Linear DNA Research Articles

Characterization and Genomic Analysis of a Novel Lytic Phage DCp1 against Clostridium perfringens Biofilms

Clostridium perfringens (C. perfringens) is one of the foremost pathogens responsible for diarrhea in foals. As antibiotic resistance increases, phages that specifically lyse bacteria are of great interest to us with regard to C. perfringens. In this study, a novel C. perfringens phage DCp1 was isolated from the sewage of a donkey farm. Phage DCp1 had a non-contractile short tail (40 nm in length) and a regular icosahedral head (46 nm in diameter). Whole-genome sequencing indicated that phage DCp1 had a linear double-stranded DNA genome with a total length of 18,555 bp and a G + C content of 28.2%. A total of 25 ORFs were identified in the genome, 6 of which had been assigned to functional genes, others were annotated to encode hypothetical proteins. The genome of phage DCp1 lacked any tRNA, virulence gene, drug resistance gene, or lysogenic gene. Phylogenetic analysis indicated that phage DCp1 belonged to the family Guelinviridae, Susfortunavirus. Biofilm assay showed that phage DCp1 was effective in inhibiting the formation of C. perfringens D22 biofilms. Phage DCp1 could completely degrade the biofilm after 5 h of interaction. The current study provides some basic information for further research on phage DCp1 and its application.

Characterization of a lytic Escherichia coli phage CE1 and its potential use in therapy against avian pathogenic Escherichia coli infections.

The high incidence of Avian pathogenic Escherichia coli (APEC) in poultry has resulted in significant economic losses. It has become necessary to find alternatives to antibiotics due to the alarming rise in antibiotic resistance. Phage therapy has shown promising results in numerous studies. In the current study, a lytic phage vB_EcoM_CE1 (short for CE1) against Escherichia coli (E. coli) was isolated from broiler feces, showing a relatively wide host range and lysing 56.9% (33/58) of high pathogenic strains of APEC. According to morphological observations and phylogenetic analysis, phage CE1 belongs to the Tequatrovirus genus, Straboviridae family, containing an icosahedral capsid (80 ~ 100 nm in diameter) and a retractable tail (120 nm in length). This phage was stable below 60°C for 1 h over the pH range of 4 to 10. Whole-genome sequencing revealed that phage CE1 contained a linear double-stranded DNA genome spanning 167,955 bp with a GC content of 35.4%. A total of 271 ORFs and 8 tRNAs were identified. There was no evidence of virulence genes, drug-resistance genes, or lysogeny genes in the genome. The in vitro test showed high bactericidal activity of phage CE1 against E. coli at a wide range of MOIs, and good air and water disinfectant properties. Phage CE1 showed perfect protection against broilers challenged with APEC strain in vivo. This study provides some basic information for further research into treating colibacillosis, or killing E. coli in breeding environments.

Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coliStrains: To Achieve Better Solubility of Putrescine Monooxygenase.

The online version contains supplementary material available at 10.1007/s12088-023-01056-x.

A single molecule TIRF-based platform to study DNA supercoiling effect on DNA processing enzymes.

