Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 µM) underlines potential interest of this nanoplatform for biomedical applications.

Pervasive transcription fine-tunes replication origin activity.

RNA polymerase (RNAPII) transcription occurs pervasively, raising the important question of its functional impact on other DNA-associated processes, including replication. In budding yeast, replication originates from Autonomously Replicating Sequences (ARSs), generally located in intergenic regions. The influence of transcription on ARSs function has been studied for decades, but these earlier studies have neglected the role of non-annotated transcription. We studied the relationships between pervasive transcription and replication origin activity using high-resolution transcription maps. We show that ARSs alter the pervasive transcription landscape by pausing and terminating neighboring RNAPII transcription, thus limiting the occurrence of pervasive transcription within origins. We propose that quasi-symmetrical binding of the ORC complex to ARS borders and/or pre-RC formation are responsible for pausing and termination. We show that low, physiological levels of pervasive transcription impact the function of replication origins. Overall, our results have important implications for understanding the impact of genomic location on origin function.

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA.

Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. Similarly, in yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a redistribution of Mcm2-7 complexes along the chromosomes, resulting in a corresponding shift in DNA replication initiation sites. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict.

Origin plasticity during budding yeast DNA replication in vitro.

The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre-replicative complexes (pre-RCs) license origins by loading Mcm2-7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2-7 DNA helicase. Budding yeast pre-RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre-RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.

Tetramolecular quadruplex stability and assembly.

Guanine quadruplexes (G4) are unusual four-stranded nucleic acid structures formed by G-rich DNA/RNA. Beyond their likely biological relevance, the self-assembly, stability, and rigidity of these structures are also interesting for nanotechnology and biotechnology applications. Therefore, efforts are carried out to understand the rules that govern stability and folding of G-quadruplexes. We focus this chapter on tetramolecular conformations which are simple tractable models. We report here the experimental parameters, molecules, and modifications that affect thermal stability and/or association kinetics of these structures. Some chemical modifications which facilitate tetramolecular quadruplex formation and can be useful for nano- or biotechnology are also described.

Substrate specificity and mutational analysis of Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease.

tRNA anticodon damage inflicted by the Kluyveromyces lactis γ-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. γ-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 3' of the wobble base of tRNA(Glu(UUC)) to generate a break with 2',3'-cyclic phosphate and 5'-OH ends. Recombinant γ-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNA(Glu). A single 2'-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to γ-toxin's substrate specificity, we tested deoxynucleoside effects at positions other than the site of transesterification. The results attest to a stringent requirement for a ribonucleoside at the uridine 5' of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting γ-toxin's cleavage activity. Whereas either the 5' half or the 3' half of the anticodon stem can be replaced en bloc with DNA without a major effect, simultaneously replacing both strands with DNA interfered strongly, signifying that γ-toxin requires an A-form helical conformation of the anticodon stem. We purified γ-toxin mutants identified previously as nontoxic in vivo and gauged their anticodon nuclease activities in vitro. The results highlight Glu9 and Arg151 as candidate catalytic residues, along with His209 implicated previously. By analogy to other endoribonucleases, we speculate that γ-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151).

How long is too long? Effects of loop size on G-quadruplex stability.

We compared here 80 different sequences containing four tracts of three guanines with loops of variable length (between 1 and 15 bases for unmodified sequences, up to 30 for fluorescently labeled oligonucleotides). All sequences were capable of forming stable quadruplexes, with T(m) above physiological temperature in most cases. Unsurprisingly, the melting temperature was systematically lower in sodium than in potassium but the difference between both ionic conditions varied between 1 and >39°C (average difference: 18.3°C). Depending on the sequence context, and especially for G4 sequences involving two very short loops, the third one may be very long without compromising the stability of the quadruplex. A strong inverse correlation between total loop length and T(m) was found in K(+): each added base leads to a 2°C drop in T(m) or ∼0.3 kcal/mol loss in ΔG°. The trend was less clear in Na(+), with a longer than expected optimal loop length (up to 5 nt). This study will therefore extend the sequence repertoire of quadruplex-prone sequences, arguing for a modification of the widely used consensus (maximal loop size of 7 bases).

DNA and RNA quadruplex ligands.

Guanine-rich nucleic acids can adopt unusual structures called guanine quadruplexes (G4) based on stacked guanine quartets. Both RNA and DNA backbones are compatible with G4 formation. As RNA and DNA quadruplexes may be recognized by ligands, it is important to understand the rules that govern the stability and specificity of these complexes. We explore the binding of a pyridine dicarboxamide derivative to various oligoribo- and oligodeoxyribo-nucleotides.

8-Amino guanine accelerates tetramolecular G-quadruplex formation.

We demonstrate here that 8-amino guanine () strongly accelerates quadruplex formation, which makes this nucleobase the most attractive replacement for guanine in the context of tetramolecular parallel quadruplexes.

Sequence effects in single-base loops for quadruplexes.

Intramolecular G-quadruplexes formed by a single DNA strand have attracted much interest due to the possibility that they may form in telomeres, oncogene promoter sequences and other biologically relevant regions of the genome. Therefore, it is important to understand the rules that govern the formation of these intramolecular structures and to determine their stabilities. We compared here 27 different sequences containing four tracts of three guanines with a medium (3) or relatively long (6-9 bases) central loop and two loops composed of a single nucleotide H (A, T or C) corresponding to the GGGHGGGN3-9GGGHGGG motif. These sequences are similar to sequence motifs that can be found in repeated and promoter sequences. Several conclusions were reached: (i) all sequences are prone to form very stable quadruplexes in potassium (Tm between 55 degrees C and 83 degrees C); (ii) these quadruplexes also form in sodium but with a strongly decreased Tm, with a 21 to 36 degrees C difference in melting temperature between Na+ and K+; (iii) a long (9 bases) central loop had a minimal deleterious impact on the stability of the quadruplex; (iv) pyrimidines are preferred over adenine in both single-base loops; (v) the folding topology is relatively robust in potassium: the CD spectra of all oligonucleotides matched the one of all-parallel stranded reference quadruplexes; (vi) conversely, in sodium the folding is diverse and sequence-dependent, as judged from CD and electrophoresis results.

Guanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes.

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromo-guanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5' end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.

Kinetics and thermodynamics of G-quadruplexes.

The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rather than being a hindrance, this kinetic inertia allows us to study association and dissociation processes independently. General rules have been extended to longer DNA motifs or sequences containing modified bases such as 8-oxo or 7-deaza guanine. Results were compared with the canonical TG4T and TG5T tetramers: we demonstrate huge differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence.