Nucleobase modified oligonucleotides (SOMAmers)

PCR could be performed with AccuPrimePfx DNA polymerase. After sequencing of the resulting natural DNA sequences, the original positions of dDs within the sequences could be identified with the “barcode-tag”²⁰².

The initial restrictions concerning the positions of dDs within the SELEX libraries were meanwhile conquered by an even more sophisticated way of sequencing²⁰³. In new versions of ExSELEX a completely randomized library containing dDs was applied in a selection of an aptamers for the von Willebrand factor A1-domain²⁰³. The Ds-containing aptamer showed significantly higher affinity to the target compared to a known aptamer consisting of natural nucleobases. The affinity of the resulting aptamer was even increased compared to aptamers resulting from the initial selection-strategy with predefined fixed positions of dDs. This result indicates that aptamers with artificial nucleobase have superior interaction capabilities compared to aptamers wit natural nucleobases^198,204.

Fig. 9. Structures of some artificial base pairs. a) Recognition of the base pair 6-amino-5-nitro- 3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (Z) and 2-amino-8-(1′-β-D-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)one (P), which is used in AEGIS-SELEX is based on H-bonds. b) The basepair 2-nitro-4-propynylpyrrole (dPx) and 7-(2-thienyl)imidazo[4,5-b]pyridine (dDs), used in ExSLEX is based on size and shape complementary. The artificial nucleotide dPa´ is used for the replacement PCR which is needed to replace the Dx bases into natural nucleotides for sequencing purposes.

approach, the resulting modified aptamer did not show superior binding behavior to thrombin compared to a previously discovered unmodified DNA aptamer¹³⁷. Nucleobase-modified SELEX-libraries are still limited to four building blocks, in contrast to oligonucleotide-libraries with an expanded genetic alphabet. Nonetheless, introducing chemical modifications into the nucleobases clearly alters their biophysical properties. Libraries harboring these nucleotides therefore do have an increased chemical diversity, compared to conventional DNA-libraries.

The company SOMAlogic has commercialized the selection approach utilizing libraries with modified pyrimidines. Mainly inspired by the hydrophobic characters of amino acid side chains, a broad range of functionalized pyrimidine bases has been synthesized (see Figure 10 a). The C5-position on pyrimidines is the position of choice for the introduction of chemical modifications, since these modified nucleotides are still accessible for enzymatically catalyzed reactions as PCR²⁰⁷.

The first selections were performed with just one modified pyrimidine, the deoxy uridine. The success-rate for those selections increased dramatically (to >80%) compared to selections with conventional oligonucleotides(<30%)^190,207. By now, two differently modified pyrimidines (deoxy-uridine as well as deoxy-cytidine) can be incorporated into oligonucleotide libraries. Oligonucleotides bearing two distinct modifications have an even further increased chemical diversity, higher target-affinity, and nuclease resistance than those with one modification ²⁰⁸. Libraries containing these modified nucleotides have been broadly applied in selections for thousands of protein targets resulting in high affinity aptamers, termed SOMAmers (Slow Off-rate Modified Aptamers)^209,210.

SOMAmers are mainly selected for diagnostic and biomarker discovery applications.

Due to the multiplicity of existing SOMAmers, the so called SOMAscan approach permits the concurrent detection of thousands of proteins in a broad range of biological matrices^211–214. A schematic representation of such a SOMAscan can be seen in Figure 10 b. SOMAmers (purple) for different proteins are equipped with individual fluorophores (F), as well as photo cleavable linkers (L) and a biotin-tag (B). The modified oligonucleotides are captured on streptavidin coated solid support and incubated with the matrix containing a diverse mixture of proteins. After the individual SOMAmers have bound their target proteins, the non-bound proteins are washed away. Captured proteins are successively biotinylated and the photo cleavable linker which attaches the SOMAmer-protein complex to the solid support is cleaved (Figure 10 b (i)-(iii)). Biotinylated proteins are captured on new streptavidin coated matrices and non-specific interactions to SOMAmers are disrupted by the addition of a polyanionic competitor (iv). Finally, the fluorescent tagged SOMAmers are released from their protein complex in a denaturating buffer (v) and hybridized to complementary sequences on a micro-array (vi). With the help of SOMAscan the concentration of several proteins in biological samples can directly be translated into SOMAmer based fluorescence intensities.

This multiplexed SOMAmer affinity assay is a novel tool in clinical diagnostics, as it helps to discover biomarkers for several diseases207,210,215.

Fig. 10. Examples of utilized pyrimidine modifications during SOMAmer-selections and the scheme of a multiplexed SOMAmer affinity assay. a) A selection of chemical building blocks which can be introduced to the C5-position of both pyrimidine bases. b) Schematic mode of operation of a SOMAscan. Proteins of interest within a biological sample are bound by the corresponding SOMAmers (i)-(iii). This binding event is converted into fluorescent signals enabling the detection and quantification of proteins of interest (iv)-(vi). Figure 8 b was modified after Gold et. al¹⁹⁰.

For SOMAmers the amount of polar contacts with the protein targets are reduced compared to conventional DNA-aptamers²¹⁶. Furthermore, the introduced protein-like side chains are often causative for target-interactions, as could be confirmed by diverse crystal structures194,216–218. Besides direct contact between nucleobase-modifications and protein-surfaces, several examples are known, wherein the modifications primarily help to stabilize the dimensional structures. Such three-dimensional structures have been previously unknown for aptamers. For example, SOMAmers containing hydrophobic modifications can fold into intramolecular zipper-like structures by π-π -stacking interactions with other modified pyrimidines (see Figure 11)^217,218. The formation of those rigid 3-dimensional structures might also be supportive for the SOMAmer´s binding properties.

Fig. 11. Presentation of the NGF-SOMAmer forming zipper-like structures. The figure was modified after Jarvis et. al. ²¹⁷ a) Cartoon illustration of the SOMAmer. Six out of nine benzyl-modified nucleotides are involved in structural stabilization of the SOMamer and do not directly interact with NGF. b, c) Within the two formed zipper-like structures, benzyl modifications show π-π -stacking interactions with adjacent modified pyrimidines. b) dU 16, dU 18 and dU 3, as well as c) dU 27, dU 11 and Bn-dU 10 form the zipper-like structures.

Disregarding the immense success of SOMAmer selections, this approach does pose some problems concerning the applicability in ordinary biochemistry labs. As can easily be envisioned, complex synthesis of the nucleoside-triphosphates and the phosphoramidites has to be performed previous to each selection-approach. The company SOMAlogic has automated the selection-process, resulting in SOMAmers for thousands of proteins. Naturally, that huge effort cannot be done in smaller laboratories. Another minor drawback of SOMAmer-selections is that a direct PCR-amplification of modified DNA-templates during SELEX is not possible. However, this problem can easily be circumvented by a two-step PCR-procedure. The modified templates are first PCR-amplified with the conventional set of nucleotides (dATP, dCTP, dTTP and dGTP). The resulting PCR-product can then be amplified with a dNTP-mixture containing modified nucleotides²¹⁹.