Facilitating RNA structure prediction with microarrays

Abstract

Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

FIGURE 1

Chemical mapping results. Conditions are: (A) 1Na⁺/4Mg²⁺/10T (1 M NaCl, 4 mM MgCl₂, 10 mM Tris-HCl pH 7.43), room temperature, (B) 0.04Na⁺/10Mg²⁺/10T (40 mM NaCl, 10 mM MgCl₂, 10 mM Tris-HCl pH 7.43), room temperature (C) 1Na⁺/4Mg²⁺/10T (1 M NaCl, 4 mM MgCl₂,10 mM Tris-HCl pH 7.43), 4 °C, and (D) 1Na⁺/250T (1 M NaCl, 250 mM Tris-HCl pH 7.8), 4 °C. Also shown are results from nuclease mapping under condition (A). Nucleases V1 and S1 are specific for double and single stranded regions, respectively. T1 is specific for single stranded G. Symbols: ● - strong DMS; ○ - medium DMS; ■ - strong CMCT; □ - medium CMCT; ▲ - strong kethoxal; △ - medium kethoxal; ◆ - strong NMIA; ◇ - medium NMIA.

FIGURE 2

Hybridization results on 2′-O-methyl heptamer microarrays for 5S rRNA from E. coli in buffer 1Na⁺/4Mg²⁺/10T (1 M NaCl, 4 mM MgCl₂, 10 mM Tris-HCl pH 7.43), room temperature. On microarrays shown, the 2′-O-methyl heptamers from left to right are: (A) row 1: 4→ 15 (no binding detected), row 2: 16→ 27, row 3: 28 → 39, row 4: 40 → 51, row 5: 52 → 60, (B) row 1: 61→ 72 (no binding detected), row 2: 73→ 84 (no binding detected), row 3: 85 → 96, row 4: 97 → 108 (no binding detected), row 5: 109 → 117 (no binding detected).

FIGURE 3

Hybridization results for 5S rRNA on 2′-O-methyl-heptamer microarrays. Conditions: (A) 1Na⁺/4Mg²⁺/10T (1 M NaCl, 4 mM MgCl₂, 10 mM Tris-HCl pH 7.43), room temperature; (B) 1Na⁺/250T (1 M NaCl, 250 mM Tris-HCl, pH 7.8), 4 °C. Note that probes 16 and 67, 38 and 93, and also 39 and 94 have identical sequences and that probe 92 likely binds to loop C. Symbols: - middle site of strong binding; - middle site of medium binding.

FIGURE 4

RNAstructure 4.11 prediction for E. coli 5S rRNA secondary structure: (A) without constraints, and (B) with constraints from hybridization results. Correct base pairs are bolded. Nucleotides constrained were: (buffer 1Na⁺/4Mg²⁺/10T) 27–29, 35, 36, 40, 41, 44; (buffer 0.15Na⁺/4Mg²⁺/10T) 27–29, 35, 36, 39, 40, 44; (buffer 0.04Na⁺/4Mg²⁺/10T) 27–29, 35 36, 39, 40, 41, 44, 90. All three sets of constraints gave the same predicted structure.

FIGURE 5

RNAstructure 4.11 predictions for form B of E. coli 5S rRNA in 1Na⁺/250T (1 M NaCl, 250 mM Tris-HCl, pH 7.8), 4 °C. (A) Using constraints from chemical maping data alone, ΔG°₃₇ = −46.8 kcal/mol, and (B) using constraints shown from oligonucleotide binding data alone, ΔG°₃₇ = −44.8 kcal/mol. The structure shown in (B) is also predicted when both chemical mapping and oligonucleotide binding data are used as constraints. Note that all binding oligonucleotides shown in Figure 3 are not used as constraints because some have potential alternative binding sites (Supporting Information Table S1). Symbols: ● - strong DMS; ○ - medium DMS; ■ - strong CMCT; □ - medium CMCT; ▲ - strong kethoxal; △ - medium kethoxal; - middle site of strong binding; - middle site of medium binding.