top of page


Aptamers are synthetic oligonucleotides that can be isolated in vitro against diverse biological targets. Aptamers assume defined 3D structures and generally bind functional sites on their respective targets. Aptamers possess the molecular recognition properties of monoclonal antibodies in terms of their high affinity and specificity. Aptamers are known for their remarkable specificity. Aptamers can discriminate targets on the basis of small differences such as presence or absence of methyl group or the D- vs L-enantiomer.

Because animals or cells are not involved in aptamer identification, toxins as well as molecules that do not elicit a good immune responses can be used to generate high-affinity aptamers. Aptamers are produced by chemical synthesis with extreme accuracy and reproducibility. They are purified under denaturing conditions to a very high degree of purity.

Little or no batch-to-batch variation is expected in aptamer production.

Reporter molecules such as fluorescein and biotin can be attached to aptamers at precise locations identified by the user. Functional groups that allow subsequent derivatization of aptamers with other molecules can also be attached during the chemical synthesis of aptamers.

Aptamers undergo denaturation, but the process is reversible. Once denatured, functional aptamers could be regenerated easily within minutes. They are stable to long-term storage and can be transported at ambient temperature.

Unlike antibodies, aptamers can fold properly and retain activity in the intracellular environment. However, the majority of aptamers with potential therapeutic utility selected to date target extracellular proteins.

The applications of aptamers range from diagnostics and biosensing, target validation, targeted drug delivery, therapeutics, templates for rational drug design to biochemical screening of small molecule leads compounds.

Single-stranded oligonucleotide sequences have an exceptional propensity to assume an array of secondary and tertiary structural motifs with different shapes. This is simply based on the ability of nucleotide bases to interact with each other through Watson-Crick base pairing.



Systematic Evolution of Ligands by Exponential Enrichment

The process starts with a chemically synthesized random sequence DNA library containing approximately 10e20 individual molecules (see picture below). A library is designed to contain randomized region (red lines) flanked by two fixed sequence regions (black). Each nucleotide position in the contiguous random region is synthesized upon delivery of a mixture of phosphoramidites containing all four building blocks: A, G, C, and T. To obtain an unbiased library with equal representation of all four nucleotides, the ratios of the four phosphoramidites in the mixture are adjusted based on the coupling efficiencies of individual monomers. Fixed sequences are used for primer binding sites in enzymatic amplification of individual sequences.

A random sequence oligonucleotide (DNA) library is exposed to the target of interest under defined buffer conditions. Sequences that bind to the target are separated from the unbound species by a suitable partitioning technique. This population of sequences recovered from the target (mixture of aptamer candidates) represents a mixture containing both high- and low-affinity binding molecules to the target. Because further screening is required to eliminate the sequences with low affinity binding, individual sequences are amplified for the next round of selection. The aptamer candidate mixture is amplified directly by PCR, provided it is DNA. The single-stranded DNA population obtained by strand separation of PCR products is incubated with a fresh sample of the target for the next round of selection.

The enrichment of the high-affinity sequences at the expense of the low-affinity binders requires several iterations of the selection process carried out under increasingly stringent conditions. Once affinity saturation is observed in an enriched library, the corresponding PCR products are used for cloning and sequencing.

All protocols for selection of aptamers are based on some type of affinity separation of binding and non-binding oligonucleotides, and when it comes to small molecules that often means chemically modifying them and then attaching them to the solid-state matrix.  In our protocol, we use small molecules in their native states, that is, unmodified and in solution-phase. It is based on attaching oligonucleotide library via an oligonucleotide complementary to their constant region (PCR primer) to a column, and then passing over the column the target in a solution, thus, isolating oligonucleotides in which the target causes displacement of this complement. After multiple runs of selection, high affinity sequences become dominant – Darwinian selection.

SELEX slide.jpg



Structure-switching biosensor

When the target (T) is absent, the aptamer binds to quencher-DNA (Q), bringing the fluorophore (F) and the quencher molecule into close proximity for maximum fluorescence quenching. When the target is introduced, the aptamer prefers to form the aptamer-target complex. Upon binding the target, appropriate folding in the random region consequently induces the structural changes, which in turn remove it from the quencher (Q).

DNA origami

DNA origami – a technique for making structures from DNA; nanoscale shapes made from one long single stranded template strand of DNA and hundreds custom short strands (staples) ordered from a DNA-synthesis company (click on the picture below for the full article in Nature, 2006). 

Additional Nature Video from 2016 about ten years since the technique was first reported and how DNA origami works and what has been achieved. 

New Picture.png

DNA Nanorobots

DNA origami, a technique for making structures from DNA, described above, can be also used to build devices, as the researches call them, the nanorobots.

DNA nanorobots can target e.g. cancer cells and deliver an antibody cargo (link to read more). Once this DNA "device" recognizes a cell, it automatically changes its shape and delivers its cargo.

The structure of the DNA nanorobots can be designed using open-source software, called Cadnano. Then the nanorobot can be built using DNA origami technique.

Here is the video of the barrel-shaped DNA nanorobots, each 35 nanometer in size, and having 12 sites on the inside for attaching payload molecules and two positions on the outside for attaching aptamers, short nucleotide strands with special sequences for recognizing molecules on the target cell. The aptamers act as clasps: once both have found their target, they spring open the device to release the payload.

Because the nanorobots can be programmed to release their payload only when the target cell is in the correct disease state, they achieve a specificity that other drug-delivery methods lack, says Hao Yan, a collaborator of Dr. Pecic from Arizona State University, Tempe in this Nature article

bottom of page