Efficient Separation of Nucleic Acids with Different Secondary Structures by Metal-Organic Frameworks.

We report the use of metal-organic frameworks (MOFs) for the selective separation of nucleic acids (DNA and RNA) with different secondary structures through size, shape, length, and capability of conformational transition. Three MOFs with precisely controlled pore environments, Co-IRMOF-74-II, -III, and -IV, composed of Co2+ and organic linkers (II, III, and IV), respectively, were used for the inclusion of nucleic acid into their pores from the solution. This was proven to be a spontaneous process from disordered free state to restricted ordered state via circular dichroism (CD) spectroscopy. Three critical factors were identified for their inclusion: (1) size selection induced by steric hindrance, (2) conformation transition energy selection induced by stability, and (3) molecular weight selection. These selection rules were used to extract nucleic acids with flexible and unstable secondary structures from complex mixtures of multiple nucleic acids, leaving those with rigid and stable secondary structures in the mother liquor. This provides the possibility to separate and enrich nucleic acids in bulk through their different structure feature, which is highly desirable in genome-wide structural measurement of nucleic acids. Unlike methods that rely on specific binding antibodies or ligand, this MOF method is capable of selecting all kinds of nucleic acids with similar secondary structure features; therefore, it is suitable for the handling of a large variety and quantity of nucleic acids at the same time. This method also has the potential to gather information about the folding stability of biomolecules with secondary structures.

Mechanism of sequence-based separation of single-stranded DNA in capillary zone electrophoresis.

Separation of DNA by length using CGE is a mature field. Separation of DNA by sequence, in contrast, is a more difficult problem. Existing techniques generally rely upon changes in intrinsic or induced differences in conformation. Previous work in our group showed that sets of ssDNA of the same length differing in sequence by as little as a single base could be separated by CZE using simple buffers at high ionic strength. Here, we explore the basis of the separation using circular dichroism spectroscopy, fluorescence anisotropy, and small angle X-ray scattering. The results reveal sequence-dependent differences among the same length strands, but the trends in the differences are not correlated to the migration order of the strands in the CZE separation. They also indicate that the separation is based on intrinsic differences among the strands that do not change with increasing ionic strength; rather, increasing ionic strength has a greater effect on electroosmotic mobility in the normal direction than on electrophoretic mobility of the strands in the reverse direction. This increases the migration time of the strands in the normal direction, allowing more time for the same-length strands to be teased apart based on very small differences in the intrinsic properties of the strands of different sequence. Regression analysis was used to model the intrinsic differences among DNA strands in order to gain insight into the relationship between mobility and sequence that underlies the separation.

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels.

Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 µm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O3 activated and an aqueous buffer mobile phase.

Non-hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single-stranded DNA.

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label-free, and economical non-hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single-stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single-base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84-mer, in which guanine to adenine single-base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.

Construction of a system for single-stranded DNA isolation.

OBJECTIVE: The system of Strep-Tactin and StrepII tag-SSB proteins binding (ST-SSB) was established to isolate the purified single-stranded DNA in a single step with low cost and high efficiency. RESULTS: We demonstrate that in the presence of large amounts of dsDNA, the ssDNA binding specificity of Escherichia coli (E. coli) single stranded DNA binding (EcSSB) protein was stronger than gene-5-protein (g5p). ST-SSB system relies on the affinity between Strep-Tactin, StrepII tag-SSB protein and ssDNA in binding buffer. Here, we successfully isolated the purified ssDNA from mixed DNA (ds- and ss-DNA form) samples and asymmetric polymerase chain reaction (aPCR) products. This system can purify ssDNA in a single tube within 1 h, and the recovery efficiency of purified ssDNA was around 60%. CONCLUSIONS: The ST-SSB system has obvious advantages of high efficiency and one-step purification to recycle any ssDNA.

Recent Progress in the Identification of Aptamers Against Bacterial Origins and Their Diagnostic Applications.

