Complementary Peptide Nucleic Acid Research Articles

Radionuclide therapy of bevacizumab-based PNA-mediated pretargeting.

The radionuclide-labeled bevacizumab (BV) is a potential therapeutic approach for vascular endothelial growth factor overexpressed tumors. Because of its large molecular weight, BV is cleared slowly in vivo, which caused damage to healthy tissues and organs. On account of this situation, using the pretargeting strategy with DNA/RNA analogs, such as peptide nucleic acid (PNA), is an effective way of treating solid tumors. The BV-PNA conjugate (BV-PNA-1) was injected intravenously as the pretargeted probe, which was specifically accumulated in a solid tumor and gradually metabolically cleared. Then the [177Lu]Lu-labeled complementary PNA strand ([177Lu]Lu-PNA-2) as the second probe was injected, and bound with BV-PNA-1 by the base complementary pairing. In this study, the BV-based PNA-mediated pretargeting strategy was systematically studied, including stability of probes, specific binding ability, biodistribution in animal model, evaluation of single photon emission computed tomography/computed tomography imaging, and therapeutic effect. Compared with group A ([177Lu]Lu-BV), the group B (BV-PNA-1 + [177Lu]Lu-PNA-2) showed lower blood radiotoxicity (22.55 ±1.62 vs. 5.18 ± 0.40%, %ID/g, P < 0.05), and similar accumulation of radioactivity in tumor (5.32 ± 0.66 vs. 6.68 ± 0.79%, %ID/g, P > 0.05). Correspondingly, there was no significant difference in therapeutic effect between groups A and B. The PNA-mediated pretargeting strategy could increase the tumor-to-blood ratio, thereby reducing the damage to normal tissues, while having a similar therapeutic effect to solid tumor. All the experiments in this study showed the potential and effectiveness of pretargeting radioimmunotherapy.

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection).

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2-4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.

Impact of shelterin complex on telomere accessibility.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5 fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Exploring a peptide nucleic acid-based antisense approach for CD5 targeting in chronic lymphocytic leukemia

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

Evaluation of an antibody-PNA conjugate as a clearing agent for antibody-based PNA-mediated radionuclide pretargeting

Radionuclide molecular imaging of cancer-specific targets is a promising method to identify patients for targeted antibody therapy. Radiolabeled full-length antibodies however suffer from slow clearance, resulting in high background radiation. To overcome this problem, a pretargeting system based on complementary peptide nucleic acid (PNA) probes has been investigated. The pretargeting relies on sequential injections of primary, PNA-tagged antibody and secondary, radiolabeled PNA probe, which are separated in time, to allow for clearance of non-bound primary agent. We now suggest to include a clearing agent (CA), designed for removal of primary tumor-targeting agent from the blood. The CA is based on the antibody cetuximab, which was conjugated to PNA and lactosaminated by reductive amination to improve hepatic clearance. The CA was evaluated in combination with PNA-labelled trastuzumab, T-ZHP1, for radionuclide HER2 pretargeting. Biodistribution studies in normal mice demonstrated that the CA cleared ca. 7 times more rapidly from blood than unmodified cetuximab. Injection of the CA 6 h post injection of the radiolabeled primary agent [131I]I-T-ZHP1 gave a moderate reduction of the radioactivity concentration in the blood after 1 h from 8.5 ± 1.8 to 6.0 ± 0.4%ID/g. These proof-of-principle results could guide future development of a more efficient CA.

Specific Disulfide Bond Formation Directed by Peptide Nucleic Acids

In this study, we successfully demonstrated a strategy for specific disulfide bond formation by hybridization of two complementary peptide nucleic acid strands. Peptides were linked to the pair of complementary peptide nucleic acids by native chemical ligation, respectively, and base pairing drove the two peptides into close proximity for interchain disulfide bond formation. Since peptide nucleic acids directed system is easier to manipulate than deoxyribonucleic acid system, this system will be more useful and have a broader application scope in the construction of complex biomolecular structures.

Intracellular delivery of a peptide nucleic acid-based hybrid of an autophagy inducing peptide with a cell-penetrating peptide.

Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.

