Intersection of calorie restriction and magnesium in the suppression of genome-destabilizing RNA–DNA hybrids

Dietary calorie restriction is a broadly acting intervention that extends the lifespan of various organisms from yeast to mammals. On another front, magnesium (Mg²⁺) is an essential biological metal critical to fundamental cellular processes and is commonly used as both a dietary supplement and treatment for some clinical conditions. If connections exist between calorie restriction and Mg²⁺ is unknown. Here, we show that Mg²⁺, acting alone or in response to dietary calorie restriction, allows eukaryotic cells to combat genome-destabilizing and lifespan-shortening accumulations of RNA–DNA hybrids, or R-loops. In an R-loop accumulation model of Pbp1-deficient Saccharomyces cerevisiae, magnesium ions guided by cell membrane Mg²⁺ transporters Alr1/2 act via Mg²⁺-sensitive R-loop suppressors Rnh1/201 and Pif1 to restore R-loop suppression, ribosomal DNA stability and cellular lifespan. Similarly, human cells deficient in ATXN2, the human ortholog of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg²⁺ in a process that is dependent on the TRPM7 Mg²⁺ transporter and the RNaseH1 R-loop suppressor. Thus, we identify Mg²⁺ as a biochemical signal of beneficial calorie restriction, reveal an R-loop suppressing function for human ATXN2 and propose that practical magnesium supplementation regimens can be used to combat R-loop accumulation linked to the dysfunction of disease-linked human genes.     

Site-specific cleavage of DNA–RNA hybrids by zinc finger/FokI cleavage domain fusions

Zinc-finger proteins of the Cys₂His₂ type bind DNA–RNA hybrids with affinities comparable to those for DNA duplexes. Such zinc-finger proteins were converted into site-specific cleaving enzymes by fusing them to the FokI cleavage domain. The fusion proteins are active and under optimal conditions cleave DNA duplexes in a sequence-specific manner. These fusions also exhibit site-specific cleavage of the DNA strand within DNA–RNA hybrids albeit at a lower efficiency (≃50-fold) compared to the cleavage of the DNA duplexes. These engineered endonucleases represent the first of their kind in terms of their DNA–RNA cleavage properties, and they may have important biological applications.     

Sequence-specific cleavage of the RNA strand in DNA–RNA hybrids by the fusion of ribonuclease H with a zinc finger

Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA–RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA–RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA–RNA hybrids. The optimization of the fusion enzyme’s specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.     

Glowworm swarm optimization for simultaneous capture of multiple local optima of multimodal functions

This paper presents glowworm swarm optimization (GSO), a novel algorithm for the simultaneous computation of multiple optima of multimodal functions. The algorithm shares a few features with some better known swarm intelligence based optimization algorithms, such as ant colony optimization and particle swarm optimization, but with several significant differences. The agents in GSO are thought of as glowworms that carry a luminescence quantity called luciferin along with them. The glowworms encode the fitness of their current locations, evaluated using the objective function, into a luciferin value that they broadcast to their neighbors. The glowworm identifies its neighbors and computes its movements by exploiting an adaptive neighborhood, which is bounded above by its sensor range. Each glowworm selects, using a probabilistic mechanism, a neighbor that has a luciferin value higher than its own and moves toward it. These movements—based only on local information and selective neighbor interactions—enable the swarm of glowworms to partition into disjoint subgroups that converge on multiple optima of a given multimodal function. We provide some theoretical results related to the luciferin update mechanism in order to prove the bounded nature and convergence of luciferin levels of the glowworms. Experimental results demonstrate the efficacy of the proposed glowworm based algorithm in capturing multiple optima of a series of standard multimodal test functions and more complex ones, such as stair-case and multiple-plateau functions. We also report the results of tests in higher dimensional spaces with a large number of peaks. We address the parameter selection problem by conducting experiments to show that only two parameters need to be selected by the user. Finally, we provide some comparisons of GSO with PSO and an experimental comparison with Niche-PSO, a PSO variant that is designed for the simultaneous computation of multiple optima. This work is partially supported by a project grant from the Ministry of Human Resources Development, India and by DRDO-IISc Mathematical Engineering Program.     

Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.     

Development of an intein-mediated split–Cas9 system for gene therapy

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.     

