Table 1.

RNA labelling conditions and HPLC-determined conversions (%)

			HPLC conversiona
			Labeling type
RNA length (nt)	Final RNA concentration (μM)b	Cond.	3′c	5′d	5′+3′e
3	100–1000	A	85–99	nd	nd
3	10–100	B	75–85	nd	nd
30–300	1–30	B	75–85	73–95	74–88
1000–2000	0.2–3	B	75–85	74–88	46–56

^aHPLC conversions were calculated by dividing the peak area of the labelled product by sum of peak areas of substrate and reaction product(s). Peak areas were measured for absorbance at 260 nm.

^bTo determine molar concentration, absorbance at 260 nm of RNA substrate solutions were measured and divided by individual molar extinction coefficient calculated for each RNA sequence. Concentration of long RNAs (>300 nt) should not exceed 3 μM for efficient labelling.

^cRNA containing 3′ ribose is oxidized (1.5 mM NaIO₄, 30 min at 25°C in dark) and subsequently subjected to reductive amination in presence of an R-EDA derivative (2 h incubation at 25°C). Conditions A: 10 mM R-EDA, 100 mM NaBH₃CN, 500 mM KH₂PO₄ pH 6.0; Condition B: 1 mM R-EDA, 20 mM NaBH₃CN, 100 mM KH₂PO₄ pH 6.0.

^dRNA containing 5′ azide moiety is subjected to SPAAC reaction (incubation with 2 mM DIBAC derivative for 2 h at 25°C).

^eRNA containing 5′ azide and 3′ ribose moieties is oxidized and subsequently subjected to simultaneous SPAAC and reductive amination reaction (Conditions B + 2 mM DIBAC derivative). nd - not determined.