Nucleic Acid Processing Research Articles

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Current pharmacotherapy of cryptosporidiosis: An update of the state-of-the-art

Introduction: Cryptosporidiosis has emerged as a major cause of diarrheal disease worldwide. It has especially serious health consequences for young, malnourished children living in endemic areas and for individuals with highly impaired T-cell function, such as HIV-positive individuals with low CD4 counts or immunosuppressed solid-organ transplant recipients. Areas covered: A selective literature search using PubMed was performed to review the available therapeutics to treat cryptosporidiosis, as well as related advances in drug development. Expert opinion: The only FDA-approved antiparasitic treatment in immunocompetent patients is nitazoxanide; however, it has failed to demonstrate convincing effectiveness among HIV-positive patients, immunosuppressed individuals and malnourished children. Thus, restoring HIV-positive patients’ cellular immune response through effective antiretroviral therapy (ART), or reducing or changing immunosuppressive drugs, is important. Several new targets have been identified for chemotherapy, and the development of drugs for these targets has progressed, including parasite kinases, nucleic acid synthesis and processing, proteases and lipid metabolism. Candidate drugs that have been shown to be effective and safe in a neonatal calf model will most likely constitute the next advance for clinical trials in humans. However, developing an effective and inexpensive vaccination, as well as complementing structural preventive measures, would most decisively reduce the global cryptosporidiosis burden.

On the Metal-Aided Catalytic Mechanism for Phosphodiester Bond Cleavage Performed by Nanozymes.

Recent studies have shown that gold nanoparticles (AuNPs) functionalized with Zn(II) complexes can cleave phosphate esters and nucleic acids. Remarkably, such synthetic nanonucleases appear to catalyze metal (Zn)-aided hydrolytic reactions of nucleic acids similar to metallonuclease enzymes. To clarify the reaction mechanism of these nanocatalysts, here we have comparatively analyzed two nanonucleases with a >10-fold difference in the catalytic efficiency for the hydrolysis of the 2-hydroxypropyl-4-nitrophenylphosphate (HPNP, a typical RNA model substrate). We have used microsecond-long atomistic simulations, integrated with NMR experiments, to investigate the structure and dynamics of the outer coating monolayer of these nanoparticles, either alone or in complex with HPNP, in solution. We show that the most efficient one is characterized by coating ligands that promote a well-organized monolayer structure, with the formation of solvated bimetallic catalytic sites. Importantly, we have found that these nanoparticles can mimic two-metal-ion enzymes for nucleic acid processing, with Zn ions that promote HPNP binding at the reaction center. Thus, the two-metal-ion-aided hydrolytic strategy of such nanonucleases helps in explaining their catalytic efficiency for substrate hydrolysis, in accordance with the experimental evidence. These mechanistic insights reinforce the parallelism between such functionalized AuNPs and proteins toward the rational design of more efficient catalysts.

The iron chaperone and nucleic acid–binding activities of poly(rC)-binding protein 1 are separable and independently essential

Poly(rC)-binding protein (PCBP1) is a multifunctional adaptor protein that can coordinate single-stranded nucleic acids and iron-glutathione complexes, altering the processing and transfer of these ligands through interactions with other proteins. Multiple phenotypes are ascribed to cells lacking PCBP1, but the relative contribution of RNA, DNA, or iron chaperone activity is not consistently clear. Here, we report the identification of amino acid residues required for iron coordination on each structural domain of PCBP1 and confirm the requirement of iron coordination for binding target proteins BolA2 and ferritin. We further construct PCBP1 variants that lack either nucleic acid- or iron-binding activity and examine their functions in human cells and mouse tissues depleted of endogenous PCBP1. We find that these activities are separable and independently confer essential functions. While iron chaperone activity controls cell cycle progression and suppression of DNA damage, RNA/DNA-binding activity maintains cell viability in both cultured cell and mouse models. The coevolution of RNA/DNA binding and iron chaperone activities on a single protein may prove advantageous for nucleic acid processing that depends on enzymes with iron cofactors.

A Tale of Loops and Tails: The Role of Intrinsically Disordered Protein Regions in R-Loop Recognition and Phase Separation.

