Aptamer discovery

Selection

FRELEX: Free-Free aptamer selection method

FRELEX is Neoventures’ patented method for aptamer selection.  In order to overcome the need to immobilize targets molecules, as required in traditional SELEX, we developed this new method to allow partitioning of bound aptamers from unbound without immobilizing aptamers or target.

The FRELEX aptamer selection method begins in phase I incubating a library of aptamers with a library of 8mers that have been immobilized on a gold chip. If the aptamers are sufficiently open they will form Watson/Crick bonds with the 8mers on the chip. We retain those sequences that bind to the 8mers. We discard all of those sequences that do not bind to the chip. This means all of the sequences retained have the capacity to bind to the 8mers. We elute these off the chip.

In phase II, we incubate the sequences retained from phase I with the target. This complex is then exposed to a fresh 8mer chip. In this phase we retain those sequences that are not bound to the chip with the logic that the reason the sequences do not bind is because they are now bound to a target molecule.

Contrasting channels in a selection strategy

We work with our clients to develop a selection strategy that fits product concept, the diagnostic device or therapeutic need.

The  following is a general schematic of a selection project with contrasting channels.

FRELEX aptamer selection

After several rounds of selection the enriched library splits into aliquots and continues positive selection against separate and contrasting targets. We perform next generation sequencing (NGS) on all selection rounds from 7 to 10.

NGS Analysis

Aptamer Next Generation Sequencing

We prepare multiple libraries for simultaneous sequencing analysis on an Illumina HiSeq 2500. We capture at least 1.000.000 sequences per library.  We assess library complexity based on distribution of copy number of these aptamer sequences. NeoVentures has developed proprietary software to characterize enrichment rates and identify candidate aptamers. We differentiate sequences that continue to curve upwards vs. those that plateau as seen in the figure below. Sequences that plateau generally have lower binding affinities.

 

aptamer next generation sequencing

 

Binding Assays

Surface Plasmon Resonance imaging (SPRi)

For large molecules we perform binding assays with surface plasmon resonance imaging (SPRi) system on a Horiba OpenPlex instrument.

horiba openplex aptamer binding

This instrument differs from BiaCore analysis. We can analyze binding to multiple spots separately and simultaneously. Thiolated aptamers are spotted onto a gold chip on a glass prism. The target molecule flows over the immobilized aptamers and determine the rate of complex formation (ka) and the rate of complex disassociation (kd).  We can determine binding affinity of antisense molecules and measure competition between aptamers for binding sites on proteins.

Isothermal Titration Calorimetry (ITC)

For small molecules we perform binding assays by Isothermal titration calorimetry (ITC) analysis on a Nano ITC by TA instruments.

nano itc aptamer binding

The instrument contains two chambers, a reference chamber and a loading chamber.

The amount of energy required to maintain the temperature of the loading chamber at the same level as the reference chamber is measured in uJ/sec. The loading chamber is loaded with one of the components of the binding reaction under analysis (usually the aptamer). Then another component (usually the target) is serially injected into the loading chamber and the heat dynamics of the reaction are measured. The loading chamber has a gold surface thus we cannot use any target molecules that bind significantly to gold. We use relatively high concentrations of targets (low mM) in order to obtain reasonable heat of binding signals. There is also a need to run several negative controls.