Table 1. Binding of nucleotides 81 to 148 of influenza A NS1 mRNA to isoenergetic probes on microarray.

Binding sitesa	Modified probe 5′ to 3′b	Base pair with GL at 3′ end	RNA bindingc		ΔG°37 predicted (kcal/mol) for complement and alternative binding sitef	Predicted ΔG°37 of modified probe/RNA for complementary binding siteg
			Buffer Id	Buffer IIe
91; [127]	UGGLGGL		M		−8.2; [−6.0]	−9.72
99	ULCDLDGLGL	C-GL		M	−6.9	−8.68
102; [115–118]	CLGDLUCLGL	U-GL		M	−7.2; [−5.2]	−9.66
109	GCLGDLAGL	C-GL		M	−8.1	−9.87
114	UCLUCLGGL	C-GL	S	S	−8.8	−9.35
115	DLUCLUCLGL	C-GL	S	S	−6.2	−9.17
116; [100–103]	GDLUCLUGL	G-GL	S	M	−6.3; [−4.7]	−9.02
127; [89–92]; [96–100]	UDLGGLGGL	U-GL	S	M	−7.7; [−7.7]; [−4.7]	−10.38
128; [90–92]; [96–101]	ULUDLGGLGL	C-GL	M	M	−7.2; [−4.5]; [−4.4]	−8.65
132; 138	CCLUCLUGL	A-GL		S	−7.5; −6.8	−10.09
[114]; [131–134]	CUCLUCLGL		S	S	[−5.8]; [−4.1]	−11.51
[116]	GGLUCLUGL		S	S	[−5.7]	−12.10
[114/115]	GLUCLUCLG		S	S	[−6.5]	−9.21
[115–118]	GGLUCLDGL		S	S	[−4.6]	−7.73
[115–118]; [99–103]	GGLUCLGGL		M	M	[−4.6]; [−4.8]	−7.73

^a- Center of binding site, where center is target RNA nucleotide complementary to third nucleotide from 5′-end of probe. Square brackets indicate possible alternative binding sites (if 5 or 6 nucleotides of probe bind) or region of binding (if predicted duplex has less than 5 canonical base pairs). Data in italics represent probes that bind in buffer II, but not in buffer I;

^b- LNA nucleotides are marked with superscript ^L, D represents 2,6-diaminopurine, nucleotides without superscript are 2′-O-methyl-nucleotides;

^c- S –strong binding, M – medium binding; underline – binding site with no ambiguity;

^d- buffer composition is 300 mM KCl, 10 mM MgCl₂, 10 mM Tris-HCl pH 7.0;

^e- buffer composition is 1 M KCl, 10 mM MgCl₂, 10 mM Tris-HCl pH 7.0;

^f- calculated by RNAstructure 4.6 program (for 1 M NaCl, assuming no structure of target RNA and unmodified probe), values correspond to binding sites listed in the first column;

^g- calculated for 100 mM NaCl buffer according to published equation [37], [38].

Probes listed underneath the double line have no perfect Watson-Crick match of the first five probe nucleotides to sequences in the target, but bind to target with at least one predicted GU wobble pair or in the case of site 114 have perfect complementarity to the target with only last five nucleotides of the probe. There are no complementary probes for sites: 129–131 and 137 on universal microarrays. Probes complementary to all other sites not listed in table do not bind strongly or moderately.