DNA topology plays an important role in regulating DNA metabolic pathways. DNA supercoiling changes the DNA properties and proteins may differentially interact with DNA under various supercoiling states. A biophysical investigation of how proteins differentiate various topological states could further our understanding of protein-DNA interactions. We developed new single molecule total internal reflection fluorescence (smTIRF) -based platform to investigate how DNA topology affects protein-DNA interactions. We used this platform to study MutS, which detects a mismatched base pair to initiate mismatch repair, and CRISPR-Cas9. We generated plasmids that are site-specifically labeled with one or two fluorophores and a biotin. We prepared them in three different topological states: “Linear”, “relaxed circular”, and “negatively supercoiled”. For MutS study, we designed a Cy5 labeled plasmid with an adjacent mismatch. We observed FRET between Cy3-MutS and Cy5 when MutS bound the mismatch. In the presence of ADP, MutS bound the negative supercoiled DNA with a longer residence time compared to the relaxed circular DNA, suggesting that negative supercoiling helps mismatch recognition. For CRISPR-Cas9, we created a dual labeled plasmid to observe R-loop formation induced by dCas9. R-loop formation rate was similar for all three DNA topological states when the guide RNA sequence matched the target DNA sequence, while the rate was highest for the negatively supercoiled DNA and decreased progressively for the relaxed circular DNA and the linear DNA when we introduced one mismatch in either the PAM (protospacer adjacent motif)-proximal position or the PAM-distal position. Our observation confirms that DNA negative supercoiling assists DNA unwinding by Cas9, and this can exacerbate off-target effects in the cell. Collectively, our smTIRF-based platform allows high throughput investigation of supercoiling effects on DNA protein interactions as demonstrated using two different biological systems.

Using CIVT-SELEX to Select Aptamers as Genetic Parts to Regulate Gene Circuits in a Cell-Free System.

The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor-operator pairs. Researchers have tried to use aptamer-ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer-thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation.

Dynamic concatenation leads to anomalous transport of ring DNA in concentrated solutions.

Circular DNA polymers have been widely studied due to their biological relevance as well as the unique anomalous transport properties they exhibit in concentrated solutions and blends with linear DNA. In cells, topoisomerase II relaxes torsional strain in circular DNA by cutting and then reconnecting one of the strands of the double-stranded polymer, enabling replication, transcription, and repair. When topoisomerase II acts on nicked circular DNA at high concentration, the relaxed rings can concatenate, forming Olympic ring structures reminiscent of kinetoplasts. The rate of topoisomerase activity as well as the concentration and size of the DNA can tune the nonequilibrium dynamics and conformations of the concatemers. Here, we visualize the fluorescent-labeled DNA rings comprising the concatemers and use particle tracking and differential dynamic microscopy to characterize the dynamics and time-varying size and shape of the actively linking and unlinking molecules. Beyond the insight our work provides to biological processes mediated by active restructuring of DNA, the complex fluids we engineer and study may have applications in the design of biocompatible active materials for cellular repair, toxin filtration and drug delivery.

Measuring jumping during DNA target search.

We are investigating the DNA target search mechanisms of type II restriction endonucleases. Here we present our work in developing a method to visualize jumping by DNA binding proteins as they search a DNA. We have previously shown that EcoRI translocates along DNA with a salt dependent diffusion coefficient. We have shown that at low salt, the protein primarily slides in contact with the DNA, whereas at higher salt, hopping increasingly contributes to the one dimensional diffusion. Whereas hopping is from one position to a nearby position in a linear DNA, jumping occurs in coiled DNA and allows a DNA binding protein to land on a distant segment of the DNA. Jumping is difficult to visualize due to the rapidly fluctuating coiled DNA. In our method, we will alternately stretch and coil a DNA using dual optical tweezers, while tracking a fluorescently labeled EcoRI. Visualizing large displacement during coiling phases will be evidence of jumping events. We have expressed and purified a cleavage deficient mutant that we are labeling with a quantum dot to allow us to track the movement of the endonuclease in real time. We intend to use this labeled mutant in our single molecule tracking experiments.

DNA mini-circles reveal effects of DNA topology on CRISPR-Cas9 functions.

