Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer

Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzinostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the β sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corroborate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic modalities towards targeted cancer therapy.

Analysis of 177Lu-octreotate therapy-induced DNA damage in peripheral blood lymphocytes of patients with neuroendocrine tumors

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) can lead to cell death, genome instability and carcinogenesis. Immunofluorescence detection of phosphorylated histone variant H2AX (γ-H2AX) is a reliable and sensitive technique to monitor external beam IR-induced DSBs in peripheral blood lymphocytes (PBL). Here, we investigated whether γ-H2AX could be used as an in vivo marker to assess normal tissue toxicity after extended internal irradiation with (177)Lu-DOTA-octreotate peptide receptor radionuclide therapy (LuTate PRRT) of neuroendocrine tumors.

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.
Results
To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.
Conclusion
We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

Azide photochemistry for facile modification of graphitic surfaces : preparation of DNA-coated carbon nanotubes for biosensing

A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms—as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions.

Postchemotherapy and tumor-selective targeting with the La-specific DAB4 monoclonal antibody relates to apoptotic cell clearance

Early identification of tumor responses to treatment is crucial for devising more effective and safer cancer treatments. No widely applicable, noninvasive method currently exists for specifically detecting tumor cell death after cytotoxic treatment and thus for predicting treatment outcomes.

High strain mechanical loading rapidly induces tendon apoptosis : an ex vivo rat tibialis anterior model

Background: The role of apoptosis, or programmed cell death, has only recently been explored in tendon.

Objective: To investigate the development of apoptosis after high strain loading of rat tendon.

Methods: The right tibialis anterior tendons of three rats were prepared for mechanical loading, and left tendons were prepared identically as non-loaded controls. Tendon was loaded with 20% strain for six hours using a 1 Hz longitudinal sine wave signal. The following were used to assess apoptosis: (a) a monoclonal mouse antibody (F7-26) to label single stranded DNA breaks; (b) a rabbit polyclonal antibody that specifically recognises the cleaved form of caspase-3.

Results: Light microscopy confirmed that the high strain protocol induced a stretch overload injury. Control tendons showed little or no staining with the F7-26 antibody, but the loaded tendons displayed numerous apoptotic cells. The percentage of apoptotic cells (20%) in the loaded tendon was significantly greater than in the control tendon (1%) (p = 0.000). The labelled cells colocalised with abnormal nuclear morphology, including nuclear fragmentation. The staining against cleaved caspase-3 was positive in loaded tendons only, and localised both to nucleus and cytoplasm.

Conclusion: This experiment extends knowledge of human tendon apoptosis by showing that apoptosis can occur in response to short term, high strain mechanical loading. This is the first report of mechanical loading of intact tendon causing excessive apoptosis.

Discovery of structural and functional features in RNA pseudoknots

An RNA pseudoknot consists of nonnested double-stranded stems connected by single-stranded loops. There is increasing recognition that RNA pseudoknots are one of the most prevalent RNA structures and fulfill a diverse set of biological roles within cells, and there is an expanding rate of studies into RNA pseudoknotted structures as well as increasing allocation of function. These not only produce valuable structural data but also facilitate an understanding of structural and functional characteristics in RNA molecules. PseudoBase is a database providing structural, functional, and sequence data related to RNA pseudoknots. To capture the features of RNA pseudoknots, we present a novel framework using quantitative association rule mining to analyze the pseudoknot data. The derived rules are classified into specified association groups regarding structure, function, and category of RNA pseudoknots. The discovered association rules assist biologists in filtering out significant knowledge of structure-function and structure-category relationships. A brief biological interpretation to the relationships is presented, and their potential correlations with each other are highlighted.

An antiviral response directed by PKR phosphorylation of the RNA helicase A

The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

RNA interference : more than a research tool in the vertebrates' adaptive immunity

