Potenciación de la intercalación de porfirina-DNA: Explorando nuevas porfirinas como intercalante de DNA para diagnóstico en PCR en tiempo real
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Fecha
2024
Autores
Valencia Mejía, Laura Melisa
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
La eficacia de las porfirina en diversas aplicaciones biomédicas, como biomarcadores, sensores y fotosensibilizadores, está bien establecida. El estudio de las interacciones del ADN con moléculas de bajo peso molecular, incluidas las porfirinas, se ha consolidado como una disciplina autónoma y en rápido desarrollo. Estos estudios han revelado resultados prometedores en la terapia génica, especialmente en el tratamiento de pacientes con cáncer.
Los modos de unión de las porfirinas al ADN son variados, incluyendo la intercalación, la unión a los surcos menor y mayor, la formación de complejos externos y la unión a cuadruplexos G de ADN. Dado el éxito comprobado de estos compuestos, la exploración de nuevas porfirinas no solo tiene el potencial de mejorar las técnicas existentes, sino también de impulsar el desarrollo de tratamientos génicos innovadores y el diseño de fármacos más precisos y eficaces.
En este contexto, se hace evidente la necesidad de desarrollar sondas moleculares más accesibles y eficientes para el diagnóstico de enfermedades infecciosas y genéticas a través de PCR en tiempo real. El uso de compuestos fluorescentes como las porfirinas permite reducir los costos y superar la escasez de materiales convencionales. De 23 porfirinas analizadas, 11 mostraron una fluorescencia adecuada. Entre estas, la 5,10,15,20‑Tetrakis(4‑carboxyphenyl) porphyrininato‑Niquel resultó ser la más fiable, sugiriendo su potencial para reemplazar al Sybr Green en la PCR en tiempo real.
La porfirina número 8, FA02-Ni (5,10,15,20‑Tetrakis(4‑carboxyphenyl) porphyrininato‑Niquel), se destacó en las pruebas de fluorescencia, mostrando una emisión constante, particularmente en la cadena doble de ADN, que era nuestro foco principal. Aunque la porfirina número 7 5,10,15,20‑Tetrakis(4‑carboxyphenyl) porphyrini mostró inicialmente altas intensidades y parecía prometedora en la curva melting, su comportamiento no corresponde al de una sonda intercalante tradicional. En contraste, la FA02-Ni (5,10,15,20‑Tetrakis(4‑carboxyphenyl) porphyrininato‑Niquel), aunque inicialmente menos notable en fluorescencia, demostró una interacción significativa con el ADN, evidenciada por incrementos de intensidad fluorescente superiores a 60,000 cuentas por encima del control, en longitudes de onda de 640 a 660 nm. Esto confirmó su eficacia como posible sonda intercalante, capaz de unirse externamente al ADN. Además, esta porfirina mostró una emisión fluorescente significativa a dos temperaturas distintas, 25°C y 45°C donde a 45 °C estaba por encima de la temperatura melting de la secuencia de ADN usada para el estudio.
The efficacy of porphyrin compounds in various biomedical applications, such as biomarkers, sensors and photosensitizers, is well established. The study of DNA interactions with low molecular weight molecules, including porphyrins, has established itself as an autonomous and rapidly developing discipline. These studies have revealed promising results in gene therapy, especially in the treatment of cancer patients. The modes of binding of porphyrins to DNA are varied, including intercalation, binding to minor and major grooves, formation of external complexes and binding to DNA G-quadruplexes. Given the proven success of these compounds, the exploration of new porphyrins not only has the potential to improve existing techniques, but also to drive the development of innovative gene treatments and the design of more precise and effective drugs. In this context, the need to develop more accessible and efficient molecular probes for the diagnosis of infectious and genetic diseases through real-time PCR is evident. The use of fluorescent compounds such as porphyrins makes it possible to reduce costs and overcome the shortage of conventional materials. Of 23 porphyrins analyzed, 11 showed adequate fluorescence. Among these, 5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrininato-Nickel proved to be the most reliable, suggesting its potential to replace Sybr Green in real-time PCR. Porphyrin number 8, FA02-Ni (5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrininato-Nickel), stood out in fluorescence testing, showing consistent emission, particularly on the DNA double strand, which was our main focus. Although porphyrin number 7 5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrini initially showed high intensities and appeared promising in the melting curve, its behavior does not correspond to that of a traditional intercalating probe. In contrast, FA02-Ni (5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrininate-Nickel), although initially less remarkable in fluorescence, showed significant interaction with DNA, evidenced by increases in fluorescent intensity greater than 60,000 counts above the control, at wavelengths from 640 to 660 nm. This confirmed its efficacy as a possible intercalating probe, capable of binding externally to DNA. In addition, this porphyrin showed significant fluorescent emission at two different temperatures, 25°C and 45°C where at 45°C it was above the melting temperature of the DNA sequence used for the study.
The efficacy of porphyrin compounds in various biomedical applications, such as biomarkers, sensors and photosensitizers, is well established. The study of DNA interactions with low molecular weight molecules, including porphyrins, has established itself as an autonomous and rapidly developing discipline. These studies have revealed promising results in gene therapy, especially in the treatment of cancer patients. The modes of binding of porphyrins to DNA are varied, including intercalation, binding to minor and major grooves, formation of external complexes and binding to DNA G-quadruplexes. Given the proven success of these compounds, the exploration of new porphyrins not only has the potential to improve existing techniques, but also to drive the development of innovative gene treatments and the design of more precise and effective drugs. In this context, the need to develop more accessible and efficient molecular probes for the diagnosis of infectious and genetic diseases through real-time PCR is evident. The use of fluorescent compounds such as porphyrins makes it possible to reduce costs and overcome the shortage of conventional materials. Of 23 porphyrins analyzed, 11 showed adequate fluorescence. Among these, 5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrininato-Nickel proved to be the most reliable, suggesting its potential to replace Sybr Green in real-time PCR. Porphyrin number 8, FA02-Ni (5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrininato-Nickel), stood out in fluorescence testing, showing consistent emission, particularly on the DNA double strand, which was our main focus. Although porphyrin number 7 5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrini initially showed high intensities and appeared promising in the melting curve, its behavior does not correspond to that of a traditional intercalating probe. In contrast, FA02-Ni (5,10,15,20-Tetrakis(4-carboxyphenyl) porphyrininate-Nickel), although initially less remarkable in fluorescence, showed significant interaction with DNA, evidenced by increases in fluorescent intensity greater than 60,000 counts above the control, at wavelengths from 640 to 660 nm. This confirmed its efficacy as a possible intercalating probe, capable of binding externally to DNA. In addition, this porphyrin showed significant fluorescent emission at two different temperatures, 25°C and 45°C where at 45°C it was above the melting temperature of the DNA sequence used for the study.
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Palabras clave
Porfirina, Sybr green, Sonda molecular, PCR en tiempo real, Curva melting