CRISPR-Cas9 is an RNA-guided nuclease that has been widely adapted for genome engineering. Prior to Cas9 cleavage, the protospacer of the target DNA must be unwound to form a three-stranded R-loop with a complementary segment of the Cas9-bound guide RNA. R-loop dictates the coordinated conformational changes allosterically controlling Cas9 nuclease activities, thus is the key in target discrimination. Current studies of Cas9 specificity focus primarily on protospacer sequences (i.e., mismatch(es) between the DNA and the RNA guide), while contributions from many collective DNA duplex properties (e.g., flexibility, topological constraints), which could significantly influence DNA unwinding and R-loop structure and dynamics, are not well-understood. In this work, DNA minicircles, which exhibit distinctive features (e.g., ring constraint, curvature), were used to delineate the role of DNA topology on Cas9 function. Two hairpins containing complementary overhangs were ligated to form a single-stranded DNA circle, which was then hybridized with linear complementary strand(s) to generate the double-stranded minicircle (dsMC). With a target imbedded in a 95 base-pair dsMC, the saturating Cas9 cleavage rate, kcat, was found to be ∼1000-fold slower as compared to the corresponding linear construct, although nicking the dsMC resulted in a rate comparable to the linear DNA. Increasing the size of the dsMC to 210 base-pair also led to a significant increase in kcat. The data clearly show that topological constraints imposed by an intact dsMC affect Cas9 cleavage. Work is ongoing to correlate these observed functional effects to the impact of dsMC on the Cas9 R-loop.

Experimental investigation of the forces dictating polymer translocation through solid-state nanopores.

After realizing direct long-read DNA and RNA sequencing, nanopores show also potential for decoding information on other polymers, including proteins and nanostructured DNA. Understanding the process by which these biopolymers are captured and traverse nanopores however remains a challenging task due to the complex nature of the non-equilibrium translocation dynamics which occur on multiple timescales and are dictated mainly by forces over which full experimental control is challenging. This unfortunately also makes verification of theoretical ideas difficult. In this work, we present solid-state nanopore data of DNA fragments for a wide range of experimental conditions in hopes of showcasing the various underlying forces and processes during capture and translocation. Namely, we specifically attempt to establish the absolute and relative magnitude of electrophoretic forces, and frictional contributions originating from hydrodynamic interactions both inside and outside nanopores. This is achieved by studying the dependence of translocation times on nanopore size (3-20 nm diameter) and DNA length (500-45,000 bp), and by measuring the time-dependent translocation velocity of DNA nanostructures, which consists of a linear DNA polymer with three helix-bundle (3HB) sub-structures patterned along its contour. The velocity profile of the patterned nanostructure is measured under different applied voltages (0.1-1 V), in different salt concentrations (0.5-4 M LiCl), and in different pore geometry. The data allow us to corroborate the predictions of tension propagation and consolidate previously published observations regarding measurements of electrophoretic pulling force and pore-polymer interactions in nanopore systems. In addition to bridging nanopore experiment and theory, these results can be used to inform and design several biosensing and digital storage applications.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

T7Max transcription system

BackgroundEfficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.ResultsHere we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.ConclusionsThe modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Recombineering-Mediated Sinorhizobium meliloti Rm1021 Gene Deletion.

Sinorhizobium meliloti Rm1021 (S. meliloti Rm1021) is a Gram-negative, soil-dwelling α-proteobacterium which serves as a model microorganism for the studies of symbiotic nitrogen fixation. The S. meliloti Rm1021 genome consists of one chromosome and two megaplasmids, pSymA and pSymB. Gene deletion is an essential tool for the elucidation of gene function and generation of mutants with improved properties. However, only two gene deletion methods, counterselectable marker sacB-based and FLP/FRT, Cre/loxP site-specific recombination, have been reported for S. meliloti Rm1021 gene deletion. Both methods require time-consuming and tedious gene cloning and conjugation steps. Herein, a λ Red recombineering-mediated gene deletion strategy is reported. The mutant was obtained via electroporating overlap-extension PCR-generated linear targeting DNA into Red-proficient cells. One gene each from the S. meliloti Rm1021 chromosome, megaplasmid SymA and pSymB was deleted, with deletion efficiency up to 100%. The straightforward and highly efficient recombineering procedure holds the promise to be a general gene manipulation method for S. meliloti Rm1021.

Loss of a Branch Sugar in the Acinetobacter baumannii K3-Type Capsular Polysaccharide Due To Frameshifts in the gtr6 Glycosyltransferase Gene Leads To Susceptibility To Phage APK37.1.

