关键词: 毛细管电泳, 核酸适配体, 8-氧代鸟嘌呤DNA糖基化酶, 竞争筛选, 多轮筛选

Abstract:

8-Oxoguanine DNA glycosylase (OGG1) is an important enzyme that plays a key role in oxidative DNA damage repair. OGG1 can specifically recognize and excise 8-oxoG (a product of oxidative damage found in double-stranded DNA) through base excision repair (BER). OGG1 is expressed in normal tissues, and in most tumor tissues. Oxidative cellular damage can produce an inflammatory reaction, alleviating some measure of constitutive OGG1 inhibition. OGG1 inhibition in cancer cells shows some promise as a new method of cancer treatment. Most current OGG1 research focuses on regulating OGG1 with targeted small molecules. To date, no aptamer screen for OGG1 has been reported. Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target with high affinity and specificity in vitro, that can be identified by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used as chemical ligands to regulate intermolecular interactions. In this study, a screen for aptamers with OGG1 affinity was performed for the first time. Capillary electrophoresis (CE) is a microanalytical technique that offers speed and high separation efficiency. In this work, two screening methods based on CE-SELEX technology were established: a one-round pressure controllable selection, and a multi-round selection. The most important criterion for successful one-round pressure controllable selection is to select a competitive target with a different CE migration time than that of the target of interest. We mixed OGG1 with a competitive target and a nucleic acid library for CE analysis. Two proteins competitively bind sequences in the library, forming independent complexes. The concentration of the competitive target is continuously increased until complexes with the target stop decreasing, indicating that the target and the ssDNA library have formed a stable complex. Complexes were collected for PCR amplification, purification, and high-throughput sequencing to obtain high affinity aptamers. This method greatly improves screening efficiency, and reduces non-specific binding to the target, which is helpful for obtaining aptamers with high affinity and specificity. One-round pressure controllable selection for high affinity OGG1 selective aptamers was performed using single strand binding protein (SSB) to competitively and tightly bind nucleic acids in the library. The competitive screening pressure was increased by increasing the SSB concentration to eliminate sequences with low affinity for OGG1 from the random oligonucleotide library. Nucleic acid sequences with high OGG1 affinity were obtainable in one step, and OGG1-ssDNA complexes were collected by creating a timed program on Beckman P/ACE MDQ capillary electrophoresis. Collection occurred from 2.2 to 2.8 min. Under identical incubation and electrophoresis conditions, multiple round selections were conducted by injecting samples of co-incubated nucleic acid library and target into the capillary. After separation under a high-voltage electric field, nucleic acid target complexes were collected, amplified by PCR, purified, and used as an enriched secondary library in the next round of screening. High affinity aptamers were generally obtained within three rounds. Comparing results of the two screening methods, the three candidate aptamer sequences found with the highest frequency were consistent, and displayed K_D values ranging from 1.71 to 2.64 μmol/L. Molecular docking analysis suggests that Apt 1 may bind to the OGG1 active pocket, which functions to repair oxidative damage. Comparison of the two screening methods indicates that one-round pressure controllable selection is more rapid and efficient, providing guidance for the design of other protein aptamer screening methods. The obtained aptamer is expected to be function effectively as an OGG1-mediated DNA repair inhibitor.

Key words: capillary electrophoresis (CE), aptamer, 8-oxoguanine DNA glycosylase, one-round pressure controllable selection, multi-round selection