1O - Suppression of KRAS-G12D and BRAF-V600E oncogene transcription with PNA-conjugates

BackgroundWe have developed a strategy to block transcription of oncogenes such as BRAF V600E and KRAS G12D directly using modified complementary peptide nucleic acid (PNA) oligomers that target oncogenesis specifically causing inhibition of tumour growth. This proof of principle strategy against BRAF V600E and KRAS G12D in vitro and in vivo should provide a new means to develop PNA-delivery peptide conjugates as targeted drug therapeutics across a broad range of oncogenes that drive cancer cell growth. MethodsObstruction of KRAS G12D and BRAF V600E expression was evaluated through suppression of cell proliferation and specific mRNA transcription. Tumour reduction was assessed through Xenograft mouse models. ResultsExposure of KRAS G12D-dependent cell line to modified PNA-peptide conjugate complementary to KRAS G12D mutation sequence also results in concentration-dependent and time-dependent inhibition of cell growth and specific and complete suppression in mRNA transcription. Cell lines expressing KRAS WT and KRAS G12C, both differing by a single nucleobase, show no suppression. Exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAF V600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAF V600E mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show tumour growth delay and necrosis compared to PNA controls. This 50mg/kg dose was well tolerated without associated weight loss. By H&E staining, tumor tissue from trials shows ablation and extensive scaring upon exposure to BRAF V600E-complementary PNA-peptide conjugate whereas saline and scramble PNA sequence controls do not. Similarly quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in TUNEL expression. ConclusionsOur results indicate that these PNA-peptide derivatives could represent a novel and promising new therapy for patients with genes specific for and causative of tumorigenesis. This strategy could be applied to a multitude of cancers either with specific translocations or mutations differing from wild-type cells even by only a single base pair. Legal entity responsible for the studyThe authors. FundingCelgene, Fortress Biotech. DisclosureAll authors have declared no conflicts of interest.

Coassembly of Complementary Peptide Nucleic Acid into Crystalline Structures by Microfluidics

AbstractThe self‐assembly of simple units into well‐ordered supramolecular polymeric structures may give rise to a broad range of desirable attributes. In the field of bionanotechnology, there are two main classes commonly used as building blocks for self‐assembly—amino acids and nucleobases. While protein, peptide, and amino acid building blocks propose broad chemical versatility, the specific Watson–Crick base pairings of nucleic acids allow rational design and precise control over the assembly process. Specifically, artificially synthesized peptide nucleic acids (PNA), short DNA mimics that have an amide backbone, can present a unique set of properties while preserving the specific base pairing of the nucleic acids. The use of complementary building blocks allows the study of supramolecular polymer coassemblies. Here, the coassembly of two paired di‐PNA building blocks provides a new layer of control, as both building blocks are required for the spontaneous assembly to occur. Utilizing a microfluidic system, the growth of the well‐ordered structure can be regulated and monitored. The crystal structure of the formed assemblies display Watson–Crick base pairing with similar distances as those found in typical DNA double helices. Moreover, the crystals exhibit intriguing optical properties that may be implemented in various materials science and nanotechnological applications.

Flow-through colorimetric assay for detection of nucleic acids in plasma

A flow-through colorimetric assay for detection of nucleic acids in plasma is reported. The proposed assay features an array of four polyvinylidene fluoride (PVDF) membranes impregnated with cationic poly (3-alkoxy-4-methylthiophene) (PT) as an optical reporter. The sensing strategy is based on monitoring the changes in optical properties of PT, upon complexation with target nucleic acids in the presence and in the absence of their corresponding complementary peptide nucleic acids (PNAs). As a proof of concept, the proposed methodology is validated using two biomarkers; lung cancer associated microRNA (mir21) and hepatitis B virus DNA (HBV-DNA). The flow-through colorimetric assay enabled detection of mir21 and HBV-DNA in plasma without requiring tedious sample pre-treatment and clean up protocols. Colorimetric responses for mir21 and HBV-DNA were obtained at nanomolar concentrations over five orders of magnitudes (from 1 nM to 10 μM), with a limit of detection of ∼0.6 nM and ∼2 nM in DI water and plasma, respectively. A logic gate system was developed to utilize the colorimetric assay responses as inputs for discrimination of mir21 and HBV-DNA and subsequently to obtain a profile of nucleic acids in samples that exceed respective clinical threshold limits, thereby enabling rapid and point of care (POC) disease diagnosis. Furthermore, the proposed methodology can be utilized for detection of a large number of nucleic acids in plasma by extending the array of PT impregnated membranes incorporated with their corresponding complementary PNAs.

