Generation of densely labeled oligonucleotides for the detection of small genomic elements

Steinek, Clemens; Guirao-Ortiz, Miguel; Stumberger, Gabriela; Tölke, Annika J.; Hörl, David; Carell, Thomas; Harz, Hartmann; Leonhardt, Heinrich (2024): Generation of densely labeled oligonucleotides for the detection of small genomic elements. Cell Reports Methods, 4 (8): 100840. ISSN 26672375

Abstract

Motivation

While three-dimensional chromatin conformations can be explored with fluorescence in situ hybridization (FISH), the visualization of small genomic loci with high spatial resolution remains challenging. For such applications, programmable oligonucleotides with high brightness are required. To further improve precision and sensitivity, secondary hybridization steps should be omitted. Here, we present a simple, quick, and inexpensive approach to generate labeled FISH probes that carry several fluorophores. Our workflow allows for the free choice of fluorophores, flexible adjustment of labeling density, and selective probe synthesis from large probe pools. With our probes, we reliably detect genomic loci below the kilobase level and examine their topological relationships.

Highlights

• Rapid, flexible, and cost-effective synthesis of densely labeled oligonucleotides
• Systematic analysis of distance-dependent dye-dye interactions
• The NOVA workflow selectively yields labeled probe sets from complex probe pools
• NOVA-FISH enables the visualization of sub-kilobase genomic loci

Summary

The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.

• Dokumententyp: Artikel (LMU)
• Organisationseinheit (Fakultäten): 19 Biologie
• DFG-Fachsystematik der Wissenschaftsbereiche: Naturwissenschaften
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 213249687
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 491502892