In recent years, RNA silencing, usage of small double stranded RNAs of ~21 – 25 base pairs to regulate gene expression, has emerged as a powerful research tool to dissect the role of unknown host cell factors in this 'post-genomic' era. While the molecular mechanism of RNA silencing has not been precisely defined, the revelation that small RNA molecules are equipped with this regulatory function has transformed our thinking on the role of RNA in many facets of biology, illustrating the complexity and the dynamic interplay of cellular regulation. As plants and invertebrates lack the protein-based adaptive immunity that are found in jawed vertebrates, the ability of RNA silencing to shut down gene expression in a sequence-specific manner offers an explanation of how these organisms counteract pathogen invasions into host cells. It has been proposed that this type of RNA-mediated defence mechanism is an ancient form of immunity to offset the transgene-, transposon- and virus-mediated attack. However, whether 1) RNA silencing is a natural immune response in vertebrates to suppress pathogen invasion; or 2) vertebrate cells have evolved to counteract invasion in a 'RNA silencing' independent manner remains to be determined. A number of recent reports have provided tantalizing clues to support the view that RNA silencing functions as a physiological response to regulate viral infection in vertebrate cells. Amongst these, two manuscripts that are published in recent issues of Science and Immunity, respectively, have provided some of the first direct evidences that RNA silencing is an important component of antiviral defence in vertebrate cells. In addition to demonstrating RNA silencing to be critical to vertebrate innate immunity, these studies also highlight the potential of utilising virus-infection systems as models to refine our understanding on the molecular determinants of RNA silencing in vertebrate cells.

Aptamer therapeutics: the 21st century's magic bullet of nanomedicine

Aptamers, also known as chemical antibodies, are short single-stranded DNA, RNA or peptide molecules. These molecules can fold into complex three-dimensional structures and bind to target molecules with high affinity and specificity. The nucleic acid aptamers are selected from combinatorial libraries by an iterative in vitro selection procedure known as systematic evolution of ligands by exponential enrichment (SELEX). As a new class of therapeutics and drug targeting entities, bivalent and multivalent aptamer-based molecules are emerging as highly attractive alternatives to monoclonal antibodies as targeted therapeutics.

Aptamers have several advantages, offering the possibility of overcoming limitations of antibodies: 1) they can be selected against toxic or non-immunogenic targets; 2) aptamers can be chemically modified by using modified nucleotides to enhance their stability in biological fluids or via incorporating reporter molecules, radioisotopes and functional groups for their detection and immobilization; 3) they have very low immunogenicity; 4) they display high stability at room temperature, in extreme pH, or solvent; 5) once selected, they can be chemically synthesized free from cell- culturederived contaminants, and they can be manufactured at any time, in large amounts, at relatively low cost and reproducibly; 6) they are smaller and thus can diffuse more rapidly into tissues and organs, leading to faster targeting in drug delivery; 7) they have lower molecular weight that can lead to faster body clearance, resulting in a low background noise for imaging and minimizing the radiation dose to the patient in diagnostic imaging. Thus, the high selectivity and sensitivity, ease of screening and production, chemical versatility as well as stability make aptamers a class of highly attractive agents for the development of novel therapeutics, targeted drug delivery vehicles and molecular imaging.

In the review, we will discuss the latest technological advances in developing aptamers, its application as a novel class of drug on its own, as well as in surface functionalization of both polymer nanoparticles or nanoliposomes in the treatment of cancer, viral and autoimmune diseases.

Simple and signal-off electrochemical biosensor for mercury(II) based on thymine-mercury-thymine hybridization directly on graphene

Abstract A simple, signal-off and reusable electrochemical biosensor was developed for sensitive and selective detection of mercury(II) based on thymine-mercury(II)-thymine (T-Hg²⁺-T) complex and the remarkable difference in the affinity of graphene with double strand DNA (ds-DNA) and single strand DNA (ss-DNA). Our system was composed of ferrocene-tagged probe DNA and graphene. Due to the noncovalent assembly, the ferrocene-tagged probe ss-DNA was immobilized on the surface of graphene nanosheets directly and employed to amplify the electrochemical signal. In the presence of Hg²⁺, the ferrocene-labeled T-rich DNA probe hybridized with target probe to form ds-DNA via the Hg²⁺-mediated coordination of T-Hg²⁺-T base pairs. As a result, the duplex DNA complex kept away from the graphene surface due to the weak affinity of graphene and ds-DNA, and the redox current decreased substantially. Meanwhile, the graphene decorated GCE surface was released for the reusability. Under the optimal conditions, the proposed sensor showed a linear concentration range from 25 pM to 10 μM with a detection limit of 5 pM for Hg²⁺ detection. The strategy afforded exquisite selectivity for Hg²⁺ against other metal ions in real environmental samples.