R-loops are non-canonical, three-stranded nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under both normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, with many of these proteins containing folded nucleic acid processing and binding domains that prevent (e.g., topoisomerases), resolve (e.g., helicases, nucleases), or recognize (e.g., KH, RRMs) R-loops. However, a significant number of these R-loop interacting Enzyme and Reader proteins also contain long stretches of intrinsically disordered regions (IDRs). The precise molecular and structural mechanisms by which the folded domains and IDRs synergize to recognize and process R-loops or modulate R-loop-mediated signaling have not been fully explored. While studying one such modular R-loop Reader, the Fragile X Protein (FMRP), we unexpectedly discovered that the C-terminal IDR (C-IDR) of FMRP is the predominant R-loop binding site, with the three N-terminal KH domains recognizing the trailing ssRNA overhang. Interestingly, the C-IDR of FMRP has recently been shown to undergo spontaneous Liquid-Liquid Phase Separation (LLPS) assembly by itself or in complex with another non-canonical nucleic acid structure, RNA G-quadruplex. Furthermore, we have recently shown that FMRP can suppress persistent R-loops that form during transcription, a process that is also enhanced by LLPS via the assembly of membraneless transcription factories. These exciting findings prompted us to explore the role of IDRs in R-loop processing and signaling proteins through a comprehensive bioinformatics and computational biology study. Here, we evaluated IDR prevalence, sequence composition and LLPS propensity for the known R-loop interactome. We observed that, like FMRP, the majority of the R-loop interactome, especially Readers, contains long IDRs that are highly enriched in low complexity sequences with biased amino acid composition, suggesting that these IDRs could directly interact with R-loops, rather than being “mere flexible linkers” connecting the “functional folded enzyme or binding domains”. Furthermore, our analysis shows that several proteins in the R-loop interactome are either predicted to or have been experimentally demonstrated to undergo LLPS or are known to be associated with phase separated membraneless organelles. Thus, our overall results present a thought-provoking hypothesis that IDRs in the R-loop interactome can provide a functional link between R-loop recognition via direct binding and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci. The absence or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the widespread R-loop-mediated DNA double strand breaks that we recently observed in Fragile X patient-derived cells.

Effects of DNA Supercoil Handedness on Catenation by Type II Topoisomerases

Type II topoisomerases interact differently with DNA substrates of opposite supercoil handedness: although the genome is globally unwound, DNA ahead of replication or transcription complexes is overwound, and therefore DNA geometry can influence where the enzyme acts during nucleic acid processes. Human topoisomerase IIa and bacterial topoisomerase IV relax positively supercoiled [(+)SC] DNA faster than negatively supercoiled [(-)SC] molecules, while human topoisomerase IIb shows no preference. An important function of topoisomerase IIa and topoisomerase IV is the decatenation of DNA prior to mitosis. Cellular studies in yeast and bacteria indicate that DNA becomes positively supercoiled through interaction with cellular factors prior to decatenation, but it is not yet known whether the enzyme displays an intrinsic preference for decatenating (+)SC DNA or if these additional cellular factors accelerate the reaction. To determine whether type II topoisomerases possess an intrinsic ability to recognize DNA supercoil handedness during catenation and decatenation, we have carried out a series of in vitro experiments. As these two reactions are in equilibrium, it is important to study the effects of DNA supercoil handedness on both of them. Our studies focused on the effects of (-)SC, (+)SC, and relaxed DNA on catenation by type II topoisomerases. Topoisomerase IIa catenated relaxed DNA ~10-fold faster than (-)SC DNA, and ~45-fold faster than (+)SC DNA. The C-terminal domain of topoisomerase IIa has been previously shown to confer the ability of the enzyme to preferentially relax (+)SC DNA over (-)SC DNA. A deletion construct of topoisomerase IIa that lacked the C-terminal domain showed that this enzyme domain is also required for the preferential catenation of relaxed and (-)SC DNA. Catenation assays with (+)SC substrates did not go to completion, suggesting that chromosomes are less likely to be catenated following positive supercoiling. Additional studies performed with topoisomerase IIa indicated that (-)SC DNA does not relax prior to catenation and, at high concentrations, the enzyme can catenate DNA with very low concentrations of ATP as compared to other strand passage reactions. Topoisomerase IIb displayed slower rates of catenation than topoisomerase IIa and did not discriminate between different supercoil handedness states. In contrast, topoisomerase IV preferentially catenated (+)SC DNA over (-)SC and relaxed DNA. Results show that topoisomerase IIa and topoisomerase IV can distinguish supercoil handedness during catenation and may help determine whether (+)SC decatenation uses a different control mechanism.

