Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways.

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

The implications of APOBEC3-mediated C-to-U RNA editing for human disease.

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans.

Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer.

Associations in cell type-specific hydroxymethylation and transcriptional alterations of pediatric central nervous system tumors.

Although intratumoral heterogeneity has been established in pediatric central nervous system tumors, epigenomic alterations at the cell type level have largely remained unresolved. To identify cell type-specific alterations to cytosine modifications in pediatric central nervous system tumors, we utilize a multi-omic approach that integrated bulk DNA cytosine modification data (methylation and hydroxymethylation) with both bulk and single-cell RNA-sequencing data. We demonstrate a large reduction in the scope of significantly differentially modified cytosines in tumors when accounting for tumor cell type composition. In the progenitor-like cell types of tumors, we identify a preponderance differential Cytosine-phosphate-Guanine site hydroxymethylation rather than methylation. Genes with differential hydroxymethylation, like histone deacetylase 4 and insulin-like growth factor 1 receptor, are associated with cell type-specific changes in gene expression in tumors. Our results highlight the importance of epigenomic alterations in the progenitor-like cell types and its role in cell type-specific transcriptional regulation in pediatric central nervous system tumors.

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants.

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a ß-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

An electrochemiluminescence microsensor based on DNA-silver nanoclusters amplification for detecting cellular adenosine triphosphate.

Adenosine triphosphate (ATP), as the primary energy source, plays vital roles in many cellular events. Developing an efficient assay is crucial to rapidly evaluate the level of cellular ATP. A portable and integrated electrochemiluminescence (ECL) microsensor array based on a closed bipolar electrode (BPE) was presented. In the BPE unit, the ECL chemicals and oxidation/reduction were separated from the sensing chamber. The ATP aptamer was assembled with single-stranded DNA (ssDNA) in the sensing chamber. ATP capture made the aptamer disassemble from the ssDNA and facilitated DNA-templated silver nanocluster (Ag NC) generation by the target-rolling circle amplification (RCA) reaction. The guanine-rich padlock sequence produced tandem periodic cytosine-rich sequences by the RCA, inducing Ag NC generation in the cytosine-rich region of the produced DNA strands through Ag+ reduction. The in situ Ag NC generation enhanced the circuit conductivity of the BPE and promoted the ECL reaction of [Ru(bpy)2dppz]2+/tripropylamine in the anodic reservoir. On this ECL microsensor, a good linear relationship of ATP was achieved ranging from 30 to 1000 nM. The ATP content in HepG2 cells was selectively and sensitively determined without complex pretreatment. The ATP amount of 25 cells could be successfully detected when a sub-microliter sample was loaded.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Distinguishing preferences of human APOBEC3A and APOBEC3B for cytosines in hairpin loops, and reflection of these preferences in APOBEC-signature cancer genome mutations.

The APOBEC3 enzymes convert cytosines in single-stranded DNA to uracils to protect against viruses and retrotransposons but can contribute to mutations that diversify tumors. To understand the mechanism of mutagenesis, we map the uracils resulting from expression of APOBEC3B or its catalytic carboxy-terminal domain (CTD) in Escherichia coli. Like APOBEC3A, the uracilomes of A3B and A3B-CTD show a preference to deaminate cytosines near transcription start sites and the lagging-strand replication templates and in hairpin loops. Both biochemical activities of the enzymes and genomic uracil distribution show that A3A prefers 3 nt loops the best, while A3B prefers 4 nt loops. Reanalysis of hairpin loop mutations in human tumors finds intrinsic characteristics of both the enzymes, with a much stronger contribution from A3A. We apply Hairpin Signatures 1 and 2, which define A3A and A3B preferences respectively and are orthogonal to published methods, to evaluate their contribution to human tumor mutations.

Prediction of methylation status using WGS data of plasma cfDNA for multi-cancer early detection (MCED).

BACKGROUND: Cell-free DNA (cfDNA) contains a large amount of molecular information that can be used for multi-cancer early detection (MCED), including changes in epigenetic status of cfDNA, such as cfDNA fragmentation profile. The fragmentation of cfDNA is non-random and may be related to cfDNA methylation. This study provides clinical evidence for the feasibility of inferring cfDNA methylation levels based on cfDNA fragmentation patterns. We performed whole-genome bisulfite sequencing and whole-genome sequencing (WGS) on both healthy individuals and cancer patients. Using the information of whole-genome methylation levels, we investigated cytosine-phosphate-guanine (CpG) cleavage profile and validated the method of predicting the methylation level of individual CpG sites using WGS data. RESULTS: We conducted CpG cleavage profile biomarker analysis on data from both healthy individuals and cancer patients. We obtained unique or shared potential biomarkers for each group and built models accordingly. The modeling results proved the feasibility to predict the methylation status of single CpG sites in cfDNA using cleavage profile model from WGS data. CONCLUSION: By combining cfDNA cleavage profile of CpG sites with machine learning algorithms, we have identified specific CpG cleavage profile as biomarkers to predict the methylation status of individual CpG sites. Therefore, methylation profile, a widely used epigenetic biomarker, can be obtained from a single WGS assay for MCED.

