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Custom Aptamer Synthesis

Aptamers are nucleic acid sequences or single-stranded oligonucleotides with selected sequences that specifically fold and bind to selected molecules or targets. The single strand of an aptamer folds into a well-defined three-dimensional (3D) structure.

Aptamers (named by Ellington and Szostak in 1990) are single-stranded DNA or RNA oligonucleotides (ssDNA and ssRNA) usually 20 to 80 nucleotides long with molecular weights ranging from 6 to 30 kDa that fold into unique 3D conformations.

If the oligonucleotide sequence of an aptamer is known, chemical solid phase oligonucleotide synthesis can be used for its production. However, aptamer synthesis can also be achieved using DNA, RNA or modified nucleic acids. Both, chemical as well as enzymatic aptamer synthesis is possible. Sometimes chemical and enzymatic synthesis are combined for the production of specific aptamers.

Aptamers bind to their targets with high affinity via van der Waals forces, hydrogen bonding, electrostatic interactions, stacking of flat moieties, and shape complementarities. Dissociation constants (Kd) can range from pico- to nanomolar.

Target molecules recognized by aptamers can be small molecules such as cocaine as well as proteins and peptides or others. Aptamers allow for the design of reagents with high affinity for desired compounds or molecules. The unique features of aptamers make them suitable for clinical applications or diagnostics.

For example, Sassanfar and Szostak in 1993 described an in vitro selection procedure to find an RNA motif that binds to ATP. A few years later, in 1996, Dieckman and others reported the structure of an ATP-binding RNA aptamer with a novel fold.  Figure 1 illustrates the aptamer fold in the structural model as determined by NMR.

Figure 1: Different models of an ATP-binding RNA aptamer as determined by NMR. Cn3D and PyMOL was used for the creation of the images using the structural pdb data form 1RAW.   


Specific detection by aptamers depends on specific interactions with the analyte and base pairing between different parts of the aptamer. The specificity of aptamers is often as high as that obtained with antibodies. Specifically designed oligonucleotide sequences can be used for the detection of a variety of different molecules.

The sequence of an aptamer can be determined using the SELEX process. The SELEX approach or “systematic evolution of ligands by exponential enrichment” allows for the evolution of aptamers (defined by Tuerk and Gold in 1990).

Random sequences are used as the input for the SELEX process that produces functional sequences. The process includes multiple rounds of exponential amplification and enrichment, allowing for the evolution of aptamers with high target-specific affinity from random oligonucleotide pools.

Several steps are needed for the generation of the final aptamer.

1

Preparation of the initial oligonucleotide pool of approximately 1014 to 1015 random sequences, 30 to 50 nucleotides in length between two primer binding sites.

2

Incubation in which random sequences in the initial pool fold into different secondary and tertiary structures and form aptamer-target complexes when optimal conditions occur.

3

Partitioning in which unbound sequences are separated from target-bound sequences using methods such as membrane filtration, affinity columns, magnetic beads, or capillary electrophoresis.

4

Amplification in which target-bound sequences are amplified by PCR, in the case of DNA aptamers, or RT-PCR, in the case of RNA aptamers. Reaction products are used as a new aptamer sub-pool for the next round of selection.

5

Sequencing of enriched aptamer sequences using Sanger sequencing or newer high-throughput sequencing methods.


Several negative-target selections are often added to the process that eliminates non-specific sequences. Often specific aptamers can be obtained after 8 to 20 rounds of selection. The whole selection process can take weeks to months.  

References

Dieckmann T, Suzuki E, Nakamura GK, Feigon J. Solution structure of an ATP-binding RNA aptamer reveals a novel fold. RNA. 1996;2(7):628-640.

Ellington, Andrew D., Szostak, Jack W.; In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-882, 1990. doi:10.1038/346818a0. 

Sassanfar, Mandana, Szostak, Jack W.; An RNA motif that binds ATP. Nature 364, 550-553. 

Milan N. Stojanovic, Paloma de Prada, andDonald W. Landry; Aptamer-Based Folding Fluorescent Sensor for Cocaine.  J. Am. Chem. Soc., 2001, 123 (21), pp 4928–4931. DOI: 10.1021/ja0038171. http://pubs.acs.org/doi/abs/10.1021/ja0038171

Sassanfar, Mandana, Szostak, Jack W.; An RNA motif that binds ATP. Nature 364, 550-553.

Hongguang Sun and Youli Zu; A Highlight of Recent Advances in Aptamer Technology and Its Application. Molecules 2015, 20(7), 11959-11980; doi:10.3390/molecules200711959. https://www.ncbi.nlm.nih.gov/pubmed/26133761

Tuerk C, Gold L.; Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505-10. https://www.ncbi.nlm.nih.gov/pubmed/2200121

Wang RE, Zhang Y, Cai J, Cai W, Gao T. Aptamer-Based Fluorescent Biosensors. Current medicinal chemistry. 2011;18(27):4175-4184.


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