Design of a high sensitive double-gate field-effect transistor biosensor for DNA detection

The study of interactions between organic biomolecules and semiconducting surfaces is an important consideration for the design and fabrication of field-effect-transistor (FET) biosensor. This paper demonstrates DNA detection by employing a double-gate field effect transistor (DGFET). In addition, an investigation of sensitivity and signal to noise ratio (SNR) is carried out for different values of analyte concentration, buffer ion concentration, pH, reaction constant, etc. Sensitivity, which is indicated by the change of drain current, increases non-linearly after a specific value (∼1nM) of analyte concentration and decreases non-linearly with buffer ion concentration. However, sensitivity is linearly related to the fluidic gate voltage. The drain current has a significant effect on the positive surface group (-NH2) compared to the negative counterpart (-OH). Furthermore, the sensor has the same response at a particular value of pH (5.76) irrespective of the density of surface group, although it decreases with pH value. The signal to noise ratio is improved with higher analyte concentrations and receptor densities.

Molecular recognition of DNA by rigid [n]-polynorbornane-derived bifunctional intercalators: synthesis and evaluation of their binding properties

We have exploited the concept of multivalency in the context of DNA recognition, using novel chemistry to synthesize a new type of bis-intercalator with unusual sequence-selectivity. Bis-intercalation has been observed previously, but design principles for de novo construction of such molecules are not known. Our compounds feature two aromatic moieties projecting from a rigid, polynorbornane-based scaffold. The length and character of the backbone as well as the identity of the intercalators were varied, resulting in mono- or divalent recognition of the double helix with varying affinity. Our lead compound proved to be a moderately sequence-selective bis-intercalator with an unwinding angle of 27 and a binding constant of about 8 M. 9-Aminoacridine rings were preferred over acridine carboxamides or naphthalimides, and a rigid [3]-polynorbornane scaffold was superior to a [5]-polynorbornane. The flexibility of the linker connecting the rings to the scaffold, although less influential, could affect the strength and character of the DNA binding.

Electrostatic complex of didodecyldimethylammonium and DNA: Langmuir monolayer, Langmuir–Blodgett film and dye recognition at air/water interface

DNA–didodecyldimethylammonium (DNA–DDDA) electrostatic complex was prepared and characterized through Fourier transformation infrared (FT-IR), 1H NMR and circular dichroism (CD) spectroscopy. When the dye molecule aqueous solutions were used as the subphase, the interaction between three dye molecules, acridine orange (AO), ethidium bromide (EB) and 5,10,15,20-tetrakis(4-N-methylpyridyl)porphine tetra(p-toluenesulfonate) (TMPyP) and the complex at air/solution interface were investigated through the surface pressure–area (π–A) isotherms, Brewster angle microscopy and UV-Vis spectroscopy, respectively. Our investigation indicates that the interaction capabilities of the three dyes to DNA–DDDA complex are different and present an order of TMPyP>AO>EB. For the interaction forms, we believe that TMPyP intercalates into the double helix of DNA, and AO adsorbs onto the surface of the DNA. As for EB, the measured signal is too weak to give a definite interaction form in the present experiment.

Long lived multi-isotype anti-HIV antibody responses following a prime-double boost immunization strategy

Despite decades of work, an effective HIV vaccine remains elusive. In an effort to elicit protective immunity, investigators have sought to define vaccines able to elicit durable HIV-specific B-cell and T-cell activities. Additionally, vaccines are sought which can induce antibodies of a variety of isotypes, as each isotype possesses unique attributes in terms of opsonization, Fc receptor binding capacity, complement fixation and location. One prominent new vaccine strategy, applied to numerous distinct antigenic systems is the prime boost-regimen, with DNA, vaccinia virus (VV), and/or purified recombinant protein. To examine the durability, location and isotype distribution of responses induced by prime-boost regimens, we tested successive immunizations with DNA, VV and protein (D-V-P), comparing three forms of protein inoculations: (i) purified protein administered intramuscularly with complete Freunds adjuvant, (ii) purified protein administered intranasally, and (iii) purified protein conjugated to oxidized mannan, administered intranasally. We found that all three protocols elicited serum antibodies of multiple isotypes, with serum IgA being most prominent among mice immunized with mannan-conjugated protein. All D-V-P protocols, regardless of protein form or route, also elicited antibody responses at mucosal surfaces. In bronchoalveolar lavage, a tendency toward IgA production was again most prominent in mice boosted with the protein–mannan conjugate. Both B-cell and T-cell responses were sustained for more than 1 year post-immunization following each form of vaccination. Contemporaneous with long-lasting serum and mucosal antibodies were antibody forming cells in the bone marrow of primed animals. Results highlight the D-V-P vaccination strategy as a promising approach for attaining durable, multi-isotype B-cell and T-cell activities toward HIV.