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. Author manuscript; available in PMC: 2021 Jun 24.
Published in final edited form as: Org Biomol Chem. 2020 Jun 24;18(24):4645–4655. doi: 10.1039/d0ob01052a

Table 2.

Thermal denaturation temperatures of DYZ-1 targeting Invader probe duplexes and duplexes between individual probe strands and cDNA.a Also shown are TA values, signal intensity, and proportion of nuclei presenting signal for the different Invader probes.

Tm [ΔTm]/(˚C)
Probe Sequence Probe duplex 5’-ON:cDNA 3’-ON:cDNA TA (˚C) Signal intensity % Coverage
15:16b 5’-Cy3-AGCCCUGTGCCCTG
3’-TCGGGACACGGGAC-Cy3		 66.0 [+5.5] 69.5 [+9.0] 74.0 [+13.5] +17.0 +++ 77
22:16 5’-Cy3-AGCCCUGTG 4 CCCTG
3’-TCGGGACAC GGGAC-Cy3		 50.5 [−10.0] 61.5 [+1.0] 74.0 [+13.5] +24.5 +++ 69
15:17 5’-Cy3-AGCCCUGTG CCCTG
3’-TCGGGACAC 4 GGGAC-Cy3		 41.5 [−19.0] 69.5 [+9.0] 62.5 [+2.0] +30.0 + 49
15:18 5’-Cy3-AGCCCUGTG CCCTG
3’-TCGGGACAC 9 GGGAC-Cy3		 32.5 [−28.0] 69.5 [+9.0] 57.5 [−3.0] +34.0 + 25
23:16 5’-Cy3-AGCCCUGTGCC 4 CTG
3’-TCGGGACACGG GAC-Cy3		 ntc 71.0 [+10.5] 74.0 [+13.5] nd ++ 76
15:24 5’-Cy3-AGCCCUGTGCC CTG
3’-TCGGGACACGG 4 GAC-Cy3		 56.0 [−4.5] 69.5 [+9.0] 62.0 [+1.5] +15.0 ++ 47
23:24 5’-Cy3-AGCCCUGTGCC 4 CTG
3’-TCGGGACACGG 4 GAC-Cy3		 54.0 [−6.5] 71.0 [+10.5] 62.0 [+1.5] +18.5 + 26
15:25 5’-Cy3-AGCCCUGTGCC CTG
3’-TCGGGACACGG 9 GAC-Cy3		 52.0 [−8.5] 69.5 [+9.0] 65.0 [+4.5] +22.0 ++ 70
15:26 5’-Cy3-AGC CCUGTGCCCTG
3’-TCG 9 GGACACGGGAC-Cy3		 ~30d [−30.5] 69.5 [+9.0] 62.0 [+1.5] ~ +41 ++ 76
19:20e 5’-Cy3-AGCGCUGAG GCCTG
3’-TCGCGACTC 4 CGGAC-Cy3		 43.5 [−20.0] 78.5 [+15.0] 64.5 [+1.0] +36.0 -f 4
19:21e 5’-Cy3-AGCGCUGAG GCCTG
3’-TCGCGACTC 9 CGGAC-Cy3		 42.0 [−21.5] 78.5 [+15.0] 60.5 [−3.0] +33.5 -f 0
a

ΔTm = change in Tm relative to unmodified dsDNA (Tm = 60.5 °C, except for ON19:ON20and ON19:ON21for which the corresponding unmodified dsDNA has a Tm = 63.5 °C). TA = Tm (5’-ON:cDNA) + Tm (3’-ON:cDNA) - Tm (Invader probe) - Tm (dsDNA). For experimental conditions, see Table 1. Cy3 = Cy3 monomer, 4= 4-hydroxybutyl monomer, 9= 9-hydroxynonyl monomer, and A/U/C= 2’-O-(pyren-1-yl)methyl-RNA monomers. For structures, see Fig. 1. nt = no transition. nd = not determined.

b

Data previously published in Reference 26.

c

Based on a single measurement due to low probe quantities.

d

Broad transition.

e

ON20and ON21do not form stable duplexes with the cDNA of ON16, whereas the duplex between ON19and the cDNA of ON15displays a Tm = 44.0 °C. Thus, ON19:ON20and ON19:ON21display TA << 0 °C for recognition of the DYZ-1 target region.

f

Signal was not observed.