Aptamer

If multiple aptamers are combined into a single assay, they can measure large numbers of different proteins in a sample.

After SELEX, the researcher might mutate or change the chemistry of the aptamers and do another selection, or might use rational design processes to engineer improvements.

Early examples include the modification of the bacteriophage Qbeta replication system and the generation of ribozymes with modified cleavage activity.

[5][13] Two years later, the Szostak lab and Gilead Sciences, acting independently of one another, used in vitro selection schemes to generate DNA aptamers for organic dyes[14] and human thrombin,[15] respectively.

In 2001, SELEX was automated by J. Colin Cox in the Ellington lab, reducing the duration of a weeks-long selection experiment to just three days.

[16][17][18] In 2002, two groups led by Ronald Breaker and Evgeny Nudler published the first definitive evidence for a riboswitch, a nucleic acid-based genetic regulatory element, the existence of which had previously been suspected.

This discovery added support to the RNA World hypothesis, a postulated stage in time in the origin of life on Earth.

DNA, RNA, XNA, and peptide aptamer chemistries can each offer distinct profiles in terms of shelf stability, durability in serum or in vivo, specificity and sensitivity, cost, ease of generation, amplification, and characterization, and familiarity to users.

XNA-based aptamers can introduce additional chemical diversity to increase binding affinity or greater durability in serum or in vivo.

One aptamer, in particular, proved effective as a recognition element in an electrochemical sensor, enabling the detection of sGP and GP1.2 in solution, as well as GP1.2 within a membrane context.The results of this research point to the intriguing possibility that certain regions on protein surfaces may possess aptatropic qualities.

Aptamer targets can include small molecules and heavy metal ions, larger ligands such as proteins, and even whole cells.

[32][33] These targets include lysozyme,[34] thrombin,[35][36] human immunodeficiency virus trans-acting responsive element (HIV TAR),[37] hemin,[38] interferon γ,[39] vascular endothelial growth factor (VEGF),[40][41] prostate specific antigen (PSA),[42][43] dopamine,[44] and the non-classical oncogene, heat shock factor 1 (HSF1).

[52] By contrast, aptamers are simple to sequence and cost nothing to maintain, as their exact structure can be stored digitally and synthesized on demand.

[66][67][68] They can function in molecular engineering applications as a way to modify proteins, such as enhancing DNA polymerase to make PCR more reliable.

[75][52] In laboratory research and clinical diagnostics, they can be used in aptamer-based versions of immunoassays including enzyme-linked immunosorbent assay (ELISA),[76] western blot,[77] immunohistochemistry (IHC),[78] and flow cytometry.

[81] Unlike antibodies, unmodified aptamers are more susceptible to nuclease digestion in serum and renal clearance in vivo.

However, phage display methods allow for selection of antibodies in vitro, followed by production from a monoclonal cell line, avoiding the use of animals entirely.

[84] The ability of aptamers to reversibly bind molecules such as proteins has generated increasing interest in using them to facilitate controlled release of therapeutic biomolecules, such as growth factors.

[87] Aptamer, known for their ability to bind specific molecules reversibly, have been used in 3D bioprinting tissues to precisely deliver growth factors to promote vascularization.

[88][89] This controlled delivery allows growth factors to be released at the right place and time, encouraging the formation of localized and complex vascular networks.

Additionally, the properties of these networks can be fine-tuned by adjusting how growth factors are released over time, making this approach a powerful tool for creating vascularized engineered tissues.

[92] The peptides that form the aptamer variable regions are synthesized as part of the same polypeptide chain as the scaffold and are constrained at their N and C termini by linkage to it.

Left: Unbound aptamer. Right: the aptamer bound to its target protein. The protein is in yellow. Parts of the aptamer that change shape when it binds its target are in blue, while the unchanging parts are in orange. The parts of the aptamer that contact the protein are highlighted in red.
Breast cancer cells incubated with aptamers that bind selectively to biomarkers on the cancer cells, but not to healthy cells. Aptamers are linked to Alexa Fluor 594 , a molecule that glows red under UV light. This type of test allows a doctor or researcher to identify cancer cells in a tissue sample from a patient .
Jack Szostak, Nobel laureate and one of the inventors of SELEX and aptamers.
The complex and diverse secondary and tertiary structure of aptamers, as shown in this schematic of an aptamer's secondary structure, is what lets them bind their target strongly and specifically. Complementary base pairing is visible in the black lines connecting some G-C and A-T bases. This is a feature of nucleic acids that does not exist in the amino acids of antibodies. It helps aptamers form these unique structures. Hairpin regions (red), which rely on this base pairing, enhance the aptamer's stability at different temperatures. This image also shows examples of chemical modifications to the base aptamer. Two unnatural bases, which enhance durability, are in yellow. The biotin molecule binds with extreme strength to streptavidin , allowing the aptamer to be anchored to other molecules or to a surface in sensors and assays.
This assay tests the ability of two different types of aptamers (V and I) to detect their respective protein targets (VEGF and IFN-y). The labels Apt1, Apt2, Apt3, and Apt4 are in decreasing order of binding strength (i.e. Apt1 is the strongest aptamer). The DD, AD, DA, and AA letters mean that they have different combinations of unnatural base pairs. This causes their difference in binding strengths. The "-" columns have no protein, and the "+" columns do have protein. Aptamer with protein (+) and without protein (-) is loaded into wells in a gel and moves down the column lanes. If target is present, they bind and travel more slowly, due to the charge on the aptamer and the mass of the protein. Otherwise, the unbound aptamer moves quickly to the end of the lane. The difference in position between the "+" and "-" bands is the "mobility shift." This allows the researcher to detect the presence or absence of the protein. The darker band in the leftmost V and I lanes show that stronger aptamer-target binding makes it easier to detect the target at a given amount of target protein in the sample. The bottom image includes denaturing urea in the gel that disrupts aptamer-target binding in the weaker I aptamers, showing that the aptamer-protein binding is indeed what caused the mobility shift.