In vivo SELEX

May 8, 2024 • Bo Fu

Introduction

The concept of in vivo SELEX emerged as researchers sought to improve the clinical relevance and effectiveness of aptamers for therapeutic and diagnostic applications. Early studies demonstrated the feasibility of selecting aptamers in living organisms, showing that it was possible to recover and amplify sequences that bind specifically to target molecules within the organism.

Traditional SELEX is performed in vitro, where nucleic acid libraries are exposed to target molecules under controlled conditions. However, this approach does not account for the complex environment within living organisms, which can affect the binding properties and functionality of aptamers. In vivo SELEX was developed to overcome these limitations by selecting aptamers directly within the biological context where they are intended to function.

In vivo SELEX is conducted within the biological environment where the aptamers are intended to function, providing a more realistic selection pressure. This method considers the full complexity of living systems, including factors such as cellular uptake, metabolism, and interaction with endogenous molecules. Aptamers selected in vivo are likely to be more robust and effective under physiological conditions compared to those selected in vitro.

One of the primary challenges of in vivo SELEX is the complexity of the living organism's environment, which includes a myriad of interactions with other biomolecules, cells, and tissues. In vivo conditions increase the likelihood of non-specific binding, making it difficult to isolate high-affinity, specific aptamers. Efficiently delivering the nucleic acid library into the organism and recovering the sequences that bind to the target is technically challenging. Besides, working with living organisms, particularly higher animals, requires careful consideration of ethical and technical issues.

Over time, advancements in molecular biology techniques, such as improved methods for library delivery and recovery, have refined the in vivo SELEX process. In vivo SELEX represents a significant advancement in the field of aptamer selection, providing a powerful tool for developing aptamers with enhanced functionality and specificity in real-world biological settings. In vivo SELEX produces aptamers that are more likely to be effective in clinical and therapeutic applications due to their selection in a physiologically relevant context. The method increases the likelihood of identifying aptamers with high specificity and affinity for their targets under realistic biological conditions. In vivo SELEX has potential applications in targeted drug delivery, diagnostics, and as therapeutic agents, particularly for diseases where in vivo functionality is critical.

Process

In vivo SELEX usually consists of the following steps: (1) Library Preparation: A diverse library of nucleic acid sequences (RNA or DNA) is generated. (2) Delivery into Organism: The nucleic acid library is introduced into the living organism, typically through injection or another method of administration. (3) Target Binding: The library circulates within the organism, and sequences that bind to the target of interest (e.g., a specific protein or cell type) are selected by their interaction with the target in its natural environment. (4) Recovery: Bound sequences are recovered from the organism, often requiring tissue or fluid extraction. (5) Amplification: The recovered sequences are amplified using PCR (for DNA libraries) or reverse transcription followed by PCR (for RNA libraries). (6) Iteration: The process is repeated for several rounds to enrich for high-affinity, specific aptamers.