ResearchPad - chemical-biology-and-nucleic-acid-chemistry https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip]]> https://www.researchpad.co/article/N7b38a4c9-ef65-4a84-927c-51b640806391 DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale.

]]>
<![CDATA[Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc–siRNA conjugates: <i>in vitro</i> and <i>in vivo</i> RNAi activity and resistance to 5′-exonuclease]]> https://www.researchpad.co/article/N2f838286-b11e-417a-be2f-2759c58c2bf5 In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2′-deoxy-2′-fluoro and 2′-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5′ end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5′ end was more stable in the presence of 5′-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5′-exonuclease degradation afforded by the ANA modification.

]]>
<![CDATA[ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes]]> https://www.researchpad.co/article/N97019b55-e130-4ece-8720-d73bcbc42864 Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC–MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

]]>
<![CDATA[Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model]]> https://www.researchpad.co/article/N80ddb538-a920-45c9-8855-fab44eac663b

Abstract

Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2′-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone—with the same or extended target sequence as nusinersen—displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29—a 2-nt longer version of nusinersen—via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.

]]>
<![CDATA[GO: a functional reporter system to identify and enrich base editing activity]]> https://www.researchpad.co/article/N8a9f1098-b74a-4fac-bc68-452350dae1bd

Abstract

Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a ‘Gene On’ (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

]]>
<![CDATA[2′-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis]]> https://www.researchpad.co/article/Ndfb443bf-8789-4e8f-b814-9f2b6d21e8e2

Abstract

The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.

]]>
<![CDATA[Dynamics of strand slippage in DNA hairpins formed by CAG repeats: roles of sequence parity and trinucleotide interrupts]]> https://www.researchpad.co/article/N215370e7-ff5a-4da3-98b1-307aefd82849

Abstract

DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5′-AGCA-3′ tetraloops, compared with alternative 5′-CAG-3′ triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)n = even, but such situation cannot occur in (CAG)n = odd, where the hairpin is “frustrated’’ and slips back and forth between states with one TR hanging at the 5′ or 3′ end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.

]]>
<![CDATA[Streamlining effects of extra telomeric repeat on telomeric DNA folding revealed by fluorescence-force spectroscopy]]> https://www.researchpad.co/article/N9c289e8f-d82b-4ab1-bacb-f7159ae4f1b2

Abstract

A human telomere ends in a single-stranded 3′ tail, composed of repeats of T2AG3. G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.

]]>
<![CDATA[A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription]]> https://www.researchpad.co/article/N77e88f3d-5e18-4037-accd-98844e0bad53

Abstract

I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif.

]]>
<![CDATA[HER3-targeted protein chimera forms endosomolytic capsomeres and self-assembles into stealth nucleocapsids for systemic tumor homing of RNA interference in vivo]]> https://www.researchpad.co/article/Nf36f5307-cfb7-4772-8f45-bb903246134f

Abstract

RNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures.

]]>
<![CDATA[Selection of self-priming molecular replicators]]> https://www.researchpad.co/article/5c9bc689d5eed0c484eea00c

Abstract

Self-priming amplification of oligonucleotides is possible based on foldback of 3′ ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.

]]>
<![CDATA[Selective recognition of c-MYC Pu22 G-quadruplex by a fluorescent probe]]> https://www.researchpad.co/article/5c9bc67ad5eed0c484ee9e53

Abstract

Nucleic acid mimics of fluorescent proteins can be valuable tools to locate and image functional biomolecules in cells. Stacking between the internal G-quartet, formed in the mimics, and the exogenous fluorophore probes constitutes the basis for fluorescence emission. The precision of recognition depends upon probes selectively targeting the specific G-quadruplex in the mimics. However, the design of probes recognizing a G-quadruplex with high selectivity in vitro and in vivo remains a challenge. Through structure-based screening and optimization, we identified a light-up fluorescent probe, 9CI that selectively recognizes c-MYC Pu22 G-quadruplex both in vitro and ex vivo. Upon binding, the biocompatible probe emits both blue and green fluorescence with the excitation at 405 nm. With 9CI and c-MYC Pu22 G-quadruplex complex as the fluorescent response core, a DNA mimic of fluorescent proteins was constructed, which succeeded in locating a functional aptamer on the cellular periphery. The recognition mechanism analysis suggested the high selectivity and strong fluorescence response was attributed to the entire recognition process consisting of the kinetic match, dynamic interaction, and the final stacking. This study implies both the single stacking state and the dynamic recognition process are crucial for designing fluorescent probes or ligands with high selectivity for a specific G-quadruplex structure.

