Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5′end of the guide strand; its complementary sequence; the 5′end of the guide strand and the 3′overhang of the passenger strand. However, most part of the 3′ two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3′end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.     

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA–DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA–DNA substrate. Binding of NS3 to the PNA–DNA substrate was similar to the DNA–DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA–RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino–DNA and phosphorothioate–DNA substrates were utilized as efficiently by NS3 as DNA–DNA substrates. These results indicate that the PNA–DNA and PNA–RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.     

New synthetic substrates of mammalian nucleotide excision repair system

DNA probes for the studies of damaged strand excision during the nucleotide excision repair (NER) have been designed using the novel non-nucleosidic phosphoramidite reagents that contain N-[6-(9-antracenylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nAnt) and N-[6-(5(6)-fluoresceinylcarbamoyl)hexanoyl]-3-amino-1,2-propandiol (nFlu) moieties. New lesion-imitating adducts being inserted into DNA show good substrate properties in NER process. Modified extended linear nFlu– and nAntr–DNA are suitable for estimation of specific excision activity catalysed with mammalian whole-cell extracts. The following substrate activity range was revealed for the model 137-bp linear double-stranded DNA: nAnt–DNA ≈ nFlu–DNA > Chol–DNA (Chol–DNA—legitimate NER substrate that contains non-nucleoside fragment bearing cholesterol residue). In vitro assay shows that modified DNA can be a useful tool to study NER activity in whole-cell extracts. The developed approach should be of general use for the incorporation of NER-sensitive distortions into model DNAs. The new synthetic extended linear DNA containing bulky non-nucleoside modifications will be useful for NER mechanism study and for applications.     

Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

Design and preparation of layered composite materials alternating between nucleic acids and proteins has been elusive due to limitations in occurrence and geometry of interaction sites in natural biomolecules. We report the design and kinetically controlled stepwise synthesis of a nano-sandwich composite by programmed noncovalent association of protein, DNA and RNA modules. A homo-tetramer protein core was introduced to control the self-assembly and precise positioning of two RNA–DNA hybrid nanotriangles in a co-parallel sandwich arrangement. Kinetically favored self-assembly of the circularly closed nanostructures at the protein was driven by the intrinsic fast folding ability of RNA corner modules which were added to precursor complex of DNA bound to the protein. The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies. The synthesis strategy for the nano-sandwich composite provides a general blueprint for controlled noncovalent assembly of complex supramolecular architectures from protein, DNA and RNA components, which expand the design repertoire for bottom-up preparation of layered biomaterials.     

Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA

Methylglyoxal, a known endogenous and environmental mutagen, is a reactive α-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a ‘primed template’ DNA and the exonuclease-deficient Klenow fragment (KF^exo–) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS–PAGE, we found that methylglyoxal induced a DNA–KF^exo– crosslink. The specific binding complex of KF^exo– and ‘primed template’ DNA was necessary for formation of the DNA–KF^exo– crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2′-deoxyguanosine was incubated with Nα-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA–DNA polymerase crosslink.     

Inferring primase-DNA specific recognition using a data driven approach

DNA–protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is both challenging and important, since it may contribute to finding the means to regulate metabolic pathways involving DNA–protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA–protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.     

Selective Glutaraldehyde-Mediated Coupling of Proteins to the 3′-Adenine Terminus of Polymerase Chain Reaction Products

Attachment of proteins to the 3′ end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ′ modifications by solid-phase synthesis for protein attachment is limited to 45–60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3′-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N⁶ position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4°C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and β2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4°C showed no evidence of DNA–DNA or DNA–protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA–3′-protein conjugates might be easily mass-produced. The protein portion of a DNA–3′-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization.     

From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.     

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo⁻ to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.     

DNA-tethered membranes formed by giant vesicle rupture

We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA–lipid conjugate developed in earlier work [Chan, Y.-H.M., van Lengerich, B., et al., 2008. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3 (2), FA17–FA21], are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA–lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.     

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non‐homologous end joining (NHEJ) as the primary conserved DNA double‐strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB‐inducing agents, generation of antibody and T‐cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X‐ray scattering (SAXS) results combined with X‐ray crystallography (MX) and cryo‐electron microscopy (cryo‐EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra‐molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo‐EM or MX structures. In the long‐range synaptic complex, X‐ray repair cross‐complementing 4 (XRCC4) plus XRCC4‐like‐factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA‐PKcs (DNA‐dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto‐phosphorylation opens DNA‐PKcs dimer licensing NHEJ via concerted conformational transformations of XLF‐XRCC4, XLF–Ku80, and LigIV^BRCT–Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short‐range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation‐of‐function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.     

Thermodynamic characterization of the multivalent binding of chartreusin to DNA

Characterization of the thermodynamics of DNA– drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 × 10⁵ M^–1 at 20°C, in a solution containing 18 mM Na⁺. The DNA–drug interaction was enthalpy driven, with a ΔH_b of –7.07 kcal/mol at 20°C. Binding enthalpies were determined by ITC in the 20–35°C range and used to calculate a binding-induced change in heat capacity (ΔCp) of –391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of ΔG_obs into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.     

