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Experimentos Húmedo-Seco-Húmedo para Aislamiento Simultáneo de Aptámeros e Identificación de Dominios de Unión

Keyi Hu¹, Yajing Gao¹, Yu Zhang¹

¹Department of Chemistry, Capital Normal University, Xisanhuan North Road 105, Beijing 100048, China.

Analytical chemistry

|February 6, 2026

Resumen

Este estudio presenta un novedoso método húmedo-seco-húmedo para aislar simultáneamente aptámeros de unión a rapamicina e identificar sus dominios de unión críticos (CBD). Esta estrategia eficiente evita el cribado de afinidad tradicional, permitiendo el descubrimiento y caracterización rápidos de aptámeros.

Área de la Ciencia:

Biotecnología
Biología Molecular
Biología Química

Sus antecedentes:

La identificación de los dominios de unión críticos (CBD) de los aptámeros generalmente requiere un cribado de afinidad exhaustivo.
Los métodos actuales para el aislamiento de aptámeros y la identificación de CBD a menudo consumen mucho tiempo y son laboriosos.

Objetivo del estudio:

Desarrollar y validar una estrategia experimental eficiente de húmedo-seco-húmedo para el aislamiento simultáneo de aptámeros y la identificación de sus CBD.
Aplicar este método para el aislamiento de aptámeros de unión a rapamicina y la caracterización de su CBD.

Principales métodos:

Se realizó un cribado SELEX (Evolución Sistemática de Ligandos por Enriquecimiento Exponencial) de 15 rondas para el enriquecimiento inicial de la biblioteca.
El análisis de datos de secuenciación de alto rendimiento, incluido el ensamblaje K-mer, RBM (Restricted Boltzmann Machine) y simulaciones mFold, identificó motivos estructurales clave de aptámeros (3S3L y 3S3L-A).
Se llevó a cabo un segundo SELEX de captura (6 rondas) en una biblioteca confinada por estructura, seguido de secuenciación para identificar aptámeros de alta afinidad y sus CBD.

Principales resultados:

El estudio aisló con éxito aptámeros de unión a rapamicina con constantes de disociación nanomolar y alta especificidad.
Se identificó un CBD de 11 nucleótidos y las mutaciones dentro de este dominio confirmaron su papel crítico en la unión.
Se desarrolló un sensor de fluorescencia de desplazamiento de hebras, logrando límites de detección nanomolar para rapamicina en suero humano.

Conclusiones:

La estrategia húmedo-seco-húmedo desarrollada ofrece un enfoque eficiente y simultáneo para el aislamiento de aptámeros y la identificación de CBD.
Este método reduce significativamente la necesidad de pruebas de afinidad laboriosas de numerosas secuencias.
El enfoque es potencialmente aplicable al descubrimiento y caracterización de aptámeros para diversos objetivos moleculares.

Palabras clave:

aptámeros rapamicina dominios de unión críticos cribado de afinidad biotecnología biología molecular biología química SELEX secuenciación de alto rendimiento sensores de desplazamiento de hebras

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