Table 1.

Thermal denaturation temperatures (T_ms) of Invader probes and duplexes between individual probe strands and cDNA, and $\Delta G_{\text{rec}}^{298}$ and $\Delta G_{\mathit{\text{rec}}}^{310}$ values.^a

Sequence 5’-GGUGGTCAA X1 CTATCUGGA 3’-CCACCAGTT X2 GATAGACCT
				Tm [ΔTm]/(˚C)
Probe	X1	X2	Invader duplex	5’-ON:cDNA	3’-ON:cDNA	$\Delta G_{\text{rec}}^{298}$ (kJ/mol)	$\Delta G_{\mathit{\text{rec}}}^{310}$ (kJ/mol)
1:2	-	-	60.5 [+1.5]	65.5 [+6.5]	68.5 [+9.5]	−39	−35
3:2	2	-	58.0 [−1.0]	63.0 [+4.0]	68.5 [+9.5]	−27	−26
1:4	-	2	54.0 [−5.0]	65.5 [+6.5]	64.5 [+5.5]	−35	−31
3:4	2	2	52.0 [−7.0]	63.0 [+4.0]	64.5 [+5.5]	−17	−18
5:2	222	-	52.0 [−7.0]	58.0 [−1.0]	68.5 [+9.5]	−43	−34
1:6	-	222	47.5 [−11.5]	65.5 [+6.5]	59.0 [0]	−44	−39
5:6	222	222	41.0 [−18.0]	58.0 [−1.0]	59.0 [0]	−39	−32
7:2	4	-	51.5 [−7.5]	58.0 [−1.0]	68.5 [+9.5]	−36	−33
1:8	-	4	47.5 [−11.5]	65.5 [+6.5]	60.0 [+1.0]	−28	−28
7:8	4	4	40.5 [−18.5]	58.0 [−1.0]	60.0 [+1.0]	−23	−22
9:2	444	-	44.0 [−15.0]	53.0 [−6.0]	68.5 [+9.5]	−38	−34
1:10	-	444	39.5 [−19.5]	65.5 [+6.5]	53.5 [−5.5]	−41	−35
9:10	444	444	37.0 [−22.0]	53.0 [−6.0]	53.5 [−5.5]	−17	−15
11:2	9	-	44.0 [−15.0]	53.5 [−5.5]	68.5 [+9.5]	−36	−32
1:12	-	9	42.0 [−17.0]	65.5 [+6.5]	55.5 [−3.5]	−40	−34
11:12	9	9	44.0 [−15.0]	53.5 [−5.5]	55.5 [−3.5]	−10	−6
13:2	999	-	45.0 [−14.0]	53.0 [−6.0]	68.5 [+9.5]	−36	−32
1:14	-	999	44.5 [−14.5]	65.5 [+6.5]	56.0 [−3.0]	−47	−40
13:14	999	999	45.0 [−14.0]	53.0 [−6.0]	56.0 [−3.0]	−15	−12

^aThermal denaturation curves (Fig. S5†) were recorded in medium salt phosphate buffer ([Na⁺] = 110 mM, [Cl⁻] = 100 mM, pH 7.0 (NaH₂PO₄/Na₂HPO₄), [EDTA] = 0.2 mM) using each ON at 0.5 μM concentration. ΔT_m is calculated relative to the corresponding unmodified DNA duplex (T_m = 59.0 °C). ΔG_rec = ΔG_5’-ON:cDNA + ΔG_3’-ON:cDNA - ΔG_{Invader probe} - ΔG_dsDNA. The corresponding ΔG²⁹⁸ and ΔG³¹⁰ values used to calculate $\Delta G_{\text{rec}}^{298}$ and $\Delta G_{\text{rec}}^{310}$ are listed in Tables S4^† and S5^†, respectively. A = adenin-9-yl, C = cytosin-1-yl, G = guanin-9-yl, T = thymin-1-yl. U/C = 2’-O-(pyren-1-yl)methyl-RNA U or C monomers. X₁ and X₂: 2= 1-amino-3-hydroxyprop-2-yl monomer, 4= 4-hydroxybutyl monomer, 9= 9-hydroxynonyl monomer (see Fig. 1 for structures of modified monomers).