Isolation of nucleic acids using liquid–liquid phase separation of pH-sensitive elastin-like polypeptides

• Published in: Scientific reports Vol. 14; no. 1; pp. 10157 - 13
• Main Authors: Díez Pérez, Telmo, Tafoya, Ashley N., Peabody, David S., Lakin, Matthew R., Hurwitz, Ivy, Carroll, Nick J., López, Gabriel P.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 02.05.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/2196/2197; 639/925/930/12; Biology; COVID-19; COVID-19 - virology; DNA - chemistry; DNA - isolation & purification; Elastin; Elastin - chemistry; Elastin-Like Polypeptides; Genetic engineering; Histidine; Humanities and Social Sciences; Humans; Hydrogen-Ion Concentration; Intrinsically Disordered Proteins - chemistry; Liquid-Liquid Extraction - methods; Molecular biology; multidisciplinary; Nanotechnology; Nucleic acids; Nucleic Acids - chemistry; Nucleic Acids - isolation & purification; Nucleotide sequence; Pandemics; PCR; Peptides - chemistry; pH effects; Phase Separation; Polypeptides; Proteins; SARS-CoV-2 - genetics; SARS-CoV-2 - isolation & purification; Science; Science (multidisciplinary); Separation; Severe acute respiratory syndrome coronavirus 2
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-024-60648-9

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid–liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.