TP63-TRIM29 axis regulates enhancer methylation and chromosomal instability in prostate cancer.

BACKGROUND: Prostate adenocarcinoma (PRAD) is the second leading cause of cancer-related deaths in men. High variability in DNA methylation and a high rate of large genomic rearrangements are often observed in PRAD. RESULTS: To investigate the reasons for such high variance, we integrated DNA methylation, RNA-seq, and copy number alterations datasets from The Cancer Genome Atlas (TCGA), focusing on PRAD, and employed weighted gene co-expression network analysis (WGCNA). Our results show that only single cluster of co-expressed genes is associated with genomic and epigenomic instability. Within this cluster, TP63 and TRIM29 are key transcription regulators and are downregulated in PRAD. We discovered that TP63 regulates the level of enhancer methylation in prostate basal epithelial cells. TRIM29 forms a complex with TP63 and together regulates the expression of genes specific to the prostate basal epithelium. In addition, TRIM29 binds DNA repair proteins and prevents the formation of the TMPRSS2:ERG gene fusion typically observed in PRAD. CONCLUSION: Our study demonstrates that TRIM29 and TP63 are important regulators in maintaining the identity of the basal epithelium under physiological conditions. Furthermore, we uncover the role of TRIM29 in PRAD development.

Structure-activity relationship study of mesyl and busyl phosphoramidate antisense oligonucleotides for unaided and PSMA-mediated uptake into prostate cancer cells.

Phosphorothioate (PS) group is a key component of a majority of FDA approved oligonucleotide drugs that increase stability to nucleases whilst maintaining interactions with many proteins, including RNase H in the case of antisense oligonucleotides (ASOs). At the same time, uniform PS modification increases nonspecific protein binding that can trigger toxicity and pro-inflammatory effects, so discovery and characterization of alternative phosphate mimics for RNA therapeutics is an actual task. Here we evaluated the effects of the introduction of several N-alkane sulfonyl phosphoramidate groups such as mesyl (methanesulfonyl) or busyl (1-butanesulfonyl) phosphoramidates into gapmer ASOs on the efficiency and pattern of RNase H cleavage, cellular uptake in vitro, and intracellular localization. Using Malat1 lncRNA as a target, we have identified patterns of mesyl or busyl modifications in the ASOs for optimal knockdown in vitro. Combination of the PSMA ligand-mediated delivery with optimized mesyl and busyl ASOs resulted in the efficient target depletion in the prostate cancer cells. Our study demonstrated that other N-alkanesulfonyl phosphoramidate groups apart from a known mesyl phosphoramidate can serve as an essential component of mixed backbone gapmer ASOs to reduce drawbacks of uniformly PS-modified gapmers, and deserve further investigation in RNA therapeutics.

Activity of nsp14 Exonuclease from SARS-CoV-2 towards RNAs with Modified 3'-Termini.

The COVID-19 pandemic has shown the urgent need for new treatments for coronavirus infections. Nucleoside analogs were successfully used to inhibit replication of some viruses through the incorporation into the growing DNA or RNA chain. However, the replicative machinery of coronaviruses contains nsp14, a non-structural protein with a 3'→5'-exonuclease activity that removes misincorporated and modified nucleotides from the 3' end of the growing RNA chain. Here, we studied the efficiency of hydrolysis of RNA containing various modifications in the 3'-terminal region by SARS-CoV-2 nsp14 exonuclease and its complex with the auxiliary protein nsp10. Single-stranded RNA was a preferable substrate compared to double-stranded RNA, which is consistent with the model of transfer of the substrate strand to the exonuclease active site, which was proposed on the basis of structural analysis. Modifications of the phosphodiester bond between the penultimate and last nucleotides had the greatest effect on nsp14 activity.

Polymer/magnetite carriers functionalized by HER2-DARPin: Avoiding lysosomes during internalization and controlled toxicity of doxorubicin by focused ultrasound induced release.

