Microarrays for identifying binding sites and probing structure of RNAs

Abstract

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure-function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.

Figure 1.

Preparation of Southern type microarrays and hybridization to tRNA^Phe. (A) schematic pattern of preparation of microarray and distribution of oligonucleotide probes on it, (B) hybridization of target RNA in 3.5 M TMACl at 4°C to microarray in reverse synthons manner, (C) hybridization of target RNA in 1 M NaCl/10 mM MgCl₂ at 4°C to microarray in a reverse synthons manner and (D) hybridization of target RNA in 3.5 M TMACl at 4°C to microarray in a standard synthons manner. Reprinted with permission from (21). Copyright 1999 Macmillan Publishers Ltd.

Figure 2.

Comparison of calculated free energies of duplexes formed by DNA (purple line), 2′-O-methylRNA (green line), 2′-O-methylRNA including 2′-O-methyl-2,6-diaminopurine riboside (red line), isoenergetic probes (LNA and 2′-O-methylRNA including 2′-O-methyl-2,6-diaminopurine riboside) (blue line) and complementary single-stranded sequence fragments of RNase P RNA from Bacillus subtilis (RNRspBs). This plot has corrections to the plot on Figure S2 of (62).

Figure 3.

Structural model for Bombyx mori 5′ RNA (R2Bm). Structure is annotated with modeling constraints generated from strong and medium binding to oligonucleotide microarrays (boxed nucleotides) and from chemical mapping (green, blue, red and orange circles, respectively, corresponding to strong reactivity with DMS, CMCT, NMIA and CMCT or DMS overlapping with NMIA). Also annotated are base-pair conservation (open boxes between nucleotides), compensatory mutations (filled boxes between nucleotides) and partition function probabilities (colored boxes or dashes between nucleotides) (34).

Figure 4.

Binding of isoenergetic oligonucleotide probes to model hairpins and pseudoknot (32). In hairpin IL1 (A) loop b terminates helix I, whereas in hairpin IL2 (B) loop c closes helix II. Inversion of the element terminating a helix correlates with inverting binding ability to the adjacent helical region. (C) Secondary structure and hybridization results for 74-pseudoknot (Pk2), a part of B. mori R2 5′RNA. Hybridization was performed in 200 mM NaCl, 5 mM MgCl₂ and 10 mM Tris-HCl, pH 8.0 at room temperature. Pk2 was hybridized to a microarray with all possible isoenergetic oligonucleotide probes complementary to Pk2. Filled in squares indicate site of strong binding to isoenergetic probe (middle nucleotide of region binding to pentamer probe), open squares indicate site of medium binding to isoenergetic probe. See the legend for meaning of other symbols.

Figure 5.

Binding sites of RNRspBs by isoenergetic probes on microarrays explored in three buffer conditions. Red squares and circles, respectively, indicate strong and medium binding in 140 mM NaCl, 80 mM HEPES, 10 mM MgCl₂ and also 135 mM KCl, 25 mM NaCl, 50 mM HEPES, 10 mM MgCl₂. Purple circles indicate medium binding only in 135 mM KCl, 25 mM NaCl, 50 mM HEPES, 10 mM MgCl₂. The red diamond indicates medium binding in 140 mM NaCl, 80 mM HEPES, 10 mM MgCl₂ and strong binding in 135 mM KCl, 25 mM NaCl, 50 mM HEPES, 10 mM MgCl₂. Green circles indicate medium binding in 140 mM NaCl, 80 mM HEPES, 10 mM MgCl₂. S indicates strong binding in 135 mM KCl, 25 mM NaHEPES, 50 mM HEPES, 1 M NaCl. M indicates medium binding in 135 mM KCl, 25 mM NaHEPES, 50 mM HEPES, 1 M NaCl. Question marks indicate ambiguous binding sites. Reprinted with modifications and permission from (64). Copyright 1999 Macmillan Publishers Ltd.

Figure 6.

Isoenergetic microarray mapping results for tRNA_i^Met (A) and tRNA_m^Met (B) in 1 M NaCl, 20 mM sodium cacodylate, 0.5 mM Na₂EDTA pH 7.5 (yellow bars), 1 M NaCl, 0.5 mM MgCl₂, 10 mM Tris-HCl pH 7.5 (red bars) and 1 M NaCl, 3.0 mM MgCl₂, 10 mM Tris-HCl pH 7.5 (blue bars). The height of the bar corresponds to strength of probe binding. Isoenergetic microarray mapping results for tRNA^Phe (C) (red bars) and unmodified transcript (blue bars) in 1 M NaCl, 0.5 mM MgCl₂, 10 mM Tris-HCl, pH 7.5 at 4°C. The height of the bar corresponds to strength of probe binding (33,63).

Figure 7.

(A) Isoenergetic microarrays mapping results for DsrA sRNA in complex with Hfq. On the left side, microarrays with hybridized DsrA RNA and complex of DsrA RNA/Hfq. On the right side, the bars are related to intensity of binding to probes (at selected positions) of DsrA RNA in the absence and presence of Hfq. On secondary structure of DsrA RNA red squares represent sites of binding that have the same intensity in the absence and presence of Hfq. (B) On the left side, mapping with isoenergetic microarrays DsrA RNA alone, complex DsrA RNA/rpoS mRNA and complex DsrA RNA/rpoS mRNA/Hfq. On the right side, schematic presentation of the interactions of DsrA RNA, rpoS mRNA and Hfq protein. The bar graph demonstrates intensity of binding to probes (at selected positions) of DsrA RNA in the absence and presence of Hfq. Various patterns hybridization of DsrA RNA is related with different positioning probes (77).