Pooja Dua

Dual Functions of Highly Potent Graphene Derivative–Poly-l-Lysine Composites To Inhibit Bacteria and Support Human Cells

Dual-function poly(L-lysine) (PLL) composites that function as antibacterial agents and promote the growth of human cell culture have been sought by researchers for a long period. In this paper, we report the preparation of new graphene derivative-PLL composites via electrostatic interactions and covalent bonding between graphene derivatives and PLL. The resulting composites were characterized by infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The novel dual function of PLL composites, specifically antibacterial activity and biocompatibility with human cells [human adipose-derived stem cells and non-small-cell lung carcinoma cells (A549)], was carefully investigated. Graphene-DS-PLL composites composed of 4-carboxylic acid benzene diazonium salt (DS) generated more anionic carboxylic acid groups to bind to cationic PLLs, forming the most potent antibacterial agent among PLL and PLL composites with high biocompatibility with human cell culture. This dual functionality can be used to inhibit bacterial growth while enhancing human cell growth.

Nucleic acid aptamers targeting cell-surface proteins.

Aptamers are chemical antibodies that bind to their targets with high affinity and specificity. These short stretches of nucleic acids are identified using a repetitive in vitro selection and partitioning technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since the emergence of this technology, many modifications and variations have been introduced to enable the selection of specific ligands, even for implausible targets. For membrane protein, the selection scheme can be chosen depending upon the availability of the system, the protein characteristics and the application required. Aptamers have been generated for a significant number of disease-associated membrane proteins and have been shown to have considerable diagnostic and therapeutic importance. In this article, we review the SELEX process used for identification of aptamers that target cell-surface proteins and recapitulate their use as therapeutic and diagnostic reagents.

Patents on SELEX and Therapeutic Aptamers

Aptamers, the oligonucleotides (DNA/RNA) that bind to target molecules with high specificity and affinity, have been a focus of therapeutic research for the last two decades. The magnitude of scientific and commercial interest shown for aptamers is not surprising because aptamers have several advantages over other curative modalities, especially antibodies. Patent activity in this field has also shown an exponential growth. Aptamers against a broad range of disease-causing pathogens and proteins have been patented. These have potential use as a biomarker, therapeutics and diagnostics. As drugs they have shown commendable results in cell and animal models, a few of them undergoing clinical trials. In this review, we discuss upon all important patents filed on therapeutic aptamers and SELEX technology employed to synthesize them. We have classified them in categories based upon their target or the diseased condition they apt for. These patents provide insight into the development that occurred in transformation of aptamers as therapeutic entities and reinforces the potential they have.

Alkaline Phosphatase ALPPL-2 Is a Novel Pancreatic Carcinoma-Associated Protein

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a very low median survival rate. The lack of early sensitive diagnostic markers is one of the main causes of PDAC-associated lethality. Therefore, to identify novel pancreatic cancer biomarkers that can facilitate early diagnosis and also help in the development of effective therapeutics, we developed RNA aptamers targeting pancreatic cancer by Cell-systematic evolution of ligands by exponential enrichment (SELEX) approach. Using a selection strategy that could generate aptamers for 2 pancreatic cancer cell lines in one selection scheme, we identified an aptamer SQ-2 that could recognize pancreatic cancer cells with high specificity. Next, by applying 2 alternative approaches: (i) aptamer-based target pull-down and (ii) genome-wide microarray-based identification of differentially expressed mRNAs in aptamer-positive and -negative cells, we identified alkaline phosphatase placental-like 2 (ALPPL-2), an oncofetal protein, as the target of SQ-2. ALPPL-2 was found to be ectopically expressed in many pancreatic cancer cell lines at both mRNA and protein levels. RNA interference-mediated ALPPL-2 knockdown identified novel tumor-associated functions of this protein in pancreatic cancer cell growth and invasion. In addition, the aptamer-mediated identification of ALPPL-2 on the cell surface and cell secretions of pancreatic cancer cells supports its potential use in the serum- and membrane-based diagnosis of PDAC.

Enhanced intracellular delivery and multi‐target gene silencing triggered by tripodal RNA structures

The development of gene interfering RNA (iRNA) molecules such as small interfering RNAs (siRNAs) and antagomirs provides promising therapeutic modalities for targeting specific mRNAs and microRNAs (miRNAs) involved in disease mechanisms. Therapeutic iRNA strategy against cancer or hypermutable viruses prefers targeting multiple genes simultaneously to achieve synergistic inhibition and to prevent resistance.

A Sol–Gel-Based Microfluidics System Enhances the Efficiency of RNA Aptamer Selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.

Long dsRNA-Mediated RNA Interference and Immunostimulation: A Targeted Delivery Approach Using Polyethyleneimine Based Nano-Carriers

RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

Long double-stranded RNA-mediated RNA interference and immunostimulation: long interfering double-stranded RNA as a potent anticancer therapeutics.

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon β. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2′F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.