Supriya Pai

Aptamers as therapeutics

Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these properties, aptamers have been slow to reach the marketplace, with only one aptamer-based drug receiving approval so far. A series of aptamers currently in development may change how nucleic acid therapeutics are perceived. It is likely that in the future, aptamers will increasingly find use in concert with other therapeutic molecules and modalities.

An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.

Proximity ligation assays with peptide conjugate ‘burrs’ for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide–oligonucleotide–phycoerythrin conjugates (‘burrs’) that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.

Using RNA aptamers and the proximity ligation assay for the detection of cell surface antigens.

The detection and typing of tumor cells based on differentially or similarly expressed antigens (biomarkers) have proven to be increasingly important for the diagnosis and treatment of various cancers. Sensitive techniques for the detection of cell surface antigens are therefore crucial for the early and accurate detection of cancer. Although techniques such as ELISA and tissue staining have proven their worth, these techniques often either require substantial amounts of starting material or are prone to high background and false negatives. The proximity ligation assay (PLA) has proven to be an exquisitely sensitive technique with very low background. Two probes that bind adjacent to one another on a protein target can be ligated, yielding a unique amplicon that can be sensitively detected by real-time PCR. We have now adapted PLA to cell surface protein targets using modified RNA aptamers, and have shown that aptamer-based cell surface PLA can successfully detect and differentiate between cells that differentially express a tumor antigen, the prostate specific membrane antigen (PSMA).

Aptamer amplification: divide and signal

BACKGROUND Aptamers are selected nucleic acids that bind their targets with affinities and specificities that are often comparable to those of monoclonal antibodies. OBJECTIVE Although aptamers have been adapted to a wide variety of assay formats, the fact that they are nucleic acids makes them uniquely useful for adaptation to amplification assays. Aptamer-based amplification assays have not previously been reviewed separately. METHODS Aptamers can be used as simple binding reagents and then detected by methods such as the polymerase chain reaction and the rolling circle amplification assay. In addition, though, aptamers can undergo programmed, ligand-dependent conformational changes, or can form unique quarternary structures that lead to amplification. RESULTS/CONCLUSION Analytical assays that involve aptamers and nuleic acid amplification technologies can be used to detect sensitively target proteins, often in the nanomolar or picomolar range. However, in many cases there are no obvious advantages to amplification assays relative to other assay formats. In all likelihood all formats are limited by the dissociation constants of the aptamers themselves. The one exception to this is the proximity ligation assay, where sequence amplification allows the detection of extremely small quantities of ligands relative to background.

Erratum: An RNA alternative to human transferrin: A new tool for targeting human cells (Molecular Therapy - Nucleic Acids (2013) 2 e79 Doi:10.1038/mtna. 2013.6)

1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA; 2Universidade do Estado de Santa Catarina, Centro de Ciências Agroveterinárias, Lages, Santa Catarina, Brazil; 3Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas, USA; 4STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, Germany. Matthew Levy 1301 Morris Park Avenue, Price Center, Room 519, Bronx, NY, 10461, USA. Correspondence: E-mail: matthew.levy@einstein.yu.edu Received 11 November 2011; revised 2 March 2012; accepted 26 March 2012