Figure 1.
Schematic of the strategy for surface immobilisation of large RNAs.
RNA (brown) is prepared by in vitro transcription and terminated by template run-off, from the corresponding DNA template (grey); the RNA incorporated between four and eight adenines at the 3′ end. A biotinylated uridine (U-biotin, filled black circle) is then ligated onto the 3′ poly-A-tail RNA by T4 RNA ligase. Excess U-biotin was then removed from the labelled RNA using a size exclusion spin column leaving it ready for sensor surface immobilisation. Following immobilisation of the biotin-labelled RNA to a streptavidin sensor surface (yellow), interactions of binding partner molecules (green) with the immobilised RNA can then be undertaken by on-slide probing or SPR analysis. A schematic sensorgram illustrative of a binding event for SPR immobilisation is shown.
Figure 2.
Analysis of the ligation reaction for Qrr2 sRNA (a) with and (b) without A-tails.
Gels were stained with the SYBR-Gold, whereas blots were probed with streptavidin-HRP to detect biotin-labelled RNA. Schematic representations of RNA species identified on the gels/blots are shown. The sequences of the RNAs are given in Table S1 in File S1.
Table 1.
Ligation yields for U-biotin to RNAs with A-tails. RNA sequences are given in Table S1 in File S1.
Figure 3.
Probing of surface-immobilised biotinylated-sRNA with partner mRNA-Cy3.
(a) Streptavidin-coated microarray slide with control spots of (1) biotin-OxyS, (2) blank surface, (3) sRNA MicA, and test spot of (4) biotin-MicA. The surface was probed with Cy3-labelled ompA. The specific ompA interaction with surface-immobilised biotin-MicA is shown by the green spot. (b) As for (a) but in this case the test spot (4) is biotin-Qrr1 and the control sRNA spot (3) is Qrr1. The surface was probed with Cy3-labelled hapR. The specific hapR interaction with surface-immobilised biotin-Qrr1 is seen by the green spot. Schematic illustrations of the interactions occurring in (a) and (b) are shown beneath the microarray slides with the streptavidin surface in yellow, sRNAs in brown and Cy3-labelled mRNA in green.
Figure 4.
SPR analysis of RNA-RNA interactions.
(a) Surface-immobilised biotin-ompA. Example sensorgrams of sequential injections of MicA (red) or OxyS (blue) from 0–10 µM; MicA data fit (black) with chi2 = 0.20 RU2. (b) Control sensorgrams of sequential injections of MicA from 0–10 µM over surface-immobilised rpoS mRNA (orange) or U-biotin reagent (green).
Table 2.
Kinetic data for sRNA-mRNA interactions from SPR analyses.
Figure 5.
SPR analysis of Qrr3-mRNA interactions.
(a) Surface-immobilised biotin-hapR. Example sensorgram of sequential injections of Qrr3 from 0–0.25 µM; data fit (black) with chi2 = 0.28 RU2. (b) Surface-immobilised biotin-vca0939. Example sensorgram of sequential injections of Qrr3 from 0–0.25 µM; data fit (black) with chi2 = 0.41 RU2.