Aptamer Identification and Characterization

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the assumption, that despite efforts to prevent the PCR bias, the enriched nucleic acid pool of the buffer-SELEX-experiment did suffer from by-product formation during PCR. This subject could have been investigated in more detail, if SELEX-rounds had been probed by NGS. Nevertheless, the buffer-SELEX-experiment did lead to the selection of Buf-2, which showed high P-domain affinity and selectivity, demonstrating that the SELEX did succeed, despite the presence of PCR by-product formation.

4.1.5 Aptamer Selection for the P-domain in Presence of Oyster Matrix

Enrichment of oligonucleotides was not noticeably achieved during the oyster- and oyster div.-SELEX-experiments, despite a mild enrichment of Buf-2. It is known that NoV VLPs bind the oyster diverticula and, depending on the NoV strain, can bind to oyster muscle tissue and glands^{55, 106}. NoV GI.1 VLPs bind oyster diverticula tissue through HBGA-A-like carbohydrate antigens¹⁰⁶. An additional study, characterizing the role of the P-domain interaction with HBGA, reported that the sole P-domain (produced in Sf9 cells) did bind to the HBGA, but that the sole shell domain did not⁴⁷. This indicates that the P-domain is responsible for the interaction with HBGA. Since the bond between NoV VLPs and oyster diverticular tissue was defined as HBGA A-like, we could assume that the P-domain bound to oyster tissue in the food matrix sample. The presence of oyster matrix during SELEX could have thereby prevented the binding of oligonucleotides to the P-domain, leading to the exclusion of P-domain binding oligonucleotides. However, it is not clear if the unassembled P-domain would bind oyster tissue in the same way as the assembled virus particles, or in the way the sole P-domain binds HBGAs.

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according to a previously published guide¹³⁸). During the screening of enriched oligonucleotides, Buf-2, Buf-5, and Buf-8 were identified as aptamer candidates since they exhibited the highest affinity to the P-domain. However, repeated FRA results for Buf-5 were inconsistent for different P-domain concentrations and not reproducible within the triplicate tested (data not shown).

Therefore, oligonucleotide Buf-5 was not further characterized. Oligonucleotide Buf-1 was also examined to understand why this candidate was enriched during the buffer- and the lettuce-SELEX-experiment, despite its merely moderate affinity to the P-domain. Aptamer selectivity was tested, by FRA with other proteins, chosen based on in-house availability. It was shown that candidates Buf-1 and Buf-2 were highly selective, based on the proteins tested in equimolar concentrations. However, Buf-8 did not bind exclusively to the P-domain, as determined in binding studies by FRA using the proteins BSA, lysozyme, and thrombin (Figure 17). Therefore, Buf-8 was not included in specificity and affinity studies.

The binding specificity of aptamer candidates Buf-1 and Buf-2 to the P-domain was assessed by adding unlabeled aptamer candidate (specific competitor) and unlabeled thrombin aptamer (non-specific competitor) to the aptamer-P-domain binding reaction. Aptamer candidate Buf-1 was inhibited by the non-specific competitor equally as it was by the specific competitor, indicating that P-domain binding of Buf-1 is not specific. This coincides with previous findings, showing that a high abundance of an oligonucleotide in the last SELEX-round does not necessarily correlate with strong binding characteristics¹⁹¹. However, P-domain binding of Buf-2 was not inhibited considerably in presence of non-specific competitor, confirming the specific P-domain affinity of this aptamer candidate.

Dextran sulfate has been used primarily as a polyanionic competitor in the development of SOMAmers (introduced in section 1.2.2) by presenting a kinetic challenge to promote the selection of aptamers with low Koff constants¹⁴⁸. Dextran sulfate has also been applied as an agent to reduce non-specific binding of DNA aptamers to their target^206-207. It is likely that a low Koff is a beneficial property for an aptamer to be used in food matrices. The FRAs employed during our study did not give kinetic information and, therefore, no information about dissociation or association rates of the aptamer candidate-P-domain complex. Therefore, target binding behavior of different aptamer candidates in presence of dextran sulfate was investigated, to estimate possible kinetic binding characteristics. Although dextran sulfate was not included during the SELEX process, food matrices and increasing number of washing steps were introduced to favor aptamers with low dissociation rates. The P-domain binding of all candidates tested in the presence of dextran sulfate was inhibited by 47-82 % with candidate Buf-2, exhibiting the least inhibition by dextran sulfate.

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These results indicate that Buf-2 could have most favorable kinetic properties of the aptamers tested. However, these observations would need to be confirmed using true kinetic techniques, such as ITC. Additionally, the role of the aptamer’s kinetic properties in relation to its suitability for application in food analytical application should be further elucidated.

Buf-2 exhibited high affinity for the P-domain as indicated by its low Kd, it also exhibited selectivity, based on the proteins tested, and bound the P-domain specifically as indicated by the binding observed in presence of specific and non-specific competitors. Buf-2, therefore, fulfilled the criteria of an aptamer as it bound its target specifically and with high affinity.

4.2.2 Structure of Aptamer Buf-2

Aptamers can adopt G-quadruplex structures which form into four-stranded circular structures as a result of stacked G-quartet structures in presence of monovalent cations such as sodium or potassium ions, e.g. physiological buffer conditions157, 208-209. G-quadruplexes result from inter- or intramolecular folding of one or multiple, molecules with high G abundance. The intramolecular folding of a G-quadruplex necessitates at least four G-tracts in one strand²¹⁰. This is the case for the potassium aptamer with the sequence: 5’-GGGTTAGGGTTAGGGTAGGG-3’²¹¹. During this work, aptamer Buf-2 exhibited a 20 nt motif with repeated occurrence of a G triplicate. Among G-quadruplex aptamers, the most common nucleotides found between the Gs are Ts, with As and Cs only being found in between the G tracts sporadiacally¹²⁴. This was consistent with the identified 20 nt motif in Buf-2, as T was found with highest abundance within the motif. For 20 nt fragment of the Buf-2 oligonucleotide with the sequence: 5’-GGGTTCGGGTTTGGGTTGGG-3’ the highest likelihood for G-quadruplex formation was projected using the open software QGRS Mapper. The result predicted the contribution of all 12 Gs. The analyzed Buf-2 variants (section 3.4.2) were chosen based on this motif.

