Antisense Peptide Nucleic Acid Research Articles

Peptide nucleic acids (PNAs) control function of SARS-CoV-2 frameshifting stimulatory element trough PNA-RNA-PNA triplex formation

The highly structured nature of the SARS-CoV-2 genome provides many promising antiviral drug targets. One particularly promising target is a cis-acting RNA pseudoknot found within a critical region called the frameshifting stimulatory element (FSE). In this study, peptide nucleic acids (PNAs) binding to stem 2 of FSE RNA inhibited protein translation and frameshifting, as measured by a cell-free dual luciferase assay, more effectively than PNAs binding to stem 1, stem 3, or the slippery site. Surprisingly, simple antisense PNAs were stronger disruptors of frameshifting than PNA tail-clamps, despite higher thermal stability of the PNA-RNA-PNA triplexes formed by the latter. Another unexpected result was a strong and sequence non-specific enhancement of frameshifting inhibition when using a cationic triplex-forming PNA in conjunction with an antisense PNA targeting key regions of the frameshifting element. Our results illustrate both the potential and the challenges of using antisense PNAs to target highly structured RNAs, such as SARS-CoV-2 pseudoknots. While triplex forming PNAs, including PNA tail-clamps, are emerging as promising ligands for RNA recognition, the binding affinity enhancements when using cationic modifications in triplex-forming PNAs must be carefully balanced to avoid loss of sequence specificity in complex biological systems.

Targeting synthesis of the Chromosome Replication Initiator Protein DnaA by antisense PNA-peptide conjugates in Escherichia coli

Initiation of chromosome replication is an essential stage of the bacterial cell cycle that is controlled by the DnaA protein. With the aim of developing novel antimicrobials, we have targeted the initiation of DNA replication, using antisense peptide nucleic acids (PNAs), directed against DnaA translation. A series of anti-DnaA PNA conjugated to lysine-rich bacterial penetrating peptides (PNA-BPPs) were designed to block DnaA translation. These anti-DnaA PNA-BPPs inhibited growth of wild-type Escherichia coli cells at low micromolar concentrations, and cells exposed to anti-DnaA PNA-BPPs exhibited characteristic hallmarks of chromosome replication inhibition. These results present one of very few compounds successfully targeting initiation of chromosome replication, an essential step in the bacterial cell cycle.

In vivo interference of pea aphid endosymbiont Buchnera groEL gene by synthetic peptide nucleic acids

The unculturable nature of intracellular obligate symbionts presents a significant challenge for elucidating gene functionality, necessitating the development of gene manipulation techniques. One of the best-studied obligate symbioses is that between aphids and the bacterial endosymbiont Buchnera aphidicola. Given the extensive genome reduction observed in Buchnera, the remaining genes are crucial for understanding the host-symbiont relationship, but a lack of tools for manipulating gene function in the endosymbiont has significantly impeded the exploration of the molecular mechanisms underlying this mutualism. In this study, we introduced a novel gene manipulation technique employing synthetic single-stranded peptide nucleic acids (PNAs). We targeted the critical Buchnera groEL using specially designed antisense PNAs conjugated to an arginine-rich cell-penetrating peptide (CPP). Within 24 h of PNA administration via microinjection, we observed a significant reduction in groEL expression and Buchnera cell count. Notably, the interference of groEL led to profound morphological malformations in Buchnera, indicative of impaired cellular integrity. The gene knockdown technique developed in this study, involving the microinjection of CPP-conjugated antisense PNAs, provides a potent approach for in vivo gene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts.

Carrier capability of halloysite nanotubes for the intracellular delivery of antisense PNA targeting mRNA of neuroglobin gene

Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently.Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis.

Subtractive modification of bacterial consortium using antisense peptide nucleic acids.

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.

Investigating the Effectiveness of mqsR-Peptide Nucleic Acid as a Novel Solution for the Eradication of Persister Cells in Clinical Isolates of Escherichia coli.