Purification and characterisation of a DNA helicase, dheI I, from Drosophila melanogaster embryos.

We have purified a DNA helicase (dhel l) from early Drosophila embryos. dhel l co-purifies with the single-stranded DNA binding protein dRP-A over two purification steps, however, the proteins can be separated by their different native molecular weight, with dhel l activity co-sedimenting with a polypeptide of approximately 200 kDa and a sedimentation coefficient of 8.6 S. The enzyme needs ATP hydrolysis and divalent cations for displacement activity. It is very salt sensitive, having a Mg2+ optimum of 0.5 mM and being inhibited by NaCl concentration > 10 mM. Dhel l moves 5'-->3' on the DNA strand to which it is bound. Unwinding activity decreases with increasing length of the double-stranded region suggesting a distributive mode of action. However, addition of dRP-A to the displacement reaction stimulates the activity on substrates with >300 nucleotides double-stranded region suggesting a specific interaction between these two proteins.     

Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelin–dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl–) were used to mimic the fluid-ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl– lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.     

Direct and random routing of a molecular motor protein at a DNA junction

With the aim of investigating how motor proteins negotiate DNA nanostructures, we produced test circuits based on recombination intermediates in which 1D translocation across a Holliday junction (HJ) could be assessed by subsequent triplex displacement signals on each DNA arm. Using the EcoR124I restriction-modification enzyme, a 3′–5′ double-strand DNA (dsDNA) translocase, we could show that the motor will tend to follow its translocated strand across a junction. Nonetheless, as the frequency of junction bypass events increases, the motor will occasionally jump tracks.     

Membrane Partitioning of Anionic,Ligand-Coated Nanoparticles Is Accompanied by Ligand Snorkeling,Local Disordering,and Cholesterol Depletion

Intracellular uptake of nanoparticles (NPs) may induce phase transitions, restructuring, stretching, or even complete disruption of the cell membrane. Therefore, NP cytotoxicity assessment requires a thorough understanding of the mechanisms by which these engineered nanostructures interact with the cell membrane. In this study, extensive Coarse-Grained Molecular Dynamics (MD) simulations are performed to investigate the partitioning of an anionic, ligand-decorated NP in model membranes containing dipalmitoylphosphatidylcholine (DPPC) phospholipids and different concentrations of cholesterol. Spontaneous fusion and translocation of the anionic NP is not observed in any of the 10-µs unbiased MD simulations, indicating that longer timescales may be required for such phenomena to occur. This picture is supported by the free energy analysis, revealing a considerable free energy barrier for NP translocation across the lipid bilayer. 5-µs unbiased MD simulations with the NP inserted in the bilayer core reveal that the hydrophobic and hydrophilic ligands of the NP surface rearrange to form optimal contacts with the lipid bilayer, leading to the so-called snorkeling effect. Inside cholesterol-containing bilayers, the NP induces rearrangement of the structure of the lipid bilayer in its vicinity from the liquid-ordered to the liquid phase spanning a distance almost twice its core radius (8–10 nm). Based on the physical insights obtained in this study, we propose a mechanism of cellular anionic NPpartitioning, which requires structural rearrangements of both the NP and the bilayer, and conclude that the translocation of anionic NPs through cholesterol-rich membranes must be accompanied by formation of cholesterol-lean regions in the proximity of NPs.     

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.     

Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease

Increasing evidence supports a role for oxidative DNA damage in aging and several neurodegenerative diseases including Alzheimer's disease (AD). Attack of DNA by reactive oxygen species (ROS), particularly hydroxyl radicals, can lead to strand breaks, DNA–DNA and DNA–protein cross-linking, and formation of at least 20 modified bases adducts. In addition, α,β-unsaturated aldehydic by-products of lipid peroxidation including 4-hydroxynonenal and acrolein can interact with DNA bases leading to the formation of bulky exocyclic adducts. Modification of DNA bases by direct interaction with ROS or aldehydes can lead to mutations and altered protein synthesis. Several studies of DNA base adducts in late-stage AD (LAD) brain show elevations of 8-hydroxyguanine (8-OHG), 8-hydroxyadenine (8-OHA), 5-hydroxycytosine (5-OHC), and 5-hydroxyuracil, a chemical degradation product of cytosine, in both nuclear and mitochondrial DNA (mtDNA) isolated from vulnerable regions of LAD brain compared to age-matched normal control subjects. Previous studies also show elevations of acrolein/guanine adducts in the hippocampus of LAD subjects compared to age-matched controls. In addition, studies of base excision repair show a decline in repair of 8-OHG in vulnerable regions of LAD brain. Our recent studies show elevated 8-OHG, 8-OHA, and 5,6-diamino-5-formamidopyrimidine in both nuclear and mtDNA isolated from vulnerable brain regions in amnestic mild cognitive impairment, the earliest clinical manifestation of AD, suggesting that oxidative DNA damage is an early event in AD and is not merely a secondary phenomenon.     

Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation.