Research Tools : DNA & RNA tools


Nanocapsule Delivery System for Ribonucleoproteins

UW–Madison researchers have engineered a biodegradable GSH-responsive nanocoating surrounding the sgRNA/Cas9 RNP complex for efficient delivery into cells. The RNP nanocapsule is a polymeric network synthesized from a mixture of (meth)acrylate monomers, acrylate crosslinkers and acrylate polyethylene glycol (PEG), built around the RNP cargo. The interactions between the RNP and the monomers include electrostatic and hydrophobic interactions and hydrogen bonding. Nanocapsule formation is completed by free radical polymerization.

Each component of this nanocapsule is essential for cellular delivery. Inclusion of the imidazole-containing monomer provided a mechanism for endosomal/lysosomal escape. Without this, the sgRNA/Cas9 RNP would be destroyed before exerting its effects. A crosslinker containing a disulfide (–S-S–) bond produced a covalently linked, yet biodegradable, shell around the cargo. And finally, to reduce potential recognition by the immune system and increase the circulation half-life, a PEG outer shell was introduced. The PEG outer shell also provides a chemical handle for attaching fluorescent dyes or targeting ligands onto the RNP nanocapsule.

S1mplex: A New Tool for Precision Gene Editing

UW–Madison researchers have developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to ‘sharpen the scalpels’ used in genome surgery. In the new approach, CRISPR-Cas9 RNPs are complexed with a nucleic acid donor template, as well as other biotinylated molecules (e.g., quantum dots).

In human pluripotent stem cells, tailored S1mplexes increased the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene editing methods, and enriched cell populations containing multiplexed precise edits up to 42-fold.

These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro/ex vivo gene editing applications in precision medicine and drug discovery, and aid in the development of increased and multiple dosing regimens for somatic gene editing in vivo.

Poly(UG) Polymerase: A Useful New RNA Tool

UW–Madison researchers have identified a poly(UG) polymerase in a roundworm called Caenorhabditis elegans. The newly discovered enzyme adds repeating UG sequences to the ends of RNA. This activity could be useful as a research tool in vitro, e.g., providing a new way to synthesize cDNA of RNAs of unknown sequence.

The gene in C. elegans that encodes the enzyme is called RDE-3. Although its sequence was already known, its polymerase activity was not.

New Protein Production Strategy for Plants

UW–Madison researchers have identified a new plant viral IRES that can facilitate the efficient expression of multiple proteins from a single mRNA. The researchers discovered the new IRES in the Triticum mosaic virus (TriMV), a wheat virus that expresses 10 proteins from a single mRNA strand.

Nanopore Antennas for Ultrahigh Speed DNA Sequencing

Building on their work, the researchers have now developed metallic nanopores for ultrahigh speed molecule sequencing. The new nanopores are electrically conductive and function as antennas, transmitting radiofrequency signals with utmost precision.

Unlike competing technology, the nanopores feature both genetically and electrically engineered components. They can be constructed of DNA attached with metal particles to enhance electromagnetic wave reception. This is achieved by replacing the side chains of the DNA molecule with sulfur groups that in turn link to gold particles. Metalized DNA strands or ‘arms’ can be added to increase antenna size and tune polarization.

Gene Editing Tool Uses New Enzyme

A UW–Madison researcher and collaborators have developed a method for RNA-directed DNA cleavage and gene editing using a new form of Cas9 enzyme. The new form, abbreviated NmCas9, comes from the Neisseria meningitides bacteria.

A CRISPR kit can be made containing RNA molecules that direct the new enzyme to bind to and cleave/nick a target sequence.

Streamlined Design for Transferring Analytes

The researchers have now improved their design and developed a microfluidic device that directly integrates with tubes, strip tubes and well plates. In this way a sample can be directly transferred from the device to downstream analysis instruments.

The device comprises a strip of wells that hold various volumes of output fluid. Following sample isolation via the researchers’ previously developed SLIDE technique, the strip containing the sample and output buffer is removed from the SLIDE and applied to a set of strip tubes in the same way that conventional covers would be applied.