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.     

Base recognition of gap sites in DNA–DNA and DNA–RNA duplexes by short oligonucleotides

To increase base recognition capability and sensitivity, we propose the separation of a commonly used single-probe system for oligonucleotide analysis into a set of three probes: a fluorophore-labeled probe, a promoter probe, and a short probe. In this study, we found that the probes of only 4 nt in length can selectively bind the corresponding gap site on complexes consisting of the target, fluorophore-labeled probe, and promoter probe, exhibiting a more than 14-fold difference in ligation between the matched and mismatched sequences. Moreover, we demonstrated that the immobilized short probes accurately recognized the sequences of the gap sites.     

Combinatorics of RNA–RNA interaction

RNA–RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called “zigzag” configuration. This paper presents the combinatorics of RNA interaction structures including their generating function, singularity analysis as well as explicit recurrence relations. In particular, our results imply simple asymptotic formulas for the number of joint structures.     

Generation of dendritic cell–tumor cell hybrids by electrofusion for clinical vaccine application

Vaccination with hybrids comprising fused dendritic cells (DCs) and tumor cells is a novel cancer immunotherapy approach designed to combine tumor antigenicity with the antigen-presenting and immune-stimulatory capacities of DCs. For clinical purposes, we have incorporated a large-scale process for the generation of clinical-grade DCs together with novel electrofusion technology. The electrofusion system provides for ease and standardization of method, efficient DC–tumor cell hybrid formation, and large-quantity production of hybrids in a high-volume (6-ml) electrofusion chamber. In addition, we have evaluated DC electrofusion with a variety of allogeneic human tumor cell lines with the rationale that these tumor cell partners would prove a ready, suitable source for the generation of DC–tumor cell hybrid vaccines. The DC production process can generate 6×10⁸ to 2×10⁹ DCs from a single leukapheresis product (~180 ml). As determined by FACS analysis, electrofusion of 6×10⁷ total cells (1:1 ratio of DC and tumor cells) resulted in a consistent average of 8–10% DC–tumor cell hybrids, irrespective of the tumor type used. Hybrids were retained in the population for 48 h postfusion and following freezing and thawing. Upon pre-irradiation of the tumor cell partner for vaccine purposes, the overall fusion efficiency was not altered at doses up to 200 Gy. Evaluation of DC–tumor cell hybrid populations for their ability to stimulate T-cell responses demonstrated that electrofused populations are superior to mixed populations of DCs and tumor cells in generating a primary T-cell response, as indicated by IFN- release. Moreover, hybrids comprising HLA-A^*0201 DCs and allogeneic melanoma tumor cells (Colo 829 cell line) stimulated IFN- secretion by antigen-specific CD8⁺ T cells, which are restricted for recognition of a melanoma gp100 peptide antigen (gp100_209–217) within the context of the DC HLA haplotype. Maturation of the DC-Colo 829 cell hybrid population served to further improve this T-cell gp100-specific response. Overall, our results are promising for the large-scale generation of electrofused hybrids comprising DCs and allogeneic tumor cells, that may prove useful in human vaccine trials.     

Cytotoxicity and DNA binding property of triphenylethylene–coumarin hybrids with two amino side chains

Novel triphenylethylene–coumarin hybrids containing two amino side chains were designed and synthesized. Some of these 3,4-diphenyl coumarins, 7b–c (the double chains at 4-position on 3-,4-phenyl, respectively), and 13b–f (the double chains at 4-position on 3-phenyl and 7-position, respectively), showed a broad-spectrum and good anti-proliferative activity against five tumor cells and low cytotoxicity in osteoblast. UV–vis, fluorescence, and circular dichroism (CD) spectroscopies and thermal denaturation exhibited that compounds 7b (R = piperidinyl), 7e (R = NEt₂), and 7f (R = 4-methylpiperazinyl) had significant interactions with Ct-DNA by the intercalative mode of binding. Structure activity relationships (SARs) analysis suggested that the location of the two amino alkyl chains would play an important role both in the compounds against tumor cells proliferation and their interactions with DNA.     

Causes of the production of multiple forms of β-galactosidase by Bacillus circulans

The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D.     

DNA–DNA kissing complexes as a new tool for the assembly of DNA nanostructures

Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology.