Artificial Dynamic RNA Structure Ensemble-Guided Rational Design of a Universal RNA Aptamer-Based Sensing Tag

Academic Background

RNA molecules play versatile roles in both natural and synthetic biological systems, with their functions strongly dependent on their dynamic and specific three-dimensional structures. RNA secondary and tertiary structures, such as hairpins, pseudoknots, and multiway junctions, are key “building blocks” for understanding RNA functions. However, most existing RNA structure studies are based on static structures, while many RNA-mediated processes involve conformational changes. Therefore, the description of dynamic structural ensembles can capture the evolutionary conservation patterns of RNA and aid in its expansion design. In recent years, fluorogenic RNA aptamers (FRAPTs) have made significant progress in biological applications, but their design lacks a deep understanding of dynamic structural ensembles and universal design principles. To overcome these limitations, researchers developed an artificial dynamic RNA structure ensemble called “SSPepper” and designed a universal fluorogenic RNA biosensing tag, “SSPepper-Apt,” for real-time imaging of small molecule metabolites and proteins. It also demonstrated excellent compatibility in CRISPR-mediated genomic imaging and gene editing.

Source of the Paper

The paper was co-authored by Jianing Hou, Pei Guo, Junyan Wang, Da Han, and Weihong Tan, affiliated with the Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, and the Hangzhou Institute of Medicine, Chinese Academy of Sciences. The paper was published on December 20, 2024, in PNAS (Proceedings of the National Academy of Sciences) under the title Artificial Dynamic Structure Ensemble-Guided Rational Design of a Universal RNA Aptamer–Based Sensing Tag.

Research Process

1. Design and Development of the Artificial Dynamic RNA Structure Ensemble “SSPepper”

To develop a universal FRAPT sensor, the researchers selected the fluorogenic RNA aptamer “Pepper,” which has well-defined structural information, as the foundation. By introducing motif mutations, the researchers created a dynamic structure ensemble called “SSPepper,” whose energy landscape was designed to include complex fluorescent conformations. The specific design steps are as follows:

1. Establishing the Correct Secondary Structure: Based on the high-resolution structure of the Pepper-HBC complex, the secondary structure of the artificial dynamic structure ensemble was established.
2. Adjusting Loop Stability: Since the region of FRAPT (Pepper) that binds to the fluorophore cannot be modified, the researchers selected representative motifs (helices and apical loops) to adjust the proportion of fluorescent conformations.
3. Introducing Slipping Stems: By modifying the length of the slipping stems, the researchers enhanced their suitability for various live-cell imaging applications.

Through these steps, the researchers dissected Pepper into three regions: the key regulatory domain (blue), the driving element (yellow), and the unchanged sequence (gray). The key regulatory domain was hypothesized to initiate structural changes at the HBC binding site, while the non-conserved P1 helix of Pepper was redesigned with slipping stems of varying lengths.

2. In Vitro Characterization of SSPepper-Apt

To verify the portability, scalability, and composability of SSPepper-Apt, the researchers seamlessly inserted the core sequences of various target-binding RNA elements, including theophylline aptamer, GTP aptamer, ATP aptamer, guanine riboswitch, SAM-III riboswitch, bovine immunodeficiency virus Tat peptide-targeting RNA, and thrombin aptamer. Through a series of experiments, the researchers validated the performance of SSPepper-Apt with different targets and found that it exhibited a wide dynamic operating range and excellent selectivity.

3. Application of SSPepper-Apt in Living Cells

The researchers further applied SSPepper-Apt in living cells, successfully monitoring the dynamic changes of theophylline and S-adenosylmethionine (SAM) and visualizing the localization of peptides and proteins. Additionally, SSPepper-Apt demonstrated excellent compatibility with the CRISPR-Cas9 system and could be used to predict gene editing efficiency.

Main Results

1. Design and Development of SSPepper: By introducing slipping stems and regulatory loops, the researchers successfully created a dynamic structure ensemble called “SSPepper,” whose proportion of fluorescent conformations could be finely tuned through loop stability.
2. In Vitro Characterization of SSPepper-Apt: SSPepper-Apt exhibited a wide dynamic operating range and excellent selectivity with different targets, enabling the detection of small molecule metabolites, peptides, and proteins.
3. Application of SSPepper-Apt in Living Cells: SSPepper-Apt successfully monitored the dynamic changes of theophylline and SAM in living cells and visualized the localization of peptides and proteins. Moreover, SSPepper-Apt demonstrated excellent compatibility with the CRISPR-Cas9 system and could be used to predict gene editing efficiency.

Conclusion

By developing the artificial dynamic RNA structure ensemble “SSPepper,” the researchers successfully designed a universal fluorogenic RNA biosensing tag, “SSPepper-Apt.” This tag is scalable, portable, composable, and reliable, enabling real-time imaging of small molecule metabolites and proteins. It also demonstrated excellent compatibility in CRISPR-mediated genomic imaging and gene editing. This research provides a universal approach for constructing functional RNA systems, avoiding the laborious process of sequence combination and expanding the application scope of synthetic biology tools.

Research Highlights

1. Design of Dynamic Structure Ensemble: By introducing slipping stems and regulatory loops, the researchers successfully created a dynamic structure ensemble called “SSPepper,” whose proportion of fluorescent conformations could be finely tuned through loop stability.
2. Development of a Universal Sensing Tag: SSPepper-Apt exhibited a wide dynamic operating range and excellent selectivity, enabling the detection of small molecule metabolites, peptides, and proteins.
3. Live-Cell Imaging and Gene Editing: SSPepper-Apt successfully monitored the dynamic changes of theophylline and SAM in living cells and visualized the localization of peptides and proteins. Additionally, SSPepper-Apt demonstrated excellent compatibility with the CRISPR-Cas9 system and could be used to predict gene editing efficiency.

Research Significance

This research provides a universal approach for constructing functional RNA systems, avoiding the laborious process of sequence combination and expanding the application scope of synthetic biology tools. SSPepper-Apt not only holds significant value in basic research but also has broad application prospects in biomedical applications, such as real-time monitoring of intracellular metabolites, protein localization, and prediction of gene editing efficiency.