Fluorescences and Light-up RNA aptamers

Light-up RNA aptamers are fluorescent RNA molecules that become fluorescent upon binding to specific fluorophores. This concept emerged from the need for non-invasive, real-time imaging tools to study RNA molecules and their interactions within living cells. Traditional fluorescent tags like GFP (green fluorescent protein) are not suitable for RNA because they are protein-based^[1]. Thus, the development of RNA-based fluorescent tags (light-up RNA aptamers) became crucial for expanding the toolkit available for RNA visualization and functional studies.

The discovery of Spinach, the first light-up RNA aptamer, by Jaffrey and colleagues in 2011 marked a significant milestone. Spinach binds to a fluorophore called DFHBI and emits green fluorescence, mimicking GFP^[2]. Following Spinach, several other light-up aptamers were developed, including Mango, Broccoli, and Corn, each binding to different fluorophores and exhibiting different fluorescence properties^[3,4,5]. Researchers optimize light-up RNA aptamers for improved brightness, stability, and minimal background fluorescence. This involves iterative cycles of selection and mutation to enhance the aptamer’s performance for better performance in cellular environments^[6,7,8].

Fluorogenic mechanisms

Light-up RNA aptamers contain specific binding sites that interact with small-molecule fluorophores. These fluorophores are non-fluorescent or weakly fluorescent in the absence of the aptamer. Upon binding to the aptamer, the fluorophore undergoes a conformational change that enhances its fluorescence properties. This binding typically occurs through a combination of hydrogen bonding, Van der Waals interactions, and stacking interactions within the RNA’s three-dimensional structure.

Understanding their mechanism of action involves exploring how these aptamers interact with their fluorophores to produce fluorescence, the structural basis of their function, and the principles guiding their design and optimization^[10].

Twisted Intramolecular Charge Transfer (TICT)

TICT refers to a mechanism where the binding of a target induces a conformational change in the RNA aptamer, leading to the activation or enhancement of fluorescence through a twisted charge-transfer state. This state involves the separation of charge within the molecule, resulting in a geometry that promotes fluorescence.

Contact Quenching (CQ)

Contact quenching in light-up RNA aptamers involves the reduction or elimination of fluorescence due to the close physical proximity of a quencher molecule to the fluorophore. This process is highly dependent on the spatial arrangement and interaction between the RNA aptamer and its target.

In the absence of the target, the fluorophore may be in close proximity to a quencher moiety within the RNA aptamer, resulting in minimal fluorescence. Binding of the target induces a conformational change in the RNA aptamer that separates the fluorophore from the quencher. The increased distance between the fluorophore and quencher reduces the efficiency of non-radiative energy transfer, allowing the fluorophore to emit fluorescence. The efficiency of contact quenching and subsequent fluorescence recovery can be modulated by the nature of the target-aptamer interaction and the specific design of the aptamer.

Spirolactonization (SP)

Spirolactonization in light-up RNA aptamers involves the formation of a spirolactone ring structure upon binding to a target. This structural change enhances the fluorescence of the aptamer, providing a clear signal upon target recognition.

In the absence of the target, the RNA aptamer remains in a conformation that does not support spirolactone formation, leading to weak or no fluorescence. Binding of the target induces a conformational rearrangement in the aptamer, promoting the formation of a spirolactone ring. The formation of the spirolactone ring stabilizes the fluorophore in a fluorescent state, significantly enhancing its emission properties. The structural rigidity and planarity introduced by the spirolactone ring reduce non-radiative decay pathways, leading to a bright fluorescence signal.

Properties of Fluorophore-Aptamer pairs

Each light-up RNA aptamer is usually specific to a particular fluorophore. The specificity arises from the precise shape and chemical environment of the binding pocket within the RNA. The sequence and structure of the aptamer dictate the compatibility with the fluorophore, ensuring selective binding and fluorescence activation. Researchers focused on optimizing these aptamers for improved brightness, stability, and minimal background fluorescence. This included engineering the aptamers and fluorophores for better performance in cellular environments.

The table below lists several fluorescent small molecular-RNA pairings for which interaction patterns have been known through crystallographic studies or NMR. (The table only shows the representative individuals of each type of fluorescent small molecule. For more details, click on small molecule to jump to the corresponding view).

References