Aptamers have gained an increasing role as the molecular recognition element (MRE) in diagnostic assay development, since their first conception thirty years ago. The process to screen for nucleic acid-based binding elements (aptamers) was first described in 1990 by the Gold Laboratory. In the last three decades, many aptamers have been identified for a wide array of targets. In particular, the number of reports on investigating single-stranded DNA (ssDNA) aptamer applications in biosensing and diagnostic platforms have increased significantly in recent years. This review article summarizes the recent (2015 to 2020) progress of ssDNA aptamer research on bacteria, proteins, and lipids of bacterial origins that have implications for human infections. The basic process of aptamer selection, the principles of aptamer-based biosensors, and future perspectives will also be discussed.

Discrimination of single-stranded DNA homopolymers by sieving out G-quadruplex using tiny solid-state nanopores.

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single-base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G-quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20-nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105-nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore-based DNA sequencing at single-base level.

Inside the Black Box: What Makes SELEX Better?

Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.

Abnormal Anionic Porphyrin Sensing Effect for HER2 Gene Related DNA Detection via Impedance Difference between MWCNTs and Single-Stranded DNA or Double-Stranded DNA.

Human epidermal growth factor receptor 2 (HER2) is a key tumor marker for several common and deadly cancers. It is of great importance to develop efficient detection methods for its over-expression. In this work, an electrochemical impedance spectroscopy (EIS) method adjustable by anionic porphyrin for HER2 gene detection has been proposed, based on the impedance difference between multi-walled carbon nanotubes (MWCNTs) and DNA. The interesting finding herein is that with the addition of anionic porphyrin, i.e., meso-tetra(4-sulfophenyl)-porphyrin (TSPP), the impedance value obtained at a glass carbon electrode (GCE) modified with MWCNTs and a single stranded DNA (ssDNA), the probe DNA that might be assembled tightly onto MWCNTs through π-π stacking interaction, gets a slight decrease; however, the impedance value from a GCE modified with MWCNTs and a double stranded DNA (dsDNA), the hybrid of the probe DNA with a target DNA, which might be assembled loosely onto MWCNTs for the screening effect of phosphate backbones in dsDNA, gets an obvious decrease. The reason may be that on the one hand, being rich in negative sulfonate groups, TSPP will try to push DNA far away from CNTs surface due to its strong electrostatic repulsion towards DNA; on the other hand, rich in planar phenyl or pyrrole rings, TSPP will compete with DNA for the surface of CNTs since it can also be assembled onto CNTs through conjugative interactions. In this way, the "loosely assembled" dsDNA will be repelled by this anionic porphyrin and released off CNTs surface much more than the "tightly assembled" ssDNA, leading to a bigger difference in the impedance value between dsDNA and ssDNA. Thus, through the amplification effect of TSPP on the impedance difference, the perfectly matched target DNA could be easily determined by EIS without any label. Under the optimized experimental conditions, this electrochemical sensor shows an excellent linear response to target DNA in a concentration range of 2.0 × 10-11⁻2.0 × 10-6 M with a limit of detection (LOD) of 6.34 × 10-11 M (S/N = 3). This abnormally sensitive electrochemical sensing performance resulting from anionic porphyrin for DNA sequences specific to HER2 gene will offer considerable promise for tumor diagnosis and treatment.

Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides.

Non-viral vector-mediated transfection is a core technique for in vitro screening of oligonucleotides. Despite the growing interests in the development of oliogonucleotide-based drug molecules in recent years, a comprehensive comparison of the transfection efficacy of commonly used commercial transfection reagents has not been reported. In this study, five commonly used transfection reagents, including Lipofectamine 3000, Lipofectamine 2000, Fugene, RNAiMAX and Lipofectin, were comprehensively analyzed in ten cell lines using a fluorescence imaging-based transfection assay. Although the transfection efficacy and toxicity of transfection reagents varied depending on cell types, the toxicity of transfection reagents generally displayed a positive correlation with their transfection efficacy. According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. We firmly believe that our findings may contribute to the future In vitro delivery and screening of single-stranded therapeutic oligonucleotides such as antisense oligonucleotides, antimiRs, and DNAzymes.

Selective Nascent Polymer Catch-and-Release Enables Scalable Isolation of Multi-Kilobase Single-Stranded DNA.