Then, by flicking or centrifuging the tubes, the sample is transferred from the cap to the tube. At this point the sample is ready for PCR or other downstream analysis.

Device Uses Air Gap for Easier Fraction Isolation

UW–Madison researchers have developed a new device for isolating desired fractions from a biological sample. The device is made of two plates separated by a gap. The first plate has droplets of bound sample/PMPs positioned on the surface. A second plate containing another reagent is positioned below. A magnet pulls the PMP/sample from the first plate, through the air gap, onto the second plate.

Regulating Stem Cell Behavior with High Throughput Mineral Coatings

UW–Madison researchers have developed methods of non-viral cell transfection and regulating cell behavior using mineral coatings. The coatings bind polynucleotides and provide a source of calcium and phosphate ions to enhance transfection.

More specifically, a mineral coating is formed by incubating a substrate in a simulated body fluid (SBF). The substrate then is loaded with a polynucleotide (e.g., plasmids, mRNA or proteins), which binds to the coating. Next, a solution of cells is deposited and cultured until a desired level of transfection occurs.

Assembly of Full-Length Genes from DNA Arrays

UW–Madison researchers have developed a method to produce full-length genes using RNA intermediaries that are produced by run-off transcription from DNA array features. Specifically, an RNA polymerase promoter is appended to the surface-bound oligonucleotides. RNA copies are produced using T7 RNA polymerase and then self-assembled into full RNA transcripts by hybridization and ligation. The RNA transcripts can readily be converted into their corresponding genes using RT-PCR (reverse transcription polymerase chain reaction). These genes then may be employed to express the encoded protein of interest.

Faster, Cheaper DNA Synthesis

UW–Madison researchers have developed a high yield method for synthesizing oligonucleotides with defined sequences and soluble in solution.

In the new method, DNA starting material is synthesized in parallel on a microarray, amplified via polymerase chain reaction (PCR) and cleaved. The cleaved fragments then can be used to assemble a double-stranded DNA target sequence.

Hundreds of thousands of oligonucleotides can be synthesized and assembled into many individual genes by this process.

Cleaving Double-Stranded DNA at User-Chosen Sites

UW–Madison researchers have developed a method of using Ref to cleave double-stranded DNA at any desired target sequence. The researchers determined that Ref is a novel HNH class and RecA-dependent endonuclease. They have shown that Ref, in combination with RecA and a single-stranded DNA targeting oligonucleotide, can specifically cause cleavage of double-stranded DNA at a site complementary to the oligonucleotide.

Device for Efficiently Extracting a Fraction Containing Nucleic Acids or Other Desired Material from a Biological Sample

UW–Madison researchers have developed a device and method for extracting and purifying a desired fraction from cultured cells, tissue samples and other biological materials. A biological sample, including both non-desired material and a fraction-bound solid phase substrate, is added to an input zone. The input zone is adjacent to a separation zone that includes an isolation buffer. A force moves the fraction-bound solid phase substrate from the input zone, through the separation zone and into an output zone, leaving the non-desired material behind. The improved purification method is simple, more efficient and produces a higher throughput than prior devices and methods. The device may be configured to allow for quantification of the fraction in the biological sample via labeling of the fraction-bound solid phase substrate.

Method for Isolating Weakly Interacting Molecules from a Fluidic Sample

UW–Madison researchers have developed a method for isolating weakly interacting molecules from a fluidic sample using an immiscible phase filtration technique. A mixture is formed using a fluidic sample and a solid phase substrate including at least one immobilized molecule. The mixture is incubated under conditions that allow the immobilized molecule to interact with a target molecule in the fluidic sample to form a solid phase, substrate-immobilized molecule-target molecule complex. The complex is immediately transferred into an immiscible phase by applying an external force to the solid phase substrate.

Immiscible phase filtration allows for the isolation and identification of weakly interacting molecules from the fluidic sample that were previously unidentifiable using traditional methods, as the analyte is isolated very quickly when the solid phase enters the immiscible phase. The method also provides the capability of providing a “snapshot” of the molecular interactions at equilibrium, which is not possible in traditional methods requiring multiple washes. 

