and Their Applications to Control Mammalian Cell Fate.

Mammalian riboswitches that can regulate transgene expression via RNA-small molecule interaction have promising applications in medicine and biotechnology, as they involve no protein factors that can induce immunogenic reactions and are not dependent on specially engineered promoters. However, the lack of cell-permeable and low-toxicity small molecules and cognate aptamers that can be exploited as riboswitches and the modest switching performance of mammalian riboswitches have limited their applications. In this study, we systematically optimized the design of a riboswitch that regulates exon skipping via an RNA aptamer that binds ASP2905. We examined two design strategies to modulate the stability of the aptamer base stem that blocks the 5' splice site to fine-tune the riboswitch characteristics. Furthermore, an optimized riboswitch was used to generate a mouse embryonic stem cell line that can be chemically induced to differentiate into myogenic cells by activating Myod1 expression and a human embryonic kidney cell line that can be induced to trigger apoptosis by activating BAX expression. The results demonstrate the tight chemical regulation of transgenes in mammalian cells to control their phenotype without exogenous protein factors.

pH-dependent binding of ATP aptamer to the target and competition strands: Fluorescent melting curve fitting study.

The pH varies in different tissues and organelles and also changes during some diseases. In this regard, the application of molecular switches that use a competition-based aptamer switch design in biological systems requires studying the thermodynamics of such systems at different pH values. In this work, we studied the binding of the classical ATP aptamer to ATP and competition strands under different pH and ionic conditions using fluorescent melting curve analysis. We have developed an original approach to processing source data from a PCR thermal cycler. It is based on constructing a thermodynamic model of the melting profile and the subsequent fit of experimental curves within this model. We have shown that this approach enables us to narrow the temperature region under study to the width of the melting region without a significant loss in the quality of the result. This impressively expands the application area of this approach compared to frequently used techniques that require mandatory measurement of the signal outside the melting region. The results obtained by the method showed that the thermodynamic parameters of the ATP aptamer and its duplexes with competition strands change depending on pH. Therefore, molecular switches that use a competition strand to the ATP aptamer may have a pH-dependent sensitivity that has not been previously considered. This should be taken into account for future rational design of similar systems.

Investigating Protein-Nucleic Acid Binding Interactions with Diethylpyrocarbonate Covalent Labeling-Mass Spectrometry.

Nucleic acids are important biomolecules that facilitate numerous cellular functions and have in recent years become promising candidates for treating disease. Consequently, there is a need for methods to characterize protein interactions with these molecules. Here, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) can provide structural information for protein-nucleic acid binding by characterizing the binding sites of two DNA aptamers specific to thrombin. Reductions in thrombin labeling are observed at the pair's binding interfaces. Furthermore, we find that binding of the aptamers causes changes in labeling at residues in the thrombin active site and known exosites for each aptamer, showcasing the sensitivity of DEPC CL-MS to significant allosteric changes.

Computational and experimental investigations of a novel aptamer targeting oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) induces the formation of atherosclerotic plaques. Apolipoprotein B100 (apoB100) is a crucial protein component in low-density lipoprotein (LDL), which includes oxLDL. The oxidation of amino acids and subsequent alterations in their structure generate oxLDL, which is a significant biomarker for the initial phases of coronary artery disease. This study employed molecular docking and molecular dynamics utilizing the MM/GBSA method to identify aptamers with a strong affinity for oxidized apoB100. Molecular docking and molecular dynamics were performed on two sequences of the aptamer candidates (aptamer no.11 (AP11: 5'-CTTCGATGTAGTTTTTGTATGGGGTGCCCTGGTTCCTGCA-3') and aptamer no.26 (AP26: 5'-GCGAACTCGCGAATCCAGAACGGGCTCGGTCCCGGGTCGA-3')), yielding respective binding free energies of -149.08 kcal/mol and -139.86 kcal/mol. Interaction modeling of the simulation revealed a strong hydrogen bond between the AP11-oxidized apoB100 complexes. In an aptamer-based gold nanoparticle (AuNP) aggregation assay, AP11 exhibits a color shift from red to purple with the highest absorbance ratio, and shows strong binding affinity to oxLDL, correlating with the simulation model results. AP11 demonstrated the potential for application as a novel recognition element in diagnostic methodologies and may also contribute to future advancements in preventive therapies for coronary artery disease.

Functional Aptamers In Vitro Evolution for Intranuclear Blockage of RNA-Protein Interaction.

Over the last 30 years, despite considerable research and endeavors aimed at harnessing aptamers as pharmaceutical molecules, the progress in developing aptamer-based drugs has been falling short of expectations. Sequential steps of affinity molecule acquisition and functional screening are typically required for discovering affinity-based macromolecule therapeutics, which can be time-consuming and limiting in candidate selection. Additionally, aptamers often necessitate tedious postselection modifications to overcome pharmacokinetic limitations, which usually impede the binding affinity. Herein, we propose a novel in vitro screening platform termed Functional Aptamers in vitro Evolution (FAIVE), which integrates affinity molecule acquisition with functional screening and introduces chemical diversity during the process. This platform aims to rapidly generate functional aptamers capable of binding to target proteins and regulating their functions. Illustrated by targeting intranuclear RNA-protein interactions involving HIV-1 Tat protein and TAR RNA, FAIVE demonstrates a selection of functional aptamers with significant intracellular blocking effects. The study also explores lipid nanoparticle delivery systems to enhance intracellular delivery efficiency, expanding aptamer targeting potential to broader intracellular and intranuclear domains. This study emphasizes the potential of FAIVE to expedite the development of aptamer-based drugs and facilitate the creation of more versatile and effective therapeutics.