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis.

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax-boric acid (pH8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP-HPLC, and also lower than 94.8% determined with RP-HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP-HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.

Novel amperometric genosensor based on peptide nucleic acid (PNA) probes immobilized on carbon nanotubes-screen printed electrodes for the determination of trace levels of non-amplified DNA in genetically modified (GM) soy

A novel amperometric genosensor based on PNA probes covalently bound on the surface of Single Walled Carbon Nanotubes – Screen Printed Electrodes (SWCNT-SPEs) was developed and validated in samples of non-amplified genomic DNA extracted from genetically modified (GM)-Soy. The sandwich assay is based on a first recognition of a 20-mer portion of the target DNA by a complementary PNA Capture Probe (CP) and a second hybridization with a PNA Signalling Probe (SP), with a complementary sequence to a different portion of the target DNA. The SP was labelled with biotin to measure current signal by means of a final incubation of an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). The electrochemical detection was carried out using hydroquinone diphosphate (HQDP) as enzymatic substrate. The genoassay provided a linear range from 250 pM to 2.5 nM, LOD of 64 pM and LOQ of 215 pM Excellent selectivity towards one base mismatch (1-MM) or scrambled (SCR) sequences was obtained. A simple protocol for extraction and analysis of non-amplified soybean genomic DNA without sample treatment was developed and validated. Our study provides insight into how the outstanding recognition efficiency of PNAs can be combined with the unique properties of CNTs in terms of signal response enhancement for direct detection of genomic DNA samples at the level of interest without previous amplification.

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.

Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with Peptide Nucleic Acid Hybridization Probes.

Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE running buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE running buffer. PNA probes are electrically neutral, while PNA-miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA-RNA hybrid was separable from the excess of the PNA probe without SSB in the running buffer, resulting in a near 10 min time window, which would allow, theoretically, separation of up to 30 hybrids. Then, we adapted to PNA-RNA hybrids our previously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modified theoretical model indicated that PNA-RNA hybrids of slightly different lengths could be separated from each other without drag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids of different lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental model included three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes had lengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; the other two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14 pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrations suggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.

Peptide Nucleic Acid Conjugated with Ruthenium-Complex Stabilizing Double-Duplex Invasion Complex Even under Physiological Conditions.

Peptide nucleic acid (PNA) can form a stable duplex with DNA, and, accordingly, directly recognize double-stranded DNA through the formation of a double-duplex invasion complex, wherein a pair of complementary PNA strands form two PNA/DNA duplexes. Because invasion does not require prior denaturation of DNA, PNA holds great potential for in cellulo or in vivo applications. To broaden the applicability of PNA invasion, we developed a new conjugate of PNA with a ruthenium complex. This Ru-PNA conjugate exhibits higher DNA-binding affinity, which results in enhanced invasion efficiency, even under physiological conditions.

Radionuclide Therapy of HER2-Expressing Human Xenografts Using Affibody-Based Peptide Nucleic Acid–Mediated Pretargeting: In Vivo Proof of Principle

Affibody molecules are small proteins engineered using a nonantibody scaffold. Radiolabeled Affibody molecules are excellent imaging probes, but their application to radionuclide therapy has been prevented by high renal reabsorption. The aim of this study was to test the hypothesis that Affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of human epidermal growth factor receptor 2 (HER2)-expressing cancer extends survival without accompanying renal toxicity. Methods: A HER2-targeting Affibody molecule ligated with an AGTCGTGATGTAGTC PNA hybridization probe (ZHER2:342-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide 177Lu (177Lu-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in 6 cycles separated by 7 d. Results: Optimal tumor targeting was achieved when 16 MBq/3.5 μg (0.65 nmol) of 177Lu-HP2 was injected 16 h after injection of 100 μg (7.7 nmol) of ZHER2:342-SR-HP1. The calculated absorbed dose to tumors was 1,075 mGy/MBq, whereas the absorbed dose to kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice was significantly longer (P < 0.05) in the treatment group (66 d) than in the control groups treated with the same amount of ZHER2:342-SR-HP1 only (37 d), the same amount and activity of 177Lu-HP2 only (32 d), or phosphate-buffered saline (37 d). Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making Affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.

Phosphoglucose Isomerase Deficiency in &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; K-12 Is Associated with Increased Spontaneous Mutation Rate

Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.