A comparative analysis of peptide-delivered antisense antibiotics using diverse nucleotide mimics

Abstract

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.

Keywords: RNA-seq; antibiotics; antimicrobial resistance; antisense oligomers; nucleic acid mimics.

FIGURE 1.

Design and synthesis of the different ASOs used in the study. (A) The different chemical modalities used in this study can be grouped into three different categories: modifications of the phosphate backbone (gray), modifications of the ribose sugar (green), and alternative backbone chemistries (blue). The gray and green boxes comprise ASOs that bear net negative charges; ASOs in the blue box are charge-neutral. (B) The KFF-PNA conjugates were synthesized as fusion peptides on a peptide synthesizer. (C) Schematic for the strain-promoted alkyne–azide click reaction between the peptide (synthesized on a peptide synthesizer) and the ASO (synthesized on an oligonucleotide synthesizer). Created with BioRender.com.

FIGURE 2.

Electrophoretic mobility shift assay. Retardation of electrophoretic mobility of a 5′ Cy5-labeled acpP target RNA (40 nt) dependent on ASO hybridization. Different ASO modalities were tested at 2.5:1, 1.25:1, 0.6:1, and 0.3:1 molar ratios of ASO:RNA as indicated. Yeast tRNA (10 µg) was spiked in samples labeled with “+.” Samples were separated on 15% native PAA gels, and one representative example of two independent experiments is shown.

FIGURE 3.

ASO:RNA duplex binding studies and in vitro translation assays. (A) Melting curves of acpP RNA–ASO complexes at equimolar concentrations. The target RNA used in this experiment is 10 nt long. (B) The capacity of the ASOs to inhibit translation of an acpP::gfp reporter transcript in vitro was analyzed at 5:1, 2.5:1, 1.25:1, and 0.6:1 molar ratios of ASO:RNA. Graphs show Image J quantitation of the GFP fusion protein in a western blot analysis of two independent experiments (replicates are color-coded in red and blue). Protein expression levels are shown relative to the water control. The corresponding western blots are shown in Supplemental Figure S5.

FIGURE 4.

Antibacterial activity of KFF-ASO conjugates. Growth of Salmonella was monitored in the absence or presence of varying concentrations of seven different KFF-ASO conjugates or the (KFF)₃KK(N₃) peptide control. PNA, PMO, and the cationic carrier peptide control are depicted in shades of yellow, whereas negASOs are in shades of blue. Water control is shown in red. Graphs are representative examples of two independent experiments.

FIGURE 5.

Global transcriptomic signatures of KFF-ASO-treated Salmonella. PCA of two independent RNA-seq experiments. The PCA plot shows a projection of the RNA-seq data onto the first two principal components. Treatment conditions separate into three clusters, as shown by the manual addition of cluster-ellipses in the PCA plot.

FIGURE 6.

Transcriptomic responses of Salmonella upon KFF-ASO treatment. Volcano plots show calculated changes in Salmonella gene expression as FDR-adjusted P-value (−log₁₀, y-axis) and fold change (FC) (log₂, x-axis). Significantly differentially regulated genes are characterized by an absolute FC > 2 (down-regulated log₂ < −1, up-regulated log₂ > 1; vertical dashed line) and an FDR-adjusted P-value < 0.001 (−log₁₀ > 3, horizontal dashed line). Significantly down-regulated genes are highlighted in blue, up-regulated genes are highlighted in red. The top three up- or down-regulated transcripts are labeled.

FIGURE 7.

Analysis of differentially expressed genes, KEGG pathways, and regulons. (A) Heatmap showing the most strongly differentially expressed genes for all KFF-ASO conjugates. Duplicate RNA-seq samples of Salmonella treated for 15 min with KFF-ASOs were normalized to untreated control samples. Log₂FCs are indicated by color. Orange and blue colors indicate up- and down-regulation, respectively. The heatmap includes the top 10 regulated transcripts per condition. Asterisks (*) denote significantly regulated (absolute log₂FC > 1 and FDR-adjusted P-value < 0.001) genes in the respective condition. (B) The heatmap shows the mean log₂FC for genes assigned to the gene sets. Only the 10 most significantly enriched/depleted gene sets per condition are visualized. Asterisks (*) denote statistical significance (FDR-adjusted P-value < 0.01) in the respective condition. The bar chart on the right shows the number of genes assigned to the respective gene set.

FIGURE 8.

Coverage plot of the acpP transcript and neighboring genes for all tested RNA-seq conditions. The coverage plot shows the abundance of mapped reads normalized by counts per million (CPM). The y-axis of all tracks shows the normalized read depth per position, ranging from 0 to 1500 CPM. Control samples without ASO, ASO modalities triggering significant (log₂FC < −1 and FDR < 0.001) acpP depletion and other ASO modalities are colored in black, red, and blue, respectively. One replicate (R1) of the RNA-seq data is shown.

FIGURE 9.

Western blot analysis of AcpP levels upon KFF-ASO treatment of Salmonella. (A) Western blot analysis of Salmonella strain SL1344 expressing AcpP::3xFLAG treated with KFF-PNA or KFF-PMO, each at 5 µM, for 30, 60, and 120 min. (B) Western blot analysis of Salmonella treated with all acpP-targeting KFF-ASO constructs at 5 µM for 120 min. (A,B) A scrambled KFF-PNA conjugate (PNA scr) and mock-treated (water) samples served as negative controls. Protein lysates were separated on 12% SDS-PAA gels, and proteins were blotted onto nitrocellulose membranes. Membranes were probed with a FLAG-specific antibody to detect the AcpP::3xFLAG fusion protein. GroEL was used as a loading control. The experiments were performed two times and exemplary images are shown.

FIGURE 10.

Outer membrane permeabilization of Salmonella. NPN fluorescence plotted over time. Different KFF-ASOs (10 µM) and controls were added after 14 min. CTAB (10 µM) and PMXB (0.4 µM) were used as positive controls, water as a negative control. A representative example of two independent experiments is shown.

Chandradhish Ghosh