Double-Strand DNA Break Repair in Vitro – Forensic and Genomic Applications

UW–Madison researchers have developed a method for the in vitro joining of two DNA fragments that have homologous DNA sequences through a simplified process of DNA double-strand break repair.  This approach enables the sequencing of poor quality or minimally available DNA that may be in a complex mixture of contaminating DNA sequences.  It requires only three proteins or their homologues: RecA protein, single-stranded DNA binding protein (SSB) and DNA polymerase I.  The proteins preferably are obtained from E. coli

In this method, a single-stranded DNA probe with some homology to the target DNA is combined with RecA protein and SSB.  Then the target duplex DNA molecule, which has a double-strand break and is not super-coiled, is added to the mixture.  The single-strand probe invades the doubled-stranded target DNA.  When DNA polymerase is added with dNTPs, it extends both strands to create a double-stranded DNA molecule in which the two fragments have joined.  PCR reactions then can be carried out using primers that bind the probe and target DNA.  This will allow amplification of STRs, if there are lesions in the DNA close to an STR.

Microfluidic Device for Rapid Nucleic Acid Isolation and Purification

UW-Madison researchers have developed a microfluidic device and method for rapidly extracting and purifying nucleic acids from biological materials. The device includes an input zone for receiving the biological sample, a phase-gate zone for holding an isolation buffer and an output zone for receiving a reagent. The isolation buffer consists of oil that acts as a phase-gate and prevents unwanted material from passing through to the output well. The device employs an isolation mechanism using paramagnetic particles (PMPs), which preferentially bind the nucleic acids, and a lysis buffer, which inhibits the polymerase chain reaction until the nucleic acids have been isolated.

The isolation method proceeds through three main steps. First, the lysis buffer and PMPs are added to the biologic sample in the input well. Then, a magnetic field is used to draw the bound nucleic acid/PMPs through the phase-gate and into the output well, a process that lasts around 15 seconds. This simple phase-gate step replaces the multiple washes required in current methods. Finally, the nucleic acid/PMP reagent is collected for processing via polymerase chain reactions.

The device and method are configured to maximize isolation and purification of nucleic acids while maintaining compatibility with existing multiwell plates and liquid-handling robotic systems. Isolation and purification using this invention can be accomplished in approximately five minutes, including separation, device loading and sample collection. This provides a vast improvement over current kits, which require multiple washing steps and 15 to 45 minutes for purification.

Protein Inhibitor of Ran Activity

UW-Madison researchers have identified a specific inhibitor of Ran activity. They discovered that the encephalomyocarditis virus leader protein, a small protein with no measurable enzymatic activity, binds directly and specifically to Ran. Expression of this leader protein in human cells inhibits Ran activity, blocking nuclear export of cellular mRNAs and leading to reduced translational activity.

Improved Methods and Materials for Transforming Plant Cells

UW-Madison researchers have developed a method of using a DNA-launching platform to introduce viral RNA into a host cell that has been engineered to support viral replication and expression. The platform encodes a modified viral RNA molecule located downstream of a DNA-dependent RNA polymerase promoter. After the platform is introduced into a host cell, it effectively “launches” the RNA molecule into the cell, where the RNA is replicated and expressed.

System for High-Throughput Analysis of Individual DNA Molecules

UW–Madison researchers have developed improved methods of using channels for tagging, characterizing and sorting individual double-stranded DNA molecules while maintaining the integrity of the biomolecules. In these methods, a single strand of the molecule is broken, or “nicked.” Because the molecule is not cleaved, the nicking process can occur before the DNA is immobilized in a channel. Fluorescently labeled, sequence-specific nucleotides also can be added at specific sites before the DNA is introduced into the channel.

Additionally, nicked DNA molecules can be kept in a low ionic strength buffer to increase their stiffness. This increased stiffness makes it possible for the molecules to remain properly aligned in channels whose height is nanometer scale but whose width is micrometer scale. Using the larger channels simplifies DNA loading, the introduction of reagents and channel construction, reducing costs and making disposable channels practical.

