Novel anti-Stokes lanthanide-doped nanoparticles and multicolor FRET mechanisms for single-molecule DNA sequencing (LantaSEQ)
Principal investigator: prof. dr hab. Artur Bednarkiewicz
Foreign co-principal investigator: Assoc. Prof. Hans Gorris Ph.D
Single-molecule DNA sequencing is highly desirable in molecular diagnostics due to:
Simplified DNA template preparation,
Higher throughput and speed,
Longer reading, leading to reduced costs compared to conventional sequencing techniques.
However, current single-molecule DNA sequencing methods have several limitations that can be overcome by exploiting the unique photophysical properties of new lanthanide-doped luminescent nanomaterials (LnNPs):
LnNPs efficiently emit visible light under NIR excitation with large anti-Stokes shifts >300 nm. Under these conditions, no other sample components are excited, and background signals from autofluorescence and light scattering are eliminated.
LnNPs are photostable and, unlike quantum dots, do not exhibit blinking. Consequently, these new nanoparticles provide a stable donor signal for FRET, which is essential for continuous readout of long DNA sequences.
LnNPs emit multiple narrow emission bands under single-wavelength excitation, allowing a single luminescent nanoparticle to serve as a FRET donor for up to four different acceptor dyes required to identify individual dNTPs.
Single-molecule sequencing using lanthanide-doped nanoparticles is a promising approach, but it has never been realized before. To achieve this, it is necessary to:
Optimize optical readout instruments for detecting single LnNPs and for wide-field microscopic imaging.
Synthesize and study new, specially designed LnNPs by optimizing dopant architecture and core–shell composition to maximize brightness and sensitivity to acceptor molecules.
Optimize polymerase surface conjugation to minimize the distance between UCNPs and polymerase.
Apply the newly developed materials, instruments, and methods to sequence test DNA molecules.