The type of capsular polysaccharide (CPS) on the cell surface of Acinetobacter baumannii can determine the specificity of lytic bacteriophage under consideration for therapeutic use. Here, we report the isolation of a phage on an extensively antibiotic resistant ST2 A. baumannii isolate AB5001 that carries the KL3 CPS biosynthesis gene cluster predicting a K3-type CPS. As the phage did not infect isolates carrying KL3 or KL22 and known to produce K3 CPS, the structure of the CPS isolated from A. baumannii AB5001 was determined. AB5001 produced a variant CPS form, K3-v1, that lacks the β-d-GlсpNAc side chain attached to the d-Galp residue in the K3 structure. Inspection of the KL3 sequence in the genomes of AB5001 and other phage-susceptible isolates with a KL3 locus revealed single-base deletions in gtr6, causing loss of the Gtr6 glycosyltransferase that adds the missing d-GlсpNAc side chain to the K3 CPS. Hence, the presence of this sugar profoundly restricts the ability of the phage to digest the CPS. The 41-kb linear double-stranded DNA (dsDNA) phage genome was identical to the genome of a phage isolated on a K37-producing isolate and thus was named APK37.1. APK37.1 also infected isolates carrying KL116. Consistent with this, K3-v1 resembles the K37 and K116 structures. APK37.1 is a Friunavirus belonging to the Autographiviridae family. The phage-encoded tail spike depolymerase DpoAPK37.1 was not closely related to Dpo encoded by other sequenced Friunaviruses, including APK37 and APK116. IMPORTANCE Lytic bacteriophage have potential for the treatment of otherwise untreatable extensively antibiotic-resistant bacteria. For Acinetobacter baumannii, most phage exhibit specificity for the type of capsular polysaccharide (CPS) produced on the cell surface. However, resistance can arise via mutations in CPS genes that abolish this phage receptor. Here, we show that single-base deletions in a CPS gene result in alteration of the final structure rather than deletion of the capsule layer and hence affect the ability of a newly reported podophage to infect strains producing the K3 CPS.

Integrated analysis of mRNA and extrachromosomal circular DNA profiles to identify the potential mRNA biomarkers in breast cancer

Extrachromosomal circular DNAs (eccDNAs) have been proved an inseparable relationship with cancer, based on the biological mechanisms of its biogenesis and impact on tumorigenesis, but still lacked of methods to analyze its function on the pathogenesis and progression of breast cancer (BC). The mRNA and eccDNA from BC cell samples (MDA-MB-453 and MCF-12A) were extracted with the removal of rRNA and linear DNA, respectively. High-throughput sequencing and bioinformatics analysis were performed to explore their expression level and molecular characterization of eccDNA. A total number of 161,062 eccDNA ranging from 33 bp to 54229 bp were detected with a median size of 1143 bp, distributed on all chromosomes and enriched on chromosome 20 the most. EccDNAs located in exons, upstream and downstream 2 kb regions were significantly increased compared with background. Analysis of eccDNA-related differentially expressed genes (eccDEGs) showed that FAT2 properly separated the two cells. CTNNB1, CACNA2D2 and CACNA1D were the hub genes with higher degrees in critical modules. All these four genes were significantly differentially expressed between breast invasive carcinoma (BRCA) tissues and normal ones. FAT2 and CTNNB1 correlated with significantly different overall survival (OS) when differentially expressed. The four genes showed a strong correlation with each other significantly and changed between tumor and normal samples. The results showed the potential of FAT2, CTNNB1, CACNA2D2 and CACNA1D as biomarkers with analysis of both DEGs and eccDEGs, which might assist in clinical medical treatment.

Enhanced production of d-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR–Cpf1 genome editing method

BackgroundCorynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum.ResultsIn this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only.ConclusionsWe developed a RecET-assisted CRISPR–Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.