Nanomedicine has revolutionized the available treatment options during the last decade, but poor selectivity of targeted drug delivery and release is still poses a challenge. In this study, doxorubicin (DOX) and magnetite nanoparticles were encapsulated by freezing-induced loading, coated with polymeric shell bearing two bi-layers of polyarginine/dextran sulphate and finally modified with HER2-specific DARPin proteins. We demonstrated that the enhanced cellular uptake of these nanocarriers predominantly occurs by SKOV-3 (HER2+) cells, in comparison to CHO (HER2-) cells, together with the controlled DOX release using low intensity focused ultrasound (LIFU). In addition, a good ability of DARPin+ capsules to accumulate in the tumor and the possibility of combination therapy with LIFU were demonstrated. A relatively high sensitivity of the obtained nanocarriers to LIFU and their preferential interactions with mitochondria in cancer cells make these carriers promising candidates for cancer treatment, including novel approaches to overcome drug resistance.

Expression of CASC8 RNA in Human Pancreatic Cancer Cell Lines.

A lot of long non-coding RNAs (lncRNAs) are expressed in human cells in a number of transcripts of different lengths and composition of exons. In case of cancer-associated lncRNAs, an actual task is to determine their specific isoforms, since each transcript can perform its own function in carcinogenesis and might have a unique expression profile in various types of tumors. For the first time, we analyzed the expression of CASC8 lncRNA in human pancreatic ductal adenocarcinoma cell lines and found an abundant isoform that was previously considered as the minor one in this type of cancer. We also revealed extremely high expression levels of all CASC8 transcripts in MIA PaCa-2 cells and, conversely, the lack of this lncRNA in PANC-1. This allows to use them as convenient models for further in vitro studies.

Determination of the Affinity of Eukaryotic DDX3 RNA Helicase to the Characteristic Elements of mRNA Secondary Structure.

DDX3 RNA helicase is involved in many processes of RNA metabolism in eukaryotic cells. Many studies of DDX3 have shown that it is also involved in the translation initiation process, both cap-dependent and IRES-dependent. However, the specificity of the secondary structure of mRNA 5'-UTRs, which require DDX3 RNA helicase for effective translation, has not yet been determined. We performed a bioinformatic analysis of the 5'-UTR secondary structures in the pool of DDX3-dependent mRNAs in silico and predicted that the length of 5'-UTRs for such mRNAs is less than the average for the genome and that there are also characteristic hairpin structures in the region of the first 50 nucleotides from the 5'-end of the mRNA.

Comparative Analysis of Long Noncoding RNA Expression in Human Hepatocyte Cell Lines and Liver.

Long noncoding RNAs (lncRNAs) are promising biomarkers and potential targets for liver cancer therapy. Stable hepatocyte lines are used in vitro to investigate functions of lncRNAs which amount in cell fluctuates during carcinogenesis. For the first time we compared gene expression of known lncRNAs in human conditional normal liver cells HepaRG and cancer cell lines Huh7 and HepG2. We showed that relative amounts of these lncRNAs in HepaRG are close to analogous variables measured for liver samples from healthy donors. Obtained data demonstrate exclusive peculiarities of HepaRG and confirm its reasonable application as a model of normal human hepatocytes for studying functions of lncRNAs.

Long Noncoding RNA LL35/Falcor Regulates Expression of Transcription Factor Foxa2 in Hepatocytes in Normal and Fibrotic Mouse Liver.

Long noncoding RNAs (lncRNA) play important roles in the regulation of transcription, splicing, translation, and other processes in the cell. Human and mouse lncRNA (DEANR1 and LL35/Falcor, respectively) located in the genomic environment in close proximity to the Foxa2 transcription factor were discovered earlier. In this work, tissue-specific expression of LL35/Falcor lncRNA has been shown in mouse liver and lungs. The use of antisense oligonucleotides allowed us to achieve LL35/Falcor lncRNA downregulation by 90%. As a result, the level of Foxa2 mRNA and protein dropped, which confirms the involvement of LL35/Falcor lncRNA in the regulation of transcription factor Foxa2. We have shown a decrease in the expression of LL35 lncRNA in liver fibrosis, which correlates with the previously published data for mRNA Foxa2. Thus, lncRNA LL35 regulates Foxa2 expression in the liver not only in normal conditions, but also during development of fibrosis, which allows one to consider lncRNA a biomarker of this pathological process.

Integrator is a key component of human telomerase RNA biogenesis.