After a nucleic acid molecule is optically analyzed and characterized in a channel, it can be captured, for example by electrostatic attraction, and collected at a defined point in the array. Then the molecule can be released and conveyed for subsequent analysis, such as sequencing.

Method and System for Delivering Nucleic Acid into a Target Cell

UW-Madison researchers have developed a DNA delivery method that uses a sequestering approach to enable spatial and temporal control over the transfection of stem cells. Oligonucleotide “handles” are covalently attached to a supporting substrate, which may be a solid surface or a two- or three-dimensional semi-solid structure, such as a hydrogel network. The oligonucleotides sequester complementary nucleic acid molecules, such as DNA, in pre-determined locations. The length of the oligonucleotides handles can be varied to control the availability of the sequestered DNA. Recipient cells are adhered to the substrate, and the nucleic acid molecules are exposed to the cells under conditions that support transfection. The culture conditions can be controlled to modulate the timing with which the DNA is made available to the cells.

Synthesis of High-Density Oligomer Microarrays

UW–Madison researchers have developed an automated method for synthesizing arrays of oligonucleotides and other chain molecules in relatively large quantities. The process involves (1) a substrate with an active surface on which the arrays will be formed and (2) a light-emitting object array having selectable light and dark areas defining the image that will be projected onto the active surface.

Projection optics made up of a system of four mirrors receive the light emitting from the object array and image the pattern. The four mirrors are arranged to form a 2X magnified image of the object array at the active surface, thus substantially increasing the area of each element in the array in which oligomers are formed.

For synthesizing oligonucleotides, a flow cell may be incorporated which encloses the active surface of the substrate and has ports for supplying DNA reagents.

Methods of Using Redox-Active Surfactants to Control Polymer Interactions

UW-Madison researchers have developed superior transfection agents that allow spatial and temporal control over the delivery of nucleic acids to cells. These redox-active surfactants switch between a first oxidation state that promotes transfection of cells and a second oxidation state that is less effective at transfecting cells. The transformation between oxidation states is triggered by applying an electrochemical potential to the transfection agent, contacting the agent with an electron donor/acceptor or exposing the agent to an oxidative/reductive environment in a tissue. Redox-active surfactants are also capable of controlling the aggregation of polymers in solution by switching between an oxidation state that facilitates aggregation and one that does not.

Assessing the Mutability of a DNA Sequence

A UW-Madison researcher has developed a quicker method for determining how susceptible a DNA sequence is to mutation. The method involves comparing the DNA sequence of interest to a mutation hotspot sequence to evaluate whether it is more or less mutable than the hotspot sequence.

For this method, the DNA sequence of interest and the hotspot sequence are each linked in-frame to a reporter gene and expressed in bacterial cells. A nonsense or frameshift mutation in either sequence results in an easily detectable loss of function in the reporter gene product. For example, if the reporter gene is a “killer gene,” the bacterial cell will survive only if there is a mutation in the DNA sequence that destroys the function of the reporter gene.

To determine if the sequence of interest is easier to mutate than the hotspot sequence, the number of surviving bacterial colonies can be compared. If more bacteria that contain the sequence of interest survive than bacteria that contain the hotspot sequence, the sequence of interest is more mutable. 

DNA Synthesis with Error Reduction

UW–Madison researchers have developed a method to reduce errors in synthesized DNA and therefore produce genes of correct sequence.

In the new process (termed ‘consensus shuffling’), DNA sequences are either assembled or digested into shorter fragment and then permitted to hybridize. The double-stranded fragments are exposed to a selective DNA filtering agent to remove mismatched sequences from the population. Finally, the surviving fragments are used as templates for PCR amplification to produce full-length sequences.

Mutated Tn5 Transposase Proteins and Their Uses

UW-Madison researchers have developed Tn5 transposase (Tnp) mutants with higher transposase activities than either wild-type Tnp or hyperactive Tnp mutants previously developed by the inventors. These new mutants are modified at several amino acids so that they have greater affinity for Tn5 end sequences than does wild-type Tnp.