Bacterial persisters are non- or slow-growing phenotypic variants that may be responsible for recalcitrance and relapse of persistent infections and antibiotic failure. In Escherichia coli, mqsRA is a well-known type II toxin-antitoxin system associated with persister cell formation. This study aimed to investigate the efficiency of an antisense peptide nucleic acid (PNA) targeting mqsRA in eliminating E. coli persisters. The study included 600 non-duplicated urine samples from adult patients with suspected urinary tract infections. The isolates were subjected to antimicrobial susceptibility testing and bacterial persister cells assay. The presence of mqsRA in the isolates was evaluated by polymerase chain reaction. Finally, expression of the mqsR and mqsA genes was assessed after exposure to normal conditions, stress, and different concentrations of mqsR-PNA (1 - 35 μM). The mqsR gene was significantly overexpressed under stress conditions, which was compensated by the PNA treatment. Complete inhibition of E. coli persister cells was achieved after overnight treatment with the anti-mqsR-PNA at concentrations ≥ 15 μM. The growth of E. coli persister cells can be inhibited by the anti-mqsR-PNA. Further studies are required to evaluate the effectiveness of this antisense PNA in both preclinical and clinical settings.

Peptide nucleic acid-zirconium coordination nanoparticles

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of >,94% w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of Zr_{6} O_{4} (OH)_{4} turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the beta-globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems.

Pyrenebutyrate Enhances the Antibacterial Effect of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pyogenes.

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane.

Combined mutations of the penA with ftsX genes contribute to ceftriaxone resistance in Neisseria gonorrhoeae and peptide nucleic acids targeting these genes reverse ceftriaxone resistance

To investigate the gene mutations associated with ceftriaxone (CRO) resistance among gonococcal isolates, and to determine the effects of the mutated genes on CRO minimum inhibitory concentrations (MICs) with transformation assays and antisense peptide nucleic acids (asPNAs). Ceftriaxone-resistant (CROR) and ceftriaxone-susceptible (CROS) isolates were identified using EUCAST and paired according to similarity in their MICs to other antimicrobials. The two groups of gonococci were sequenced and analysed. Mutated genes that showed a statistical difference between the two groups were transformed into gonococcal reference strains to determine their functions. AsPNAs were designed and transformed into the former transformant to further confirm the effects of the mutated genes. Twenty-two paired CROR and CROS isolates were obtained. The incidence of the penA-A501T and penA-G542S mutations individually, as well as combined mutations (penA-A501T and ftsX-R251H, penA-G542S and ftsX R251H), was statistically different between the two groups. The MIC of ATCC43069 (A43) increased 2 times following transformation with penA-A501T, and the MICs of A43 and ATCC49226 (A49) increased 32 times and 2 times following transformation with penA-A501T and ftsX-R251H, respectively. Antisense PNA-P3 reduced the MIC of the A43 transformant most significantly when transformed individually. PNA-P3 and PNA-F1 (asPNAs of the penA and ftsX) restored CRO susceptibility. PenA-A501T and penA-G542S mutations are important in CRO resistance among gonococci isolates. The ftsX-R251H mutation is also related to CRO resistance, and combined mutations of ftsX-R251H and penA-A501T comediate a significant reduction in CRO susceptibility. The combined application of PNA-P3 and PNA-F1 could effectively reverse the resistance to CRO in N. gonorrhoeae.

High-order framework nucleic acid for targeted-delivery of antisense peptide nucleic acids to overcome drug resistance.

A sophisticated high-order framework nucleic acid (FNA) was engineered for the targeted delivery and responsive release of environment tolerant antisense peptide nucleic acids (asPNAs). The dendritic FNA-asPNAs system was constructed via simple one-pot modular assembly and demonstrated a good synergistic effect with chemotherapy on drug resistant cancer cells.

DNA Nanoribbon for Efficient Anti-miRNA Peptide Nucleic Acid Delivery and Synergistic Enhancement of Cancer Cell Apoptosis.