CD spectroscopy enables the distinction between different DNA structures, by measuring the difference in absorption of right- and left-handed circularly polarized light of chiral molecules, which is called circular dichroism²¹². CD spectroscopy was, therefore used to investigate the structure of Buf-2 and of three variants of Buf-2. In addition to CD spectroscopy investigation, FRA of the four molecules were completed to find the P-domain binding motif of Buf-2 and investigate the possibility of truncating the aptamer. The positive and negative peaks of the molecules’ CD spectra were compared with recently published CD spectra of aptamers with parallel, antiparallel formation, and the B form of DNA²¹³ (Table 12).

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Table 12. Maxima and minima in CD spectra of different aptamer structures. The wavelength maxima and minima of aptamer structures are shown, as reported in a recent study²¹³ and the maxima and minima of the Buf-2 variants are shown as recorded during the CD analysis (section 3.6.3).

Structure/molecule Positive Peak [nm] Negative Peak [nm]

Antiparallel G-quadruplex 295 (bulge 240) 265

Parallel G-quadruplex 260 240

B-form DNA 270 240

Buf-2 285, 260 240

Buf-2 variant 1 285,260 235

Buf-2 variant 2 285 250

Buf-2 variant 3 290,240 265

The Buf-2 variant 3 showed the typical spectrum of an antiparallel G-quadruplex, which has also been observed for the thrombin binding aptamer TBA¹⁵⁴. However, the spectra of 2 and Buf-2 variant 1are not consistent with this spectrum. Both their CD spectra show identical bands, despite the difference in amplitude. The CD spectra of Buf-2, Buf-2 variant 1 and Buf-2 variant 2 shared a common positive peak at 285 nm, which matches neither maxima of the G-quadruplex structures, nor the B-form DNA. Negative peaks for the three molecules were observed at 235 nm, 240 nm, and 250 nm. CD-spectra of B-from DNA and the parallel G-quadruplex aptamers both typically show a trough in the CD spectrum at 240 nm; it is therefore not possible to make a distinction between the B-from DNA and the parallel G-quadruplex based on this negative peak.

However, the CD spectra of Buf-2 and Buf-2 variant 1 each showed a shoulder peak in the CD spectrum at 260 nm. The combination of the positive peak at 260 nm and the negative peak at 240 nm indicated the presence of a parallel G-quadruplex structure. The additional peak observed in both spectra at 285 nm, and the fact that an antiparallel Q-quadruplex structure had already been identified for Buf-2 variant 3 (20 nt motif) leads to the conclusion that Buf-2 is a parallel/antiparallel G-quadruplex hybrid. G-quadruplex hybrids were first described for telomeric regions in 2006²¹⁴. The CD spectrum shown in the 2006 study (Figure 37 (B)) shows multiple CD spectra. The G-quadruplex hybrid was termed Tel26 and in presence of K⁺ shows a spectrum (shown in light pink) very similar to the Buf-2 CD spectrum. The Tel26 spectrum exhibits the same maximum peak around 290nm, the shoulder peak around 265 nm, and a trough at 240 nm.

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A B

Figure 37. Proposed folding and CD spectrum of parallel/antiparallel G-quadruplex as described previously. (A) Schematic diagram of the folding topology of the unimolecular human telomeric hybrid G-quadruplex in potassium solution. Red ball: guanine, Red box (anti) guanine, magenta box: (syn) guanidine, green ball: adenine, blue ball:

thymidine. (B) CD spectra of multiple telomeric molecules in 100 mM Na⁺, or K⁺ solutions at 25 °C. For Tel26, a parallel/antiparallel hybrid structure was proposed in K⁺ solution. The according CD spectrum is shown in a in a light pink line²¹⁴ (this figure has been used for the thesis in agreement with Oxford University Press and Copyright Clearance Center).

The formation of the hybrid G-quadruplex described in the study by Ambrus et al. 2006 is induced by the presence of potassium ions, as shown in Figure 37 (A)²¹⁴. In the case of Buf-2 however, the hybrid formation seems to be related to the presence of the 5’- end, as the shoulder peak at 260nm is only visible in the Buf-2 and the Buf-2 variant 1 CD spectra (Figure 20).

The FRA of the Buf-2 and its three molecule variants revealed that only the 40 nt Buf-2 molecule bound the P-domain. This could signify two things. One possibility is the binding motif is located within the 5’- or 3’- end of the molecule, but both need to be present to form the required structure to bind the P-domain. Secondly, it is possible that the molecule adopts an unpredicted structure upon target binding (induced fit). Such an induced fit upon target binding has been proposed other for aptamers, among these is the OTA binding aptamer, which folds into an antiparallel G-quadruplex upon binding to OTA²¹⁵.

The actual confirmation of Buf-2, as well as the aptamer structure when bound to the target molecule, would need to be assessed in further studies using NMR and UV thermal denaturation.

Additionally, the P-domain binding of Buf-2 under different buffer conditions, focusing on the K⁺ and Na⁺ concentration could be assessed using CD spectroscopy to investigate the ion-dependency of the aptamer structure.

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