Scalable methods currently are lacking for isolation of long ssDNA, an important material for numerous biotechnological applications. Conventional biomolecule purification strategies achieve target capture using solid supports, which are limited in scale and susceptible to contamination owing to nonspecific adsorption and desorption on the substrate surface. We herein disclose selective nascent polymer catch and release (SNAPCAR), a method that utilizes the reactivity of growing poly(acrylamide-co-acrylate) chains to capture acrylamide-labeled molecules in free solution. The copolymer acts as a stimuli-responsive anchor that can be precipitated on demand to pull down the target from solution. SNAPCAR enabled scalable isolation of multi-kilobase ssDNA with high purity and 50-70 % yield. The ssDNA products were used to fold various DNA origami. SNAPCAR-produced ssDNA will expand the scope of applications in nanotechnology, gene editing, and DNA library construction.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).

Introducing an artificial photo-switch into a biological pore: A model study of an engineered α-hemolysin.

In recent years, engineered biological pores responsive to external stimuli have been fruitfully used for various biotechnological applications. Moreover, the strategy of tethering photo-switchable moieties into biomolecules has provided an unprecedented temporal control of purposely designed nanodevices, as demonstrated, for example, by the light-mediated regulation of the activity of enzymes and biochannels. Inspired by these advancements, we propose here a de novo designed nanodevice featuring the α-hemolysin (αHL) membrane channel purposely functionalized by an artificial "on/off" molecular switch. The switch, which is based on the photo-isomerization of the azobenzene moiety, introduces a smart nano-valve into the natural non-gated pore to confer tunable transport properties. We validated through molecular dynamics simulations and free energy calculations the effective inter-conversion of the engineered αHL pore between two configurations corresponding to an "open" and a "closed" form. The reported switchable translocation of a single-stranded DNA fragment under applied voltage supports the promising capabilities of this nanopore prototype in view of molecular sensing, detection and delivery applications at single-molecule level.

Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production.

The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concentration of produced ssDNA on the other hand. Using a growth rate of 0.15 h-1 and a multiplicity of infection of 0.05 pfu cfu-1 in the fed-batch production process, the concentration of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L-1 . Thus, our results help enabling M13 production for industrial uses in nanobiotechnology. Biotechnol. Bioeng. 2017;114: 777-784. © 2016 Wiley Periodicals, Inc.

Separation of single-base sequential isomers of single-stranded DNA by capillary electrophoresis and its application in the discrimination of single-base DNA mutations.

Separation of single-base substitution sequential DNA isomers remains one of the most challenging tasks in DNA separation by capillary electrophoresis. We developed a simple, versatile capillary electrophoresis technique for the separation of single-base sequential isomers of DNA having the same chain length. This technique is based on charge differences resulting from the different protonation (acid dissociation) properties of the four DNA bases. A mixture of 13 single-base sequential isomers of 12-mer single-stranded DNA was separated by using an electrophoretic buffer solution containing 20 mM phosphoric acid (pH 2.0) and 8 M urea. We demonstrated that our method could separate all possible mutation patterns under identical experimental conditions. In addition, application of our method to the separation of the polymerase chain reaction product of a 68-mer gene fragment and its single-base isomers indicates that in combination with the appropriate genomic DNA extraction techniques, the method can detect single-base gene mutations.

Sequence-based separation of single-stranded DNA at high salt concentrations in capillary zone electrophoresis.

DNA separation by fragment length can be readily achieved using sieving gels in electrophoresis. Separation by sequence has not been as simple, generally requiring adequate differences in native or induced conformation between single or hybridized strands or differences in thermal or chemical stability of hybridized strands. Previously, it was shown that four single-stranded DNA (ssDNA) 76-mers that differ by only a few A-G substitutions could be separated based solely on sequence by adding guanosine-5'-monophosphate to the running buffer in capillary zone electrophoresis (CZE). The separation was attributed to interactions of the ssDNA with self-assembled guanine-tetrad structures; however, subsequent studies of an expanded set of ten 76-mers showed that the separation was a more general phenomenon that occurred at high salt concentrations. With the long-term goal of using experimental and computational methods to provide insight into the basis of the separation, a set of ssDNA 15-mers was designed including a poly(dT) 15-mer and nine variants. Separations were performed using fluorescent-labeled ssDNA in CZE with laser-induced fluorescence detection. Results show that separation improves with increasing buffer concentration and decreasing temperature, due at least in part to longer separation times. Migration times increase with increasing purine content, with A having a much larger effect that G. Circular dichroism spectra of the mixtures of the strands suggest that the separation is not due to changes in conformation of the ssDNA at high salt concentrations.