Cooperative Dynamics of REC-Nuc Lobes Prime Cas12a for DNA Processing

The CRISPR-Cas12a system is the basis of the DETECTR technology, which allows extraordinarily rapid detection of viruses including SARS-CoV-2 that is spreading across multiple countries. Here, a collective ensemble of multi-microsecond molecular dynamics is used to characterize the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids.

Correlative Single-Molecule Force-FRET Measurements of DNA Hairpin Conformational Dynamics

The biological function of many macromolecules such as proteins and nucleic acids is tightly coupled to their conformation and their conformational dynamics. Therefore, studying how macromolecules fold and undergo conformational changes is crucial to understand the underlying mechanisms of biological processes. In particular DNA/RNA hairpins play essential regulatory roles in vivo, since they can regulate the interaction with nucleic acid processing proteins in processes such as transcription, recombination or replication. Therefore, understanding the principles of their structure formation is important for elucidating those biological roles. Single-molecule force spectroscopy represents an ideal tool for such studies because of its unique capability to isolate individual biomolecules and observe conformational transitions in real-time, while providing the control over the population distribution as opposed to approaches in solution. Here we report a study on FRET-labelled DNA hairpin folding transitions using high-resolution optical tweezers combined with fluorescence microscopy. We present equilibrium measurements at unprecedent trap distance stability, which allows to measure conformational state distributions over hours without altering the energy landscape. Measuring over longer time periods while keeping the trap distance within nanometer precision is crucial to capture the rare conformational states that might be overlooked in measurements over a timescale that is limited by instrumental drift. These measurements were enabled by integrating trap-distance and temperature stabilization feedback loops to achieve the ultimate stability in distance between the traps. Kinetic, energetic and structural properties of these states could be determined quantitatively from a correlative information source – force and fluorescence data, thereby providing new insights into folding pathways and energy landscape. This study shows how correlating force spectroscopy measurements with fluorescence microscopy could complement the investigation of the prevalence and energy landscape of certain conformational states with extra three-dimensional structural information of the macromolecule of interest.

CoV-er all the bases: Structural perspectives of SARS-CoV-2 RNA synthesis.

The ongoing Covid-19 pandemic has spurred research in the biology of the nidovirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Much focus has been on the viral RNA synthesis machinery due to its fundamental role in viral propagation. The central and essential enzyme of the RNA synthesis process, the RNA-dependent RNA polymerase (RdRp), functions in conjunction with a coterie of viral-encoded enzymes that mediate crucial nucleic acid transactions. Some of these enzymes share common features with other RNA viruses, while others play roles unique to nidoviruses or CoVs. The RdRps are proven targets for viral pathogens, and many of the other nucleic acid processing enzymes are promising targets. The purpose of this review is to summarize recent advances in our understanding of the mechanisms of RNA synthesis in CoVs. By reflecting on these studies, we hope to emphasize the remaining gaps in our knowledge. The recent onslaught of structural information related to SARS-CoV-2 RNA synthesis, in combination with previous structural, genetic and biochemical studies, have vastly improved our understanding of how CoVs replicate and process their genomic RNA. Structural biology not only provides a blueprint for understanding the function of the enzymes and cofactors in molecular detail, but also provides a basis for drug design and optimization. The concerted efforts of researchers around the world, in combination with the renewed urgency toward understanding this deadly family of viruses, may eventually yield new and improved antivirals that provide relief to the current global devastation.

