Fig. 1: FRET-FISH implementation and optimization.

a Scheme of FRET-FISH. A FRET-FISH probe consisting of alternating oligos labeled with donor (D) or acceptor (A) fluorescent dyes is hybridized to its DNA target in fixed cells. If most D and A oligos are farther than the Förster distance, R₀, no FRET is detected. If a substantial number of D and A oligos are closer than R₀, FRET is detected. b FRET-FISH probe designs tested. Each probe contains ~200 pairs of D and A primary oligos containing a sequence complementary to the genomic target (T) flanked by two orthogonal adapter sequences (L and R) used as docking sites for PCR primers during probe production and for complementary detection oligos (L* and R*) conjugated to FRET donor (green) and acceptor (orange) dyes. In design 2, L and R adapter sequences are extended with a 6 nt stabilizing sequence (LSS and RSS), allowing annealing of the RSS of one D oligo with the LSS of the next A oligo. In design 3, the LSS and RSS sequences are added to the 5′ and 3′ end of the L* and R* detection oligos, respectively. c Distributions of FRET-FISH scores obtained with a probe targeting the MYC gene in human HAP1 cells, for each probe design shown in (b). D, Probes containing only donor oligos. A, probes containing only acceptor oligos. D+A, probes containing both donor and acceptor oligos. n, number of FRET signals analyzed. P, Wilcoxon test, two-tailed. Violins extend from the minimum to the maximum value and horizontal lines represent (from top to bottom) the 75th percentile, the median, and the 25th percentile of the distributions. d Maximum z-projections exemplifying donor (Cy3 excitation, Cy3 emission), acceptor (Cy5 excitation, Cy5 emission) and FRET (Cy3 excitation, Cy5 emission) signals in two nuclei of HAP1 cells hybridized with a FRET-FISH probe targeting MYC designed based on Design 3 in (b). Blue, DNA stained with Hoechst 33342. Scale bar, 10 μm. e–j FRET-FISH score distributions for six different types of FRET-FISH probes targeting the Ogt locus in female mouse embryonic fibroblasts (MEFs). Probe design is represented above each histogram. G, group of D or A oligos. S, spacing between consecutive oligos. Cy3 and Cy5 were used as FRET donor and acceptor dyes, respectively. Red lines, kernel density estimation function. n, number of FRET signals analyzed. Source data for all the plots shown in the figure are provided as a separate Source Data file.