]]>
<![CDATA[Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases]]> https://www.researchpad.co/article/5c9bc680d5eed0c484ee9f19

Abstract

Six 1′,5′-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

]]>
<![CDATA[Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity]]> https://www.researchpad.co/article/5c5f1bc0d5eed0c48469ba07

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar–phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA ‘seed’ region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2′-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2′-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2′-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.

]]>
<![CDATA[Tethered imidazole mediated duplex stabilization and its potential for aptamer stabilization]]> https://www.researchpad.co/article/5c26b336d5eed0c48475f874

Abstract

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pKa. Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and the Hoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pKa-motif responsible for these duplex-stabilizing and pKa-modulating properties. Here, the robustness and utility of this pKa-motif was explored by introducing multiple imidazole-tethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pKa-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pKa values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazolium-tethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the l-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.

]]>
<![CDATA[5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells]]> https://www.researchpad.co/article/5c09e5e5d5eed0c484347b2b

Abstract

5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA–protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130–14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and –1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.

]]>
<![CDATA[Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin]]> https://www.researchpad.co/article/5b29caa7463d7e27ab10a85c

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)3) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.

]]>
<![CDATA[Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry]]> https://www.researchpad.co/article/5aec9628463d7e3f0f73f9d6

Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.

]]>
<![CDATA[Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex]]> https://www.researchpad.co/article/5b47919a463d7e7359e8065b

Abstract

Proliferating cell nuclear antigen (PCNA) is a multifunctional protein present in the nuclei of eukaryotic cells that plays an important role as a component of the DNA replication machinery, as well as DNA repair systems. PCNA was recently proposed as a potential non-oncogenic target for anti-cancer therapy. In this study, using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we developed a short DNA aptamer that binds human PCNA. In the presence of PCNA, the anti-PCNA aptamer inhibited the activity of human DNA polymerase δ and ϵ at nM concentrations. Moreover, PCNA protected the anti-PCNA aptamer against the exonucleolytic activity of these DNA polymerases. Investigation of the mechanism of anti-PCNA aptamer-dependent inhibition of DNA replication revealed that the aptamer did not block formation, but was a component of PCNA/DNA polymerase δ or ϵ complexes. Additionally, the anti-PCNA aptamer competed with the primer-template DNA for binding to the PCNA/DNA polymerase δ or ϵ complex. Based on the observations, a model of anti-PCNA aptamer/PCNA complex-dependent inhibition of DNA replication was proposed.

]]>
<![CDATA[Periodic-shRNA molecules are capable of gene silencing, cytotoxicity and innate immune activation in cancer cells]]> https://www.researchpad.co/article/5af83585463d7e029a53a0d8

Large dsRNA molecules can cause potent cytotoxic and immunostimulatory effects through the activation of pattern recognition receptors; however, synthetic versions of these molecules are mostly limited to simple sequences like poly-I:C and poly-A:U. Here we show that large RNA molecules generated by rolling circle transcription fold into periodic-shRNA (p-shRNA) structures and cause potent cytotoxicity and gene silencing when delivered to cancer cells. We determined structural requirements for the dumbbell templates used to synthesize p-shRNA, and showed that these molecules likely adopt a co-transcriptionally folded structure. The cytotoxicity of p-shRNA was robustly observed across four different cancer cell lines using two different delivery systems. Despite having a considerably different folded structure than conventional dsRNA, the cytotoxicity of p-shRNA was either equal to or substantially greater than that of poly-I:C depending on the delivery vehicle. Furthermore, p-shRNA caused greater NF-κB activation in SKOV3 cells compared to poly-I:C, indicating that it is a powerful activator of innate immunity. The tuneable sequence and combined gene silencing, immunostimulatory and cytotoxic capacity of p-shRNA make it an attractive platform for cancer immunotherapy.

]]>