In silico analysis of aptamer-RNA conjugate interactions with human transferrin receptor.

The human transmembrane protein Transferrin Receptor-1 is regarded as a promising target for the systemic delivery of therapeutic agents, particularly of nucleic acid therapeutics, such as short double stranded RNAs. This ubiquitous receptor is involved in cellular iron uptake, keeping intracellular homeostasis. It is overexpressed in multiple cancer cell types and is internalized via clathrin-mediated endocytosis. In previous studies, a human transferrin receptor-1 RNA aptamer, identified as TR14 ST1-3, was shown to be capable of effectively internalizing into cells in culture and to deliver small, double stranded RNAs in vitro and in vivo, via systemic administration. To understand, at the molecular level, the aptamer binding to the receptor and the impact of conjugation with the therapeutic RNA, a multi-level in silico protocol was employed, including protein-aptamer docking, molecular dynamics simulations and free energy calculations. The competition for the binding pocket, between the aptamer and the natural ligand human Transferrin, was also evaluated. The results show that the aptamer binds to the same region as Transferrin, with residues from the helical domain showing a critical role. Moreover, the conjugation to the therapeutic RNA, was shown not to affect aptamer binding. Overall, this study provides an atomic-level understanding of aptamer association to human Transferrin Receptor-1 and of its conjugation with a short model-therapeutic RNA, providing also important clues for futures studies aiming to deliver other oligonucleotide-based therapeutics via Transferrin Receptor.

Substrate-Switched Dual-Signal Self-Powered Sensing System Based on Dual-Nanozyme Activity of Bimetal-Doped CeO2 Nanospheres for Electrochemical Assay of Aflatoxin B1.

The long-term operation feature of enzymatic biofuel cell-based self-powered biosensor (EBFC-SPB) endows them with the potential to execute dual-signal biosensing without having to integrate an extra signal acquisition device. Herein, cobalt and manganese codoped CeO2 nanospheres (CoMn-CeO2 NSs) with glucose-oxidase-like and peroxidase-like activities have been developed as substrate-switched dual-channel signal transduction components in EBFC-SPB for a dual-signal assay of aflatoxin B1 (AFB1). The CoMn-CeO2 NSs modified with aptamer are anchored to a complementary DNA-attached bioanode of EBFC-SPB by base complementary pairing, which catalyze the glucose oxidation together with the glucose oxidase (GOx) on the bioanode. Once the AFB1 appears, CoMn-CeO2 NSs will be released from the bioanode due to the binding specificity of the aptamer, resulting in a decreased catalytic efficiency and the first declining stage of EBFC-SPB. Accompanied by the introduction of H2O2, the residual CoMn-CeO2 NSs on the bioanode switch to peroxidase-like activity and mediate the production of benzo-4-chlorohexadienone (4-CD) precipitate, which increases the steric hindrance and yields another declining stage of EBFC-SPB. By assessing the variation amplitudes during these two declining stages, the dual-signal assay of AFB1 has been realized with satisfying results. This work not only breaks ground in dual-signal bioassays but also deepens the application of nanozymes in EBFC-SPB.

Aptamer-Based Enforced Phosphatase-Recruiting Chimeras Inhibit Receptor Tyrosine Kinase Signal Transduction.

Aberrant phosphorylation of receptor tyrosine kinases (RTKs) is usually involved in tumor initiation, progression, and metastasis. However, developing specific and efficient molecular tools to regulate RTK phosphorylation remains a considerable challenge. In this study, we reported novel aptamer-based chimeras to inhibit the phosphorylation of RTKs, such as c-Met and EGFR, by enforced recruitment of a protein tyrosine phosphatase receptor type F (PTPRF). Our studies revealed that aptamer-based chimeras displayed a generic and potent inhibitory effect on RTK phosphorylation induced by growth factor or auto-dimerization in different cell lines and modulated cell biological behaviors by recruiting PTPRF. Furthermore, based on angstrom accuracy of the DNA duplex, the maximum catalytic radius of PTPRF was determined as ∼25.84 nm, providing a basis for the development of phosphatase-recruiting strategies. Taken together, our study provides a generic methodology not only for selectively mediating RTK phosphorylation and cellular biological processes but also for developing novel therapeutic drugs.

Blocker-SELEX: a structure-guided strategy for developing inhibitory aptamers disrupting undruggable transcription factor interactions.

Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges in modulating their protein interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs do not favor the use of traditional tools. Aptamers possess large molecular weights, expansive blocking surfaces and efficient cellular internalization, making them compelling tools for modulating TF interactions. Here, we report a structure-guided design strategy called Blocker-SELEX to develop inhibitory aptamers (iAptamers) that selectively block TF interactions. Our approach leads to the discovery of iAptamers that cooperatively disrupt SCAF4/SCAF8-RNAP2 interactions, dysregulating RNAP2-dependent gene expression, which impairs cell proliferation. This approach is further applied to develop iAptamers blocking WDR5-MYC interactions. Overall, our study highlights the potential of iAptamers in disrupting pathogenic TF interactions, implicating their potential utility in studying the biological functions of TF interactions and in nucleic acids drug discovery.