A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs

Summary All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying the adaptation to such changes. sRNAs base-pair with their target mRNAs, allowing rapid modulation of the proteome. This post-transcriptional regulation is usually facilitated by RNA chaperones, such as Hfq. sRNAs have a potential as synthetic regulators that can be modulated by rational design. In this study, we use a library-based approach and oxacillin susceptibility assays to investigate the importance of the seed region length for synthetic sRNAs based on RybB and SgrS scaffolds in Escherichia coli. In the presence of Hfq we show that 12 nucleotides are sufficient for regulation. Furthermore, we observe a scaffold-specific Hfq-dependency and processing by RNase E. Our results provide information for design considerations of synthetic sRNAs in basic and applied research.


Figure S1 |
Figure S1 | RybB seed region length library for targeting of acrA mRNA, related to Figure 1.The acrA 5' UTR has a length of 79 nt, indicated by lowercase letters.The acrA open reading frame is indicated by the black arrow and capital letters.The RybB seed region length (SRL) library includes the start codon and extends to the 5' end of the transcript in 2-nt increments.The library consists of 42 different lengths, SRL0 represents a RybB scaffold that has no seed region and cannot bind to the acrA mRNA.The smaller sRNAs presumably do not bind, but are indicated at their cognate position to visualize the concept of the SRL library.For convenience, only SRL0 to SRL6 and SRL78 to SRL82 are visualized (dots indicate increasing seed region lengths).The translation initiation region (TIR) is underlined in the acrA mRNA.The acrA mRNA is truncated for visualization.

Figure S2 |
Figure S2 | Northern blot analysis of the RybB SRL-library, related to Figure 2-4.Northern blot analysis reveals a distinct processing pattern with the first band appearing with SRL26, the second with SRL52, the third with SRL64 and the fourth with SRL72.Intensities of synthetic sRNAs and processing patterns do not correlate with functionality (cf.

Figure
Figure 2B).Red asterisk indicate sRNAs which are not expressed (SRL0), are potentially a mixed population (SRL24) or are the wrong size (SRL36).5S rRNA serves as a loading control.

Figure S3 |
Figure S3 | Determination of synthetic RybB sRNAs functionality in Δhfq, related to Figure 3. Liquid growth analysis of the SRL-library indicates no clear regulation for the synthetic sRNAs by accessing the area under the curve(AUC) in the absence and presence of oxacillin (25 µg/mL).The Δhfq strain has a reduced viability, the AUC is decreased by approximately 2-fold even in the absence of oxacillin when compared to the wild type (wt).An increased SRL of the sRNAs does not show an effect.Wild type (wt), Δhfq and ΔacrA with an empty plasmid serve as positive and negative controls.Oxacillin susceptibility assay was performed in quadruplicate.Wilcoxon-Mann-Whitney-Test was applied for statistical testing against the Δhfq reference strain; p<0.05 (non-parametric, unpaired).

Figure S4 |
Figure S4 | MIC determination for the hfq deletion strain, related to Figure 3 and Figure 7. Stationary-phase cultures were diluted 1,000-fold and loaded into 96-well plates.Oxacillin was present at the indicated concentrations(2.5-folddilution series starting at 500 μg/mL).A well without oxacillin was used as growth control.The 96-well plates were incubated at 37°C under continuous shaking for 24 h.A representative experiment is shown.

Figure S5 |Figure 3 .
Figure S5 | Solid growth oxacillin susceptibility assay for selected candidates in wild type and Δhfq, related to Figure 3. Upper panels show sRNA functionality in the wild type (wt).The lower panels show oxacillin susceptibility for Δhfq expressing the indicated synthetic RybB sRNAs.No regulation of the acrA target can be observed in Δhfq, whichis consistent with the liquid media susceptibility assay (Figure3A).Based on the reduced viability of Δhfq in contrast to the wild type, the concentration of oxacillin for Δhfq assays was reduced by half (FigureS4).A representative replicate is shown.

Figure S6 |
Figure S6 | Liquid oxacillin susceptibility assay for minimal seed region length of synthetic SgrS sRNAs, related to Figure 5. Oxacillin susceptibility assay was performed in quadruplicate.Wilcoxon-Mann-Whitney-Test was used for statistical testing of the difference between growth with and without oxacillin; p<0.05 (non-parametric, unpaired).

Figure S7 |
Figure S7 | Structure predictions of selected synthetic SgrS sRNAs, related to Figure 6.The seed regions of SgrS SRL35-37 and SRL41-43 are in a flexible stem loop structure with a comparably low base-pairing probability (seed regions are indicated by black arrows).The stem-loop structures of SgrS SRL37 and 41 have a higher basepairing probability than the remaining SgrS sRNAs.Structures were generated using RNAfold [S1].

Figure S8 |
Figure S8 | Determination of synthetic SgrS sRNAs functionality in Δhfq, related to Figure 7. Liquid growth analysis of the SgrS SRL-library indicates no strong regulation for the synthetic sRNAs by accessing the area under the curve (AUC) in the absence and presence of oxacillin (25 µg/mL).Increasing the SRL shows a mild correlation with reduced AUC in the presence of oxacillin.Notably, the best performing SgrS sRNAs (SRL36 and SRL42) are not the best regulating sRNAs in the absence of Hfq.Wild type (wt), Δhfq and ΔacrA with an empty plasmid serve as respective positive and negative controls.Oxacillin susceptibility assay was performed in quadruplicate.Wilcoxon-Mann-Whitney-Test was used for statistical testing of the difference between growth with and without oxacillin; p<0.05 (non-parametric, unpaired).