5-Formylcytosine mediated DNA-peptide cross-link induces predominantly semi-targeted mutations in both Escherichia coli and human cells.

Histone proteins can become trapped on DNA in the presence of 5-formylcytosine (5fC) to form toxic DNA-protein conjugates. Their repair may involve proteolytic digestion resulting in DNA-peptide cross-links (DpCs). Here, we have investigated replication of a model DpC comprised of an 11-mer peptide (NH2-GGGKGLGK∗GGA) containing an oxy-lysine residue (K∗) conjugated to 5fC in DNA. Both CXG and CXT (where X = 5fC-DpC) sequence contexts were examined. Replication of both constructs gave low viability (<10%) in Escherichia coli, whereas TLS efficiency was high (72%) in HEK 293T cells. In E. coli, the DpC was bypassed largely error-free, inducing only 2 to 3% mutations, which increased to 4 to 5% with SOS. For both sequences, semi-targeted mutations were dominant, and for CXG, the predominant mutations were G→T and G→C at the 3'-base to the 5fC-DpC. In HEK 293T cells, 7 to 9% mutations occurred, and the dominant mutations were the semi-targeted G → T for CXG and T → G for CXT. These mutations were reduced drastically in cells deficient in hPol η, hPol ι or hPol ζ, suggesting a role of these TLS polymerases in mutagenic TLS. Steady-state kinetics studies using hPol η confirmed that this polymerase induces G → T and T → G transversions at the base immediately 3' to the DpC. This study reveals a unique replication pattern of 5fC-conjugated DpCs, which are bypassed largely error-free in both E. coli and human cells and induce mostly semi-targeted mutations at the 3' position to the lesion.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Clinical evaluation of pediatric patients with recurrent respiratory papillomatosis.: A longitudinal study at a Saudi Arabian tertiary care center.

OBJECTIVES: To study the clinical evaluation of recurrent respiratory papillomatosis (RRP) patients and the factors associated with the improvement in the Derkay's score as a measure of disease severity. METHODS: A retrospective cohort that included all juvenile RRP patients who were admitted to King Abdulaziz University Hospital, Riyadh, Saudi Arabia, between September 2015 and June 2022 and underwent surgical debulking. RESULTS: A total of 16 patients were eligible to join our study. Among them, 7 patients were males. Hoarseness of voice was the most frequent symptom. The median period of the follow-up was 56 months. Complete remission was achieved in 31.3%. The univariate linear regression model revealed that the cidofovir-treated patients had a significant reduction in the change value of Derkay's score compared to those without treatment (regression coefficient= -5.83, 95% confidence interval [CI]: [-11.5 to -0.143], p=0.045). Also, the increased first Derkay's score decreased the change value and subsequently increased the improvement chance of the disease (regression coefficient= -0.424, 95% CI: [-0.764 to -0.083], p=0.018). However, in the multivariate regression model, both variables showed non-significant results. CONCLUSION: cidofovir treatment and higher Derkay's scores affected the disease improvement.

Using NMR to Monitor TET-Dependent Methylcytosine Dioxygenase Activity and Regulation.

The active removal of DNA methylation marks is governed by the ten-eleven translocation (TET) family of enzymes (TET1-3), which iteratively oxidize 5-methycytosine (5mC) into 5-hydroxymethycytosine (5hmC), and then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET proteins are frequently mutated in myeloid malignancies or inactivated in solid tumors. These methylcytosine dioxygenases are α-ketoglutarate (αKG)-dependent and are, therefore, sensitive to metabolic homeostasis. For example, TET2 is activated by vitamin C (VC) and inhibited by specific oncometabolites. However, understanding the regulation of the TET2 enzyme by different metabolites and its activity remains challenging because of limitations in the methods used to simultaneously monitor TET2 substrates, products, and cofactors during catalysis. Here, we measure TET2-dependent activity in real time using NMR. Additionally, we demonstrate that in vitro activity of TET2 is highly dependent on the presence of VC in our system and is potently inhibited by an intermediate metabolite of the TCA cycle, oxaloacetate (OAA). Despite these opposing effects on TET2 activity, the binding sites of VC and OAA on TET2 are shared with αKG. Overall, our work suggests that NMR can be effectively used to monitor TET2 catalysis and illustrates how TET activity is regulated by metabolic and cellular conditions at each oxidation step.

High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma.

Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5' side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genes RPL13A and RPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription.

Chemical Methods to Identify Epigenetic Modifications in Cytosine Bases.