Nobel Prize in Chemistry 2020: The great breakthrough from frontier research in microbiology

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for their joint development of the highly efficient CRISPR-Cas9 genome editing tools, which have taken the life sciences into a new era. The CRISPR-Cas system was first discovered in bacteria in 1987. After more than 20 years of arduous exploration of these enigmatic systems that were found in more than 90% archaea and about half bacteria, their antiviral function was finally confirmed in 2007. This has inspired more researchers to focus on the CRISPR-Cas systems, of which the general anti-virus mechanisms were also uncovered within the next few years, that the crRNA containing the virus sequence information guides the Cas protein(s) to recognize the corresponding virus and cleave their DNA or RNA. Among those pioneers in this field, Charpentier and Doudna mainly focused on the type II CRISPR-Cas9 system, which is much simpler than the wide-spread type I systems. In 2012, Doudna and Charpentier had elucidated the fine structure of the anti-virus effector complex of the CRISPR-Cas9 system, which contains a single Cas9 protein and two RNA components (crRNA and tracrRNA). It was this work that led to the breakthrough in developing the CRISPR-Cas9 genome editing tools. Significantly, they innovatively connected the tracrRNA and crRNA into a single RNA chimera (sgRNA), greatly simplifying the genome editing tools to only two components, the sgRNA and the single Cas9 protein. Compared with the genome editing tools developed in the past (Meganuclease, ZFN and TALEN), CRISPR-Cas9 has significant advantages such as easy programming, high efficiency and a wide application range. These merits make CRISPR-Cas9 so far the most widely used genome editing technique. In basic research, CRISPR-Cas9 genome editing has been used to elucidate the functions of unknown genes or pathways. In application, this technique has greatly improved the efficiency of animal and plant breeding, and made it possible to cure some genetic diseases and even cancers. Moreover, based on this CRISPR-Cas9 platform, other advanced genome editing tools have also been developed, such as base editing and prime editing, etc. It is also believed that, in addition to CRISPR-Cas systems, there are still many other undiscovered antiviral systems in microbes, as well as a large number of unknown nucleic acid recognition and processing elements, which are expected to optimize or upgrade the current genome editing tools, or even innovate entirely new genome editing tools. The CRISPR-Cas genome editing tools were developed from the bacterial anti-virus immune systems, thus the 2020 Nobel Prize in Chemistry is essentially a reward to the frontier research in microbiology. In this article, we will briefly introduce i) the timeline for the development of genome editing tools; ii) the historical background of the CRISPR-Cas research; iii) the breakthrough works for the birth of CRISPR-Cas9 genome editing technology. The future frontier research in microbiology and big data mining to further optimize or innovate genome editing tools is also prospected.

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a.

CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system’s specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.

RNA Editing in Interferonopathies.

The type I interferonopathies comprise a heterogenous group of monogenic diseases associated with a constitutive activation of type I interferon signaling.The elucidation of the genetic causes of this group of diseases revealed an alteration of nucleic acid processing and signaling.ADAR1 is among the genes found mutated in patients with this type of disorders.This enzyme catalyzes the hydrolytic deamination of adenosines in inosines within a double-stranded RNA target (RNA editing of A to I). This RNA modification is widespread in human cells and deregulated in a variety of human diseases, ranging from cancers to neurological abnormalities.In this review, we briefly summarize the knowledge about the RNA editing alterations occurring in patients with mutations in ADAR1 gene and how these alterations might cause the inappropriate IFN activation.

APOBEC3-dependent kataegis and TREX1-driven chromothripsis during telomere crisis.

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1–5. Here we examine the mechanism underlying chromothripsis and kataegis using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In absence of TREX1, the genome alterations induced by telomere crisis primarily involve Breakage-Fusion-Bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.

UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor.