Antisense peptide nucleic acid (asPNA), an effective antisense drug, has been employed as a gene therapy agent and a useful tool in molecular biology. Gaining control over the delivery of asPNA to target tissues has been a major hindrance to its wide application in clinical practice. A simple and efficient DNA nanoribbon (DNR)-based drug delivery process has been designed in this study that releases the asPNA agent to inhibit oncogenic microRNAs (miRNAs). Furthermore, we demonstrated how the AS1411 aptamer that binds nucleolin on the cell membranes works as a control mechanism capable of identifying target cancer cells and enhancing the enrichment capacity of DNR. With the biodegradability of DNR, we can efficiently initiate the release of asPNA into the cytoplasm, particularly targeting the intended miR-21 and synergistically increasing programmed cell death 4 (PDCD4) expression to enhance cell apoptosis. We assume that this well-defined delivery mechanism will aid in designing antisense site-specific treatments for various diseases, including cancer.

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models. PNAs are frequently coupled to cell-penetrating peptides (CPPs) to improve spontaneous cellular PNA uptake. In this study, different CPPs were investigated for their capability to support translocation of antisense PNAs into S. pneumoniae. HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs efficiently reduced the viability of S. pneumoniae strains TIGR4 and D39 in vitro. Two essential genes, gyrA and rpoB, were used as targets for antisense PNAs. Overall, the antimicrobial activity of anti-gyrA PNAs was higher than that of anti-rpoB PNAs. Target gene transcription levels in S. pneumoniae were reduced following antisense PNA treatment. The effect of HIV-1 TAT- and (RXR)4XB-anti-gyrA PNAs on pneumococcal survival was also studied in vivo using an insect infection model. Treatment increased the survival of infected Galleria mellonella larvae. Our results represent a proof of principle and may provide a basis for the development of efficient antisense molecules for treatment of S. pneumoniae infections. IMPORTANCE Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems. For this reason, S. pneumoniae has been added to the WHO "global priority list" of antibiotic-resistant bacteria for which novel antimicrobials are most urgently needed. In this study, we investigated whether CPP-coupled antisense PNAs show antibacterial activity in S. pneumoniae. We demonstrated that HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs were able to kill S. pneumoniae in vitro. The specificity of the antimicrobial effect was verified by reduced target gene transcription levels in S. pneumoniae. Moreover, CPP-antisense PNA treatment increased the survival rate of infected Galleria mellonella larvae in vivo. Based on these results, we believe that efficient antisense PNAs can be developed for the treatment of S. pneumoniae infections.

Machine Learning Guides Peptide Nucleic Acid Flow Synthesis and Sequence Design.

Peptide nucleic acids (PNAs) are potential antisense therapies for genetic, acquired, and viral diseases. Efficiently selecting candidate PNA sequences for synthesis and evaluation from a genome containing hundreds to thousands of options can be challenging. To facilitate this process, this work leverages machine learning (ML) algorithms and automated synthesis technology to predict PNA synthesis efficiency and guide rational PNA sequence design. The training data is collected from individual fluorenylmethyloxycarbonyl (Fmoc) deprotection reactions performed on a fully automated PNA synthesizer. The optimized ML model allows for 93% prediction accuracy and 0.97 Pearson's r. The predicted synthesis scores are validated to be correlated with the experimental high-performance liquid chromatography (HPLC) crude purities (correlation coefficient R2 = 0.95). Furthermore, a general applicability of ML is demonstrated through designing synthetically accessible antisense PNA sequences from 102315 predicted candidates targeting exon 44 of the human dystrophin gene, SARS-CoV-2, HIV, as well as selected genes associated with cardiovascular diseases, type II diabetes, and various cancers. Collectively, ML provides an accurate prediction of PNA synthesis quality and serves as a useful computational tool for informing PNA sequence design.

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors.

Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with the analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.

Development of Surface Chemical Strategies for Synthesizing Redox-Responsive Diatomite Nanoparticles as a Green Platform for On-Demand Intracellular Release of an Antisense Peptide Nucleic Acid Anticancer Agent.