Chemical modifications to Cytosine bases are among the most studied epigenetic markers and their detection in the human genome plays a crucial role in gaining more insights about gene regulation, prognosis of genetic disorders and unraveling genetic inheritance patterns. The Cytosine methylated at the 5th position and oxidized derivatives thereof generated in the demethylation pathways, perform separate and unique epigenetic functions in an organism. As the presence of various Cytosine modifications is associated with diverse diseases, including cancer, there has been a strong focus on developing methods, both chemical and alternative approaches, capable of detecting these modifications at a single-base resolution across the entire genome. In this comprehensive review, we aim to consolidate the various chemical methods and understanding their chemistry that have been established to date for the detection of various Cytosine modifications.

Silver-Coordinated Watson-Crick Pairing-Driven Three-Dimensional DNA Walker for Locus-Specific Detection of Genomic N6-Methyladenine and N4-Methylcytosine at the Single-Molecule Level.

N6-Methyladenine (6mdA) and N4-methylcytosine (4mdC) are the two most dominant DNA modifications in both prokaryotes and eukaryotes, but standard hybridization-based techniques cannot be applied for the 6mdA/4mdC assay. Herein, we demonstrate the silver-coordinated Watson-Crick pairing-driven three-dimensional (3D) DNA walker for locus-specific detection of genomic 6mdA/4mdC at the single-molecule level. 6mdA-DNA and 4mdC-DNA can selectively hybridize with the binding probes (BP1 and BP2) to form 6mdA-DNA-BP1 and 4mdC-DNA-BP2 duplexes. The 6mdA-C/4mdC-A mismatches cannot be stabilized by AgI, and thus, 18-nt BP1/BP2 cannot be extended by the catalysis of KF exonuclease. Through toehold-mediated strand displacement (TMSD), the signal probe (SP1/SP2) functionalized on the gold nanoparticles (AuNPs) can competitively bind to BP1/BP2 in 6mdA-DNA-BP1/4mdC-DNA-BP2 duplex to obtain SP1-18-nt BP1 and SP2-18-nt BP2 duplexes. The resulting DNA duplexes can act as the substrates of lambda exonuclease, leading to the cleavage of SP1/SP2 and the release of Cy3/Cy5 and 18-nt BP1/BP2. The released 18-nt BP1/BP2 can subsequently serve as the walker DNA, moving along the surface of the AuNP to activate dynamic 3D DNA walking and releasing abundant Cy3/Cy5. The released Cy3/Cy5 can be quantified by single-molecule imaging. This nanosensor exhibits high sensitivity with a limit of detection (LOD) of 9.80 × 10-15 M for 6mdA-DNA and 9.97 × 10-15 M for 4mdC-DNA. It can discriminate 6mdA-/4mdC-DNA from unmodified genomic DNAs, distinguish 0.01% 6mdA-/4mdC-DNA from excess unmethylated DNAs, and quantify 6mdA-/4mdC-DNA at specific sites in genomic DNAs of liver cancer cells and Escherichia coli plasmid cloning vector, providing a new platform for locus-specific analysis of 6mdA/4mdC in genomic DNAs.

Oral USC-093, a novel homoserinamide analogue of the tyrosinamide (S)-HPMPA prodrug USC-087 has decreased nephrotoxicity while maintaining antiviral efficacy against human adenovirus infection of Syrian hamsters.

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. Presently, there is no drug specifically approved for the treatment of adenovirus infections by the FDA. The state-of-the-art treatment of such infections is the off-label use of cidofovir, an acyclic nucleotide phosphonate. While cidofovir inhibits adenovirus replication, it has dose-limiting kidney toxicity. There is an apparent need for a better compound to treat adenovirus infections. To this end, we have been developing acyclic nucleotide phosphonate prodrugs that utilize an amino acid scaffold equipped with a lipophilic modifier. Here, we compare the antiviral potential of two prodrugs of HPMPA that differ only in the amino acid-based promoiety: USC-087, based on an N-hexadecyl tyrosinamide, and USC-093, based on an N-hexadecyl serinamide. Oral administration of both compounds was very efficacious against disseminated HAdV-C6 infection in immunosuppressed Syrian hamsters, suppressing virus replication and mitigating pathology even when treatment was withheld until 4 days after challenge. We saw only marginal efficacy after respiratory infection of hamsters, which may reflect suboptimal distribution to the lung. Importantly, neither compound induced intestinal toxicity, which was observed as the major adverse effect in clinical trials of brincidofovir, a prodrug of cidofovir which also contains a C-16 modifier. Notably, we found that there was a significant difference in the nephrotoxicity of the two compounds: USC-087 caused significant kidney toxicity while USC-093 did not, at effective doses. These findings will be valuable guidepoints in the future evolution of this new class of potential prodrugs to treat adenovirus infections.