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

Host nucleases are implicated in antiviral response through the processing of pathogen-derived nucleic acids. Among many host RNases, decapping enzymes DCP1 and 2, and 5′→3′ exonuclease XRN1, which are components of the RNA decay machinery, have been extensively studied in prokaryotes, plants, and invertebrates but less so in mammalian systems. As a result, the implication of XRN1 and DCPs in viral replication, in particular, the spatio-temporal dynamics during RNA viral infections remains elusive. Here, we highlight that XRN1 and DCPs play a critical role in limiting several groups of RNA viral infections. This antiviral activity was not obvious in wild-type cells but clearly observed in type I interferon (IFN-I)-deficient cells. Mechanistically, infection with RNA viruses induced the enrichment of XRN1 and DCPs in viral replication complexes (vRCs), hence forming distinct cytoplasmic aggregates. These aggregates served as sites for direct interaction between XRN1, DCP1/2, and viral ribonucleoprotein that contains viral RNA (vRNA). Although these XRN1-DCP1/2-vRC-containing foci resemble antiviral stress granules (SGs) or P-body (PB), they did not colocalize with known SG markers and did not correlate with critical PB functions. Furthermore, the presence of 5′ mono- and 5′ triphosphate structures on vRNA was not required for the formation of XRN1-DCP1/2-vRC-containing foci. On the other hand, single-, double-stranded, and higher-ordered vRNA species play a role but are not deterministic for efficient formation of XRN1-DCP1/2 foci and consequent antiviral activity in a manner proportional to RNA length. These results highlight the mechanism behind the antiviral function of XRN1-DCP1/2 in RNA viral infections independent of IFN-I response, protein kinase R and PB function.

Structural modification of ellipticine derivatives with alkyl groups of varying length is influential on their effects on human DNA topoisomerase II: a combined experimental and computational study

The compounds reducing tumor cell viability and disrupting DNA topoisomerase reactions have been widely used in anticancer drug development. Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is a potent intercalating agent that interferes with nucleic acid processing through interaction with DNA topoisomerase II. Although ellipticine is a well-characterized compound, it is not a widely-accepted drug due to the adverse effects detected upon administration. We have previously reported two novel ellipticine derivatives, N-methyl-5-demethyl ellipticine (ET-1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2) as potent compounds targeting DNA topoisomerase II. This study covers an extended synthesis, characterization, and activity data for five new salts of N-methyl 5-demetyl ellipticine (Z-1, Z-2, Z-4, Z-5 and Z-6) having several organic halides and their effects on human topoisomerase II enzymes. Moreover, combined in silico studies were conducted for better understanding of modes of action of studied molecules at the binding pocket of target. Our results showed that three of the derivatives (Z-1, Z-2, and Z-6) have considerable effect on the catalytic activity of human topoisomerase II implying the influence of alkyl groups added to the parental structure of ellipticine.

Free energy of cylindrical polyions: Analytical results.

Within the Poisson-Boltzmann (PB) framework useful for a wealth of charged soft matter problems, we work out the Coulombic grand potential of a long cylindrical charged polyion in a binary electrolyte solution of arbitrary valency and for low salt concentration. We obtain the exact analytical low-salt asymptotic expression for the grand potential, derived from the known properties of the exact solutions to the cylindrical PB equation. These results are relevant for understanding nucleic acid processes. In practice, our expressions are accurate for arbitrary polyion charges, provided their radius is smaller than the Debye length defined by the electrolyte.

Multi-level patterning nucleic acid photolithography

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage.

Detection and characterization of TDP-43 in human cells and tissues by multiple reaction monitoring mass spectrometry

Transactive response DNA-binding protein 43 kDa (TDP-43) is a highly conserved and widely expressed protein in human tissues that regulates nucleic acid processing. In frontotemporal dementia and amyotrophic lateral sclerosis, however, TDP-43 forms insoluble aggregates in central nervous tissues. These pathological deposits of TDP-43 have been primarily studied by ligand binding, namely western blot analysis, and, thus, methods with greater structural resolution are needed to aid in our understanding of the pathological processes associated with TDP-43 misfolding and aggregation. Toward this goal, we have developed a selective and multiplex method for the detection and characterization of TDP-43 using liquid chromatography tandem mass spectrometry. As proof-of-concept, the method was applied to the detection and characterization of TDP-43 in human cell lines and human brain tissue.

Rational design of an XNA ligase through docking of unbound nucleic acids to toroidal proteins.

Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.