Characterization of pharmacological properties of isolated single-stranded DNA aptamers against angiotensin II.

Nucleic acid aptamers can be served as drugs, carriers and diagnostic probes in living systems. Before recruiting aptamers, their pharmacological characteristics should be determined. Here we intended to investigate four important properties of isolated ssDNA anti-angiotensin II aptamers (FLC112 and FLC125) including hemolytic activity, cytotoxicity, immunogenicity and serum stability through in vitro and in vivo models. The hemolytic effect and cytotoxicity potential of aptamers were measured through hemolysis test and MTT assay respectively. In the following test, the humoral immune responses to aptamers in BALB/c mice were assessed. The human serum stability of aptamers was also determined using real-time PCR (qPCR). The results of this study revealed that the FLC112 aptamer with its unique structure had slightly higher cytotoxicity and hemolysis effect (9.14% and 0.1 ± 0.037% respectively) relative to FLC125 (8.07% and 0.08 ± 0.045% respectively) at the highest concentration (5 µM). FLC112 showed ignorable immune response in mice and barely higher than FLC125. Serum stability test confirmed that FLC112 with 12 h had more nuclease stability than FLC125 with 8 h. Aptamer molecule analysis revealed that the structure, sequense composition and motifs are the determinative parameters in aptamer pharmacological properties.

Identification, sequencing and comparative analysis of pBp15.S plasmid from the newly described entomopathogen Bacillus pumilus 15.1.

The Bacillus pumilus 15.1 strain, a recently described entomopathogenic strain active against Ceratitis capitata, contains at least two extrachromosomal elements, pBp15.1S and pBp15.1B. Given that B. pumilus is not a typical entomopathogenic bacterium, the acquisition of this extrachromosomal DNA may explain why B. pumilus 15.1 is toxic to an insect. One of the plasmids present in the strain, the pBp15.1S plasmid, was sub-cloned, sequenced and analyzed using bioinformatics to identify any potential virulence factor. The pBp15.1S plasmid was found to be 7785 bp in size with a GC content of 35.7% and 11 putative ORFs. A replication module typical of a small rolling circle plasmid and a sensing and regulatory system specific for plasmids was found in pBp15.1S. Additionally, we demonstrated the existence of ssDNA in plasmid preparations suggesting that pBp15.1S replicates by the small rolling circle mechanism. A gene cluster present in plasmid pPZZ84 from a distantly isolated B. pumilus strain was also present in pBp15.1S. The plasmid copy number of pBp15.1S in exponentially growing B. pumilus cells was determined to be 33 copies per chromosome. After an extensive plasmid characterization, no known virulence factor was found so a search in the other extrachromosomal elements of the bacteria is needed.

Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction.

Single-stranded DNA (ssDNA) intermediates are formed in multiple cellular processes, including DNA replication and recombination. Here, we describe a quantitative polymerase chain reaction (qPCR)-based assay to quantitate ssDNA intermediates, specifically the 3' ssDNA product of resection at specific DNA double-strand breaks induced by the AsiSI restriction enzyme in human cells. We protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing immunofluorescence-based methods.

Enzymatic preparation of multimilligram amounts of pure single-stranded DNA samples for material and analytical sciences.

We present a method for high-yield production of multimilligram amounts of pure single-stranded DNA employing rolling circle amplification (RCA) and processing by restriction enzymes. Pure and homogeneous samples are produced with minimal handling time, reagents, and waste products. The RCA method is more than twice as efficient in dNTP incorporation than conventional polymerase chain reaction in producing end product. The validity and utility of the method are demonstrated in the production of a uniformly (13)C/(15)N-labeled 38-nt cocaine aptamer DNA used in nanosensing devices.