Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2

Abstract

Despite the importance of nucleic acid testing in managing the COVID-19 pandemic, current detection approaches remain limited due to their high complexity and extensive processing. Here, we describe a molecular nanotechnology that enables direct and sensitive detection of viral RNA targets in native clinical samples. The technology, termed catalytic amplification by transition-state molecular switch (CATCH), leverages DNA-enzyme hybrid complexes to form a molecular switch. By ratiometric tuning of its constituents, the multicomponent molecular switch is prepared in a hyperresponsive state—the transition state—that can be readily activated upon the binding of sparse RNA targets to turn on substantial enzymatic activity. CATCH thus achieves superior performance (~8 RNA copies/μl), direct fluorescence detection that bypasses all steps of PCR (<1 hour at room temperature), and versatile implementation (high-throughput 96-well format and portable microfluidic assay). When applied for clinical COVID-19 diagnostics, CATCH demonstrated direct and accurate detection in minimally processed patient swab samples.

Analytical device miniaturization for the detection of circulating biomarkers

“…Upon binding of specific nucleic acids (DNA or RNA), the DNA complex dissociates to activate strong enzymatic activity. Through ratiometric tuning of molecular components within individual switches and collaborative coupling among multiple catalytic networks, this technology can sensitively detect SARS-CoV-2 RNA in clinical swab lysates, bypassing the technical requirements and processing steps of PCR detection (for example, sample purification, reverse transcription and thermal cycling), while achieving comparable sensitivity to PCR detection (detection limits as low as single copies) 166 , 167 .…”

Microfluidics-based strategies for molecular diagnostics of infectious diseases

“…Sun et al [21] fabricated a microfluidic chip that can multiply amplify specific nucleic acid sequences of five pathogens, including SARS-CoV-2 by LAMP, and detected them at the end of the reactions by a smartphone in 1 h. As another example, Sundah et al [112] formed a molecular switch [catalytic amplification by the transition-state molecular switch (CATCH)], which can directly and sensitively detect SARS-CoV-2 RNA targets with a smartphone. CATCH is compatible with portable LOCC and achieves superior performance (approximately 8 RNA copies/μl; < 1 h at room temperature) [112]. In addition, some driving forces are also used in LOCC equipment for molecular diagnostics, such as vacuum, stretching, and electric fields.…”

DNA‐Mediated Control of Protein Function in Semi‐Synthetic Systems

“…The activated polymerase then extended a DNA hairpin and incorporated biotin labeled dNTPs into the hairpin for subsequent attachment of HRP as the signaling molecule. This has led to sensitive assays for the visual detection of human papillomavirus and SARS‐CoV‐2 associated nucleic acids [50,58] . In another work where presence of target gave rise to active polymerase, Shao et al.…”

“…This has led to sensitive assays for the visual detection of human papillomavirus and SARS-CoV-2 associated nucleic acids. [50,58] In another work where presence of target gave rise to active polymerase, Shao et al showed a system where elongating the hairpin led to the destruction of a hemin containing G-quadruplex peroxidase domain. [59] The loss of peroxidase activity resulted in a detectable difference in electrical signal between positive and non-positive samples.…”