Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications.

Many biological processes (physiological or pathological) are relevant to membrane proteins (MPs), which account for almost 30% of the total of human proteins. As such, MPs can serve as predictive molecular biomarkers for disease diagnosis and prognosis. Indeed, cell surface MPs are an important class of attractive targets of the currently prescribed therapeutic drugs and diagnostic molecules used in disease detection.

Computational Modeling of RNA Aptamers: Structure Prediction of the Apo State.

RNA aptamers are single-stranded oligonucleotides that bind to specific molecular targets with high affinity and specificity. To design aptamers for new applications, it is critical to understand the ligand binding mechanism in terms of the structure and dynamics of the ligand-bound and apo states. The problem is that most of the NMR or X-ray crystal structures available for RNA aptamers are for ligand-bound states.

Ligands with polyfluorophenyl moieties promote a local structural rearrangement in the Spinach2 and Broccoli aptamers that increases ligand affinities.

The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand how nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI [(Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one] that reach out from the Spinach2 ligand binding pocket.

Aptamer Applications in Neuroscience.

Being the predominant cause of disability, neurological diseases have received much attention from the global health community. Over a billion people suffer from one of the following neurological disorders: dementia, epilepsy, stroke, migraine, meningitis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, prion disease, or brain tumors. The diagnosis and treatment options are limited for many of these diseases.

Sampling Performance of Multiple Independent Molecular Dynamics Simulations of an RNA Aptamer.

Using multiple independent simulations instead of one long simulation has been shown to improve the sampling performance attained with the molecular dynamics (MD) simulation method. However, it is generally not known how long each independent simulation should be, how many independent simulations should be used, or to what extent either of these factors affects the overall sampling performance achieved for a given system. The goal of the present study was to assess the sampling performance of multiple independent MD simulations, where each independent simulation begins from a different initial molecular conformation.

Common Secondary and Tertiary Structural Features of Aptamer-Ligand Interaction Shared by RNA Aptamers with Different Primary Sequences.

Aptamer selection can yield many oligonucleotides with different sequences and affinities for the target molecule. Here, we have combined computational and experimental approaches to understand if aptamers with different sequences but the same molecular target share structural and dynamical features. NEO1A, with a known NMR-solved structure, displays a flexible loop that interacts differently with individual aminoglycosides, its ligand affinities and specificities are responsive to ionic strength, and it possesses an adenosine in the loop that is critical for high-affinity ligand binding.

Aptamer-enabled uptake of small molecule ligands.

The relative ease of isolating aptamers with high specificity for target molecules suggests that molecular recognition may be common in the folds of natural RNAs. We show here that, when expressed in cells, aptamers can increase the intracellular concentrations of their small molecule ligands. We have named these aptamers as DRAGINs (Drug Binding Aptamers for Growing Intracellular Numbers).

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl.

The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule.

Aptamers in analytics.

Nucleic acid aptamers are promising alternatives to antibodies in analytics. They are generally obtained through an iterative SELEX protocol that enriches a population of synthetic oligonucleotides to a subset that can recognize the chosen target molecule specifically and avidly. A wide range of targets is recognized by aptamers.

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells.

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization.

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures.

Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B.

Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5.

Investigating the malleability of RNA aptamers.

Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics. Here we discuss the integration of biochemical analysis with NMR spectroscopy and computational modeling to explore the relation between ligand binding and structural malleability of some well-studied aptamers.