Oligodeoxynucleotide in diagnostics

With the aids of the molecular biology, it is now possible to have an assay for imaging the specific molecular processes, such as the gene expression. Imaging gene expression enables us to determine the locations of particular gene expressing. Besides, we can even monitor the level and duration of that gene expression. Imaging the gene expression with the generic approaches to the gene expression of interest has been researched for years. The conventional imaging techniques use the radiolabeled substrate to interact with the protein of the gene to image the expression. To be convenient to the users, more and more general methods without the need for radiolabeled substrates are developed to image the gene expression. In this section, we would like to focus on the aptamer approach for imaging protein of the gene expression.

To date, there are only a few groups using the dODN to image the proteins that related to the specific gene expression. Among the diagnostic oligonucelotieds, aptamer has pave a way for the dODN to develop the protein imaging. Therefore, methods to introduce aptamer into target, principle, and applications of aptamer for imaging gene

• Fundamentals of gene expression

Even today, the process of gene expression (figure 1.3) and the components of gene regulation are only partially understood. Every cell has its own gene expression feature, which leads to the wide variety of functions or phenotypes. The development, maturation, proliferation, and even the oncogenesis of the cell is attributed to the kinds of gene expression. Promoter is a sequence motifs (~100 nucleotide bases) at the specific region of the gene that the transcription initiates. It is involved in the RNA polymerase binding and the transcription initiation. In the eukaryotic cell, the promoters do not always work alone. The other point that worth to be mentioned is that it can be influenced by the enhancers. The enhancer has no certain location, it exist in the either the upstream or the downstream from he promoter. Sometimes, the enhancer is even located within the transcription unit. The role of the enhancer is either increasing the initiation efficiency or participating in a specific regulation. After interacting with the promoter, the complex of the proteins and the RNA polymerase starts to join the transcription process. To image the gene expression, two main types of promoters are worth to be understood. They are constitutive and inducible promoters. The former can be used to produce continuous gene transcription, and the latter can be used to externally control the level of transcription. For example, constitutive promoters are cytomegalovirus, CMV (exogenous gene) and GAPDH (endogenous gene). Inducible promoters are tetracycline regulable (exogenous gene) and c-jun (endogenous gene).

Figure 1.3 An illustration briefly describes the gene expression in cell.

• Aptamer probes targets to the protein

Gene expression imaging with oligodeoxynucleotide can be achieved wither by the mRNA monitoring or down stream protein probing. In the case of using aptamer to probe the protein, the affinity between the sequence and the target protein is dominated not by the base pairing, but three-dimensional steric interactions between the molecules.

An aptamer possess a dissociation constants in a range between the micro-molar to pico-molar, which is comparable to that of the antibody. The systematic evolution of ligands by exponential enrichment (SELEX) has been used both to characterise the interaction of nucleic acids pool with proteins and to generate the new aptamers (Conrad et al., 1995, Buerger and Groner, 2003, Tavitian, 2003). The library contains the random sequence is used to screen for the high affinity probe targeting to a specific protein. The aptamers is able to recognise a single or multiple targets, including proteins, small molecules, or even the complex of the heterogeneous targets. The aptamers is able to be transported into the cell theoretically, thus the down stream of genes can potentially be targeted. Almost two decades ago, the first use of an aptamer to

target the neutrophil elastase (a surface protein) to achieve the in vivo inflammation imaging with a gamma camera is reported (Charlton et al., 1997). The used model is a rat with reverse passive Arthus reaction (local hypersensitive reaction on skin). In this case, the aptamer approached the maximum target-to-background ratio of 4 in 2 hours.

Afterward, the aptamer is fast removed with the blood clearance. From now on, more and more researches in this area are reported to facilitate other imaging applications in which the protein is targeted with the aptamers. These approaches allow the user to image the endogenous gene expression, by selecting the aptamers target toward the protein of that specific gene. In a following research, the aptamers are labeled with

99mTc for imaging purposes and to investigate aptamer pharmacokinetics toward the Tenascin C, which is an extracellular stroma protein present in tumour and neovasculature (Hicke and Stephens, 2000). Due to a long tumour half life and fast clearance from the body, ^99mTc-labeled aptamers yielded a tumour-to-blood ratio of 50 within 3 hours. And the clear images of U251 and MDA-MB-435 are obtained with a gamma camera (Hicke et al., 2006). Like what has been mentioned above, since there are a few group doing the research of dODN imaging, the aptamer based system become a reliable reference. on the same basis of protein recognition, methods and knowledges from the aptamer imaging approach lead the dODN imaging research toward the right direction.

To date, the significant progress in the molecular imaging has been made, thus facilitate the developing of assays for in vivo gene expression imaging. Even though, there is still room for improvement to develop a specific, quantitative, highly sensitive, and practical assays for clinical applications. Generally, oligonucleotide-based imaging approaches will need to enhance the key features, including the stability of probes, prevention of the non-specific interaction, and minimising the background signal. Once the successful has

been developed, it will provide a general method to imaging gene expression. As well as to identify the early changes in gene expression signalling between the normal cellular function to an altered state of disease. Oligonucleotide-based imaging approaches have been proven in many preliminary animal applications and pilot human studies. Studies with transgenic animals, in vivo inducible expression, bicistronic vectors, and the monitoring of endogenous gene expression are all widely investigated and accompanied with the meaningful result. In the nest step, multi-modality reporters and the complex of reporters and probes will facilitate the the development process of in vitro and in vivo assays, which will allows the researchers to study the more complicated biological processes.