Redox-responsive silica drug delivery systems are synthesized by aeco-friendly diatomite source to achieve on-demand release of peptide nucleic acid (PNA) in tumor reducing microenvironment, aiming to inhibit the immune checkpoint programmed cell death 1 receptor/programmed cell death receptor ligand 1 (PD-1/PD-L1) in cancer cells. The nanoparticles (NPs) are coated with polyethylene glycol chains as gatekeepers to improve their physicochemical properties and control drug release through the cleavable disulfide bonds (S-S) in a reductive environment. This study describes different chemical conditions to achieve the highest NPs' surface functionalization yield, exploring both multistep and one-pot chemical functionalization strategies. The best formulation is used for covalent PNA conjugation via the S-S bond reaching a loading degree of 306 ± 25µg PNA mg-1 DNPs . These systems are used for in vitro studies to evaluate the kinetic release, biocompatibility, cellular uptake, and activity on different cancer cells expressing high levels of PD-L1. The obtained results prove the safety of the NPs up to 200µg mL-1 and their advantage for controlling and enhancing the PNA intracellular release as well as antitumor activity. Moreover, the downregulation of PD-L1 observed only with MDA-MB-231 cancer cells paves the way for targeted immunotherapy.

The novel putative target to the eradication of Acinetobacter baumannii persister cells

BackgroundBacterial persister cells are dormant, slow-growing or growth-arrested phenotypic variants of normal cells in bacterial populations and are suggested that occurrence of the persisters leads to the recalcitrance and relapse of different chronic infections. Acinetobacter baumannii associated-infections are often correlated with high rates of drug-tolerant persisters, triggering a leading human health concern. MethodsIn our study, mqsR (as the toxin) and mqsA (as antitoxin) were considered as novel antimicrobial targets and silencing with antisense peptide nucleic acid (PNA) assay to eradicate the A. baumannii persister cells. Firstly, mqsR/A genes were molecularly evaluated. The functionality was assessed by normal and stress conditions in A. baumannii persister cells. ResultsExpression of mqsR significantly increased the under stress. A high expression of mqsR TA type II system was detected in A. baumannii persister cells. The mqsR gene fully silenced by PNA and A. baumannii persisters is eradicated. ConclusionsAntisense mqsR-PNA in 15 and 20 μM concentrations could eradicate A. baumannii persister cells. Moreover, it is proposed that other TA loci in A. baumannii are surveyed by antisense PNA to detect their functionality. However, conferring to importance of persisters in human infections, ex vivo, in vivo, preclinical and clinical settings could be highlighted.

Antisense Peptide Nucleic Acid-Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria.

Precision antisense antibacterial agents may be developed into novel antibiotics in the fight against multidrug-resistant Gram-negative bacteria. In this study, a series of diaminobutanoic acid (DAB) dendrons are presented as novel carriers for the delivery of antisense antibacterial peptide nucleic acids (PNAs). The dendron–PNA conjugates targeting the essential acpP gene exhibit specific antisense antimicrobial bactericidal activity against Escherichia coli and Klebsiella pneumoniae at one-digit micromolar concentrations, while showing low toxicity to human cells. One compound selected from a structure–activity relationship series showed high stability in mouse and human serum (t1/2 ≫ 24 h) as well as in vivo activity against a multidrug-resistant, extended spectrum beta-lactamase-producing E. coli in a murine peritonitis model. The compound was also well tolerated in mice upon i.v. administration up to a dose of 20 mg/kg, and in vivo fluorescence imaging indicated clearance via renal excretion with slight accumulation in the kidneys and liver. Thus, DAB-based dendrons constitute a promising new chemistry platform for development of effective delivery agents for antibacterial drugs with possible in vivo use.

The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes.

The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.

Automated Flow Synthesis of Peptide-PNA Conjugates.

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5′ untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 μM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.

Potentiating antibiotic efficacy via perturbation of non-essential gene expression

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.