A linear DNA encoding the SARS-CoV-2 receptor binding domain elicits potent immune response and neutralizing antibodies in domestic cats

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of the COVID-19 pandemic, has been shown to infect a wide range of animal species, especially mammals, and besides human-to-human transmission, human-to-animal transmission has also been observed in some wild animals and pets, especially in cats. It has been demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a clinical trial conducted in domestic cats to assess safety and immunogenicity of a linear DNA (linDNA) vaccine encoding the receptor-binding domain (RBD) from SARS-CoV-2 (Lin-COVID-eVax). Lin-COVID-eVax proved to be safe, with no significant adverse events, and was able to elicit both RBD-specific antibodies and Tcells. Also, the linDNA vaccine induced neutralizing antibody titers against ancestral SARS-CoV-2 virus and its variants. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.

A novel tetranuclear Cu(ii) complex for DNA-binding and in vitro anticancer activity.

A novel tetranuclear Cu(ii) complex (TNC) was successfully synthesized and characterized by X-ray single crystal diffraction. The interaction of the complex with calf thymus DNA (CT-DNA) has been studied by UV-vis absorption titration, fluorescence technology and molecular docking. The results indicated that TNC could bind to the DNA through an intercalative mode. The agarose gel electrophoresis experiment showed that TNC could cleave supercoiled plasmid DNA into linear DNA. The anticancer activity of TNC was tested on four cancer cell lines: MCF7, A549, 4T1 and HepG2. The results indicated that TNC shown significant activity against all of above cell lines.

A newly isolated bacteriophage vB8388 and its synergistic effect with aminoglycosides against multi-drug resistant Klebsiella oxytoca strain FK-8388.

The bacteriophage vB8388 can lyse multi-drug resistant Klebsiella oxytoca strain FK-8388 and maintain stability in a wide range of temperatures (from 4°C to 80°C) and pHs (3-11). Bioinformatics analysis showed that vB8388 is a linear double-stranded DNA virus that is 39,750 long with 50.65% G+C content and 44 putative open reading frames (ORFs). Phage vB8388 belongs to the family Autographviridae and possesses a non-contractile tail. The latency period of vB8388 was approximately 20min. The combination of phage vB8388 and gentamicin, amikacin, or tobramycin could effectively inhibit the growth of K. oxytoca strain FK-8388, with a decrease of more than 4 log units within 12h in vitro. Phage vB8388 showed a strong synergistic effect with gentamicin that could enhance the anti-biofilm effect of vB8388. The phage+gentamicin combination also showed synergy in vivo in the larval infection model of Galleria mellonella. In conclusion, the findings of this study suggest the potential of phage+antibiotic combination therapy to be used as an alternative therapeutic approach for treating infectious diseases caused by multidrug-resistant bacteria.

Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.

Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.

Spectrophotometric and nucleic acid-binding properties of halloysite clay nanotubes and kaolinite

Halloysite particles (HNTs) are naturally occurring aluminosilicate nanotubes of low toxicity that have shown great promise for drug and biomolecule delivery into human and animal cells. Kaolinite particles retain the same layered structure as HNT, but do not form nanotubes. In this study, the spectrophotometric and sedimentation properties of the two clays in aqueous solutions and their abilities to associate with both small and large nucleic acids have been investigated. Both clays scattered ultraviolet light strongly and this characteristic of HNT was not affected by either vacuum treatment to remove trapped gases or by sonication. Vacuum treatment increased the binding of small nucleic acids to HNT and this association was further enhanced by addition of divalent metal ions. By contrast, only small RNAs were bound efficiently by kaolinite in the presence of Mg2+ ions. Large linear double-stranded DNAs and circular plasmid DNAs bound poorly to kaolinite under all conditions, but these nucleic acids could form strong associations with HNT. Differences in binding data were largely consistent with measurements of the available surface areas of each clay. These results demonstrate that interactions with each clay are critically dependent on both the type and the conformation of each nucleic acid.