Computer-Based Determination of Haplotype

UW–Madison researchers have developed a computer-based method for determining haplotypes. Their system uses genotype data obtained from a wide range of mapping techniques (e.g., experiments with single molecules, families or populations).

The new system identifies polymorphic markers (SNPs, etc.) at one or more contiguous genetic loci and then considers which haplotypes are most likely to account for the observed data. The procedure is nearly linear in the number of markers examined, and therefore more accurate and efficient.

Nuclease Inhibitors and Methods for Their Use

UW-Madison researchers have identified a class of anionic oligomers and polymers that inhibit nucleases, particularly ribonucleases. The inhibitors are polymers of vinyl sulfonate. When nucleases are brought into contact with them in vitro, the polymers are capable of reversibly inhibiting or inactivating the nucleases. The researchers also have developed a method for purifying commercial buffers of these inhibitors.

Rapid, Capillary-Based Synthesis of DNA and Other Large Chain Molecules

A UW–Madison researcher has developed a rapid, efficient and cost-effective method for the synthesis of large chain molecules such as DNA. The method involves synthesizing oligomers within a quartz capillary or column filled with quartz microspheres ranging in diameter from a few microns to hundreds of microns rather than on the surface of glass slides using an optical system formed by a DLP chip and a UV lamp. The synthesis taking place within the quartz column is patterned after DNA synthesis in the MicroArray Synthesizer (MAS) component of the Automated Gene Synthesizer (AGS).

A series of LEDs are located along the quartz capillary to illuminate different portions of the column. The microspheres or other carrier particles within the column are coated with a photoprotecting group that makes a linker between the surface of the sphere and the oligomers. Upon exposure to light from an LED, the photoprotecting group is removed. When a base flows through the column, it will attach to the regions that have been deprotected by light from specific, activated LEDs. Then the LEDs are turned off and reagents that reprotect the regions are applied. This process of selective illumination, deprotection and attachment of a new base is repeated until the desired sequences have been formed on the carrier particles at each section. The synthesized molecules then may be removed for use or further processing.

Tn5 Transposase Showing Enhanced Activity

UW-Madison researchers have developed Tn5 transposase (Tnp) mutants with higher transposase activities than either the wild-type Tnp or their previously developed hyperactive Tnp mutants. These new mutants are modified at several amino acids so that they have greater affinity for a Tn5 outside end sequence than wild-type Tnp.

Method for Converting Single-Copy BAC Vectors into Conditional High-Copy BAC Vectors

UW-Madison researchers have developed a method for converting a single-copy BAC vector into a conditional high-copy BAC vector. Conversion is achieved by introducing a conditional origin of replication (ori) into a single-copy BAC vector in a host cell. The conditional ori is introduced by site-specific recombination between the single-copy BAC vector and a vector containing the conditional ori, producing a high-copy BAC vector that can be conditionally amplified by activating the ori.

Double Transposition Methods for Manipulating Nucleic Acids

UW-Madison researchers have developed a simple two-step procedure for fusing any two genes, making it possible to bring together in a single molecule the structures and functions of diverse molecules. The technology involves two sequential in vitro DNA transposition events catalyzed by two hyperactive variants of Tn5 transposase, in which a DNA sequence flanked by short specific end sequences is "moved" from one site to a second random site. These two transposases, TnP EK/LP and Tnp sC7v2.0, have distinct end recognition specificities for short end DNA (IE and OE sequences) and can be used to generate libraries of random fusions between any two target genes. The resulting IE/OE linker sequence will result in predictable short linker sequence inserts in either direction.

This methodology generates libraries of plasmids that encode chimeric proteins composed of the N-terminal part of one protein and the C-terminal part of a second protein with a short linker inserted at the fusion junction. The total size of a chimeric protein can vary from zero to the total length of both proteins plus the linker. Use of this technology can generate proteins of novel specificities and/or that produce novel metabolic products.

