Advanced Small Molecule Aptamer Selection via Capture SELEX to Enhance Boronophenylalanine (BPA) Monitoring for BNCT Precision Medicine

Cancer treatment, particularly for head and neck cancers, presents numerous challenges due to the complexity of the disease and the limitations of conventional therapeutic approaches. Traditional treatments are often invasive, leading to severe side effects that significantly impact patients' quality of life. Additionally, the inability of many drugs to penetrate the blood-brain barrier further complicates the effective treatment of cancers in this region. These challenges highlight the need for advanced therapeutic strategies to precisely target deep-seated tumors while minimizing collateral damage to healthy tissues. In recent years, Boron Neutron Capture Therapy (BNCT) has emerged as a promising approach to addressing these challenges. BNCT utilizes boron-conjugated compounds, particularly Boronophenylalanine (BPA), which selectively accumulate in cancer cells. When exposed to neutron irradiation, these boron-containing compounds undergo nuclear reactions that release high-energy particles. These particles target and destroy malignant cells with high precision while sparing surrounding healthy tissues. This unique mechanism positions BNCT as a transformative technique for overcoming the limitations of traditional cancer therapies. Despite its advantages, the current BNCT protocol faces significant challenges. Monitoring boron concentrations during therapy heavily relies on techniques such as Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). However, these methods are time-consuming (often exceeding 25 minutes per sample) and lack the capability for real-time monitoring of BPA levels in whole blood, which hinders the adaptability and optimization of treatment protocols. Such limitations underscore the need for more efficient and precise methods to support BNCT. To address the challenges in monitoring Boronophenylalanine (BPA) during Boron Neutron Capture Therapy (BNCT), we developed a novel method to efficiently select highly specific aptamers. This method integrates three key components: capture SELEX, Next-Generation Sequencing (NGS), and Circular Dichroism (CD) spectroscopy. These steps collectively ensure aptamer specificity, binding efficiency, and mechanistic understanding, providing a robust framework for advancing BNCT monitoring and therapeutic applications. Capture SELEX represents the foundation of this method. Unlike traditional approaches that require immobilizing small molecules on hard substrates, our capture SELEX protocol avoids this limitation, streamlining the aptamer selection process. Including select aptamer for small molecule, Novel NGS strategy, and CD spectrum analysis. Using fructose-BPA as the target, the protocol facilitated the identification of aptamers capable of binding specific functional regions of the molecule. This process not only improves efficiency but also broadens the scope of molecules that can be targeted with aptamers, marking a significant advancement in small-molecule aptamer selection techniques. For small molecules, building on the aptamers identified through capture SELEX, Next-Generation Sequencing (NGS) was employed to analyze the aptamer pool. NGS provided high-resolution data, allowing the identification of sequences with strong specificity for distinct regions of fructose-BPA, including the fructose moiety, boron group, and amino acid components. The sequencing data were further analyzed to elucidate the binding preferences and affinities of the selected aptamers, offering insights into the molecular interactions at play. This innovative use of NGS extends beyond simple aptamer identification, enabling a detailed exploration of the aptamer-target relationship and supporting the design of more precise therapeutic tools. Finally, the specificity and binding efficiency of the selected aptamers were validated through Circular Dichroism (CD) spectroscopy. CD analysis confirmed the conformational changes in the aptamers upon binding to fructose-BPA, providing critical evidence of their functionality. By correlating CD spectroscopy results with NGS data, we identified aptamers that demonstrated strong and specific interactions with targeted regions of the molecule. This comprehensive approach underscores the novelty of combining spectroscopic validation with sequence analysis, ensuring the reliability and applicability of the selected aptamers. Together, these integrated methodologies—capture SELEX, NGS, and CD spectroscopy—represent a groundbreaking approach for selecting and validating aptamers tailored to small molecules like BPA. This work not only addresses the limitations of traditional monitoring techniques for BNCT but also establishes a foundation for real-time, precision medicine platforms, paving the way for more effective and adaptable cancer therapies. Figure 1