Telomeres are special DNA-protein structures that are located at the ends of linear eukaryotic chromosomes. The telomere length determines the proliferation potential of cells. Telomerase is a key component of the telomere length maintenance system. While telomerase is inactive in the majority of somatic cells, its activity determines the clonogenic potential of stem cells as a resource for tissue and organism regeneration. Reactivation of telomerase occurs during the process of immortalization in the majority of cancer cells. Telomerase is a ribonucleoprotein that contains telomerase reverse transcriptase and telomerase RNA components. The RNA processing mechanism of telomerase involves exosome trimming or degradation of the primary precursor. Recent data provide evidence that the competition between the processing and decay of telomerase RNA may regulate the amount of RNA at the physiological level. We show that termination of human telomerase RNA transcription is dependent on its promoter, which engages with the multisubunit complex Integrator to interact with RNA polymerase II and terminate transcription of the human telomerase RNA gene followed by further processing.

mRNA-Based Therapeutics - Advances and Perspectives.

In this review we discuss features of mRNA synthesis and modifications used to minimize immune response and prolong efficiency of the translation process in vivo. Considerable attention is given to the use of liposomes and nanoparticles containing lipids and polymers for the mRNA delivery. Finally we briefly discuss mRNAs which are currently in the clinical trials for cancer immunotherapy, vaccination against infectious diseases, and replacement therapy.

Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study.

Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259.

Oligonucleotide inhibitors of telomerase: prospects for anticancer therapy and diagnostics.

The activity of telomerase allows eukaryotic cells to have unlimited division potential. On its functioning, telomerase synthesizes short DNA repeats at the 3'-end of DNA within chromosomes that ensures genome stability during cell division. Telomerase is active in the majority of cancer cell types and is virtually absent in somatic cells with rare exceptions. This difference allows us to consider inhibition of telomerase activity as a possible approach to antitumor therapy. Telomerase is a nucleoprotein composed of two main components: the reverse transcriptase (hTERT), which is a catalytic subunit, and telomerase RNA (hTR), which encodes a template for synthesis of repeats. The biogenesis and features of telomerase seem very promising for its inhibition due to complementary interactions. In this review, we analyze putative pathways of oligonucleotide influence on telomerase and consider the known native and modified oligonucleotide inhibitors of telomerase, as well as possible mechanisms of their action. We also discuss the application of telomerase-targeted oligonucleotide conjugates for in vivo imaging of tumor cells.

[Modification of nucleic acids using [3+2] dipolar cycloaddition of azides and alkynes].

The use of the reaction of azide and alkyne cycloaddition for the synthesis of nucleic acid conjugates and DNA oligomer analogues is considered. The data on chemical and enzymatic techniques of azides and alkynes introduction into DNA are summarized.

[New derivatives of azobenzene for the directed modification of proteins].

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA.

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.

DNA-methyltransferase SsoII as a bifunctional protein: features of the interaction with the promoter region of SsoII restriction-modification genes.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.

Analysis of DNA-protein interactions in complexes of transcription factor NF-kappaB with DNA.

We have applied bioinformatic analysis of X-ray 3D structures of complexes of transcription factor NF-kappaB with DNAs. We determined the number of possible Van der Waals contacts and hydrogen bonds between amino acid residues and nucleotides. Conservative contacts in the NF-kappaB dimer-DNA complex composed of p50 and/or p65 NF-kappaB subunit and DNA sequences like 5 -GGGAMWTTCC-3 were revealed. Based on these results, we propose a novel scheme for interactions between NF-kappaB p50 homodimer and the kappaB region of the immunoglobulin light chain gene enhancer (Ig-kappaB). We applied a chemical cross-linking technique to study the proximity of some Lys and Cys residues of NF-kappaB p50 subunit with certain reactive nucleotides into its recognition site. In all cases, the experimentally determined protein-DNA contacts were in good agreement with the predicted ones.

Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents.

The review describes key aspects of the synthesis and biological activities of conjugates of oligonucleotides and their analogues with synthetic peptides, in particular aimed towards gene silencing applications. The common methods of synthesis of oligonucleotide-peptide conjugates (OPCs) and PNA-peptide conjugates (PPCs) are described, which include both total solid-phase and fragment coupling approaches. In addition, various applications of conjugates as gene silencing agents are outlined. These include antisense and steric block applications in mammalian cells of OPCs, PPCs and phosphorodiamidate morpholinooligonucleotide (PMO)-peptide conjugates, gene silencing in bacteria, various DNA targeting applications, and recent reports of gene silencing activities of siRNA-peptide conjugates. A table listing all peptides used as oligonucleotide conjugates for gene silencing applications is also included.

Affinity modification of the restriction endonuclease SsoII by 2'-aldehyde-containing double stranded DNAs.

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.