Expression Vector with Dual Control of Replication and Transcription

UW-Madison researchers have constructed a controlled BAC expression system that can produce large amounts of genomic polypeptide upon induction. Their modified pBAC system includes both a conditionally amplifiable origin of replication(oriV) with a broad host range, and a tightly regulated, inducible transcriptional promoter linked to a gene of interest. The conditional oriV and the inducible promoter can be activated jointly or separately by signals in the host cell.

Specifically, BAC copy number is controlled by a single inducing protein, TrfA, which induces replication from oriV. Gene transcription is controlled by an arabinose-inducible promoter, which can be induced to produce many copies of the inserted gene. Thus, this pBAC includes all of the elements necessary for transcribing large quantities of polynucleotides in a controlled fashion.

Markerless Gene Replacement Plasmids for E. coli

UW-Madison researchers have discovered a simple and efficient gene replacement method that permits targeted introduction of markerless deletions, insertions and point mutations into the E. coli chromosome. In this method, the mutant allele is carried on a circular plasmid that integrates into the chromosome at a homologous locus, resulting in a direct duplication. Resolution of this cointegrate via intramolecular recombination is controlled by introducing a unique double-stranded break into the chromosome by the meganuclease I-SceI. The enzyme recognizes an 18-base pair sequence and generates a double-stranded break with a four-base 3` hydroxyl overhang. The method can be used in recombination-proficient E. coli and produces markerless replacements at high efficiency.

Super Bac 1 - An Inducible Vector With Shuttle Capability

UW-Madison researchers have now engineered an improved BAC vector, called Super BAC 1, for use in genomic studies. They have modified a widely used BAC to include an oriV gene from the IncP plasmid RK2. In the presence of a specific transcription factor, oriV provides arabinose-inducible replication, allowing a 50 to 150-fold amplification of BAC copy number upon addition of arabinose. As a second modification, they have added the oriT gene, also from plasmid RK2, to the BAC. This gene confers on E. coli the capacity for conjugal mating into Bacillus when the tra gene is also supplied in trans. To maintain the transferred BAC in Bacillus, the researchers have included the rep60 gene from a Bacillus plasmid. This Bacillus-specific origin of replication maintains the plasmid at five to six copies per cell in this alternate host.

Promoter-Trap Plasmid for Identifying Promoters

UW-Madison researchers have developed a promoter-trap vector for use in Gram-positive bacteria such as Bacillus cereus. The promoter-trap vector was constructed to contain genes for ampicillin and chloramphenicol resistance and can replicate in E. coli and B. cereus. A multiple cloning site containing EcoRI, Sac-I, Kpn-I, SmaI, BamHI, and XbaI restriction sites was inserted at the 5’ end of a promoter-less, green fluorescent protein (GFP) marker gene. Expression of this modified GFP can be quantified by measuring fluorescence intensity and is amenable to flow cytometry and cell sorting.

RNAPtag Plasmids for Construction of Tagged RNA Polymerase Strains

To enhance our ability to study various aspects of this enzyme, UW-Madison researchers developed a universal plasmid scaffold, called RNAPtag, that allows the insertion of a tag into the 3` end of rpoC, the gene that encodes the β` subunit of RNA polymerase. RNAPtag also encodes a kanamycin resistance cassette followed by DNA that is naturally found downstream of rpoC. These features enable easy recombination of the tagged rpoC gene into the E. coli genome, facilitating the production of E. coli strains in which all the RNA polymerase made by the cell contains a tag of interest. Such strains are useful in the study of RNA polymerase.

Titin Minigene: Cardiac Research Tool

To study the mechanisms of Rbm20-mediated exome skipping, the researchers have produced a number of minigene constructs (described in Li 2013). The construct comprises exons 64-70 of the titin gene.

Fusion Protein Expression Vectors, CMV-GST and CMV-tac-GST

UW-Madison researchers have developed two plasmid vectors that allow expression of the GST pi protein. These vectors were tested in bacterial and mammalian hosts, where they allowed expression of the GST protein as expected. Pieces of DNA can be inserted into a cloning site in these vectors. The encoded recombinant gene then can be expressed in bacterial and mammalian cells, where it will direct the synthesis of a fusion protein in which the amino terminal portion is a GST and the carboxy terminal end is the protein of interest.