Decoy oligodeoxynucleotide engineering

The dODN is usually designed base on the promoter sequence in gene. Take the the dODN that targets to the STAT3 for example, it is based on the Serum Inducible Element (SIE) of the human c-fos promoter (Leong et al., 2003a). The oligonucleotide with 10-20 base pair was designed to bind the DNA binding domain of the STAT3. It abrogates the function of ability to bind to DNA response elements and induce transcription of target genes, STAT3 and elicits the anti-tumour effects through the inhibition of STAT3 down stream gene expressions. Into the cells, the introduced oligonucleotides faces the challenge of rapid degradation in serum. To conquer this

most common method is the phosphorothioation, which replaces the non-bridging oxygen with sulphur in the phosphate linkages (Brown et al., 1994). In addition to enhance the stability, researches have shown that the phosphorothioation also increases the cellular uptake efficiency and protein binding affinity. However, the modification also results in the increased nonspecific binding compared to the unmodified dODN (Brown et al., 1994).

• Means to determine the specificity

Generally, the consensus sequence of the dODN is derived as an integrated site from the transcription binding sites in the promoter of several genes. A tool for searching the appropriate sequence is, for example, the MatInspector (Cartharius et al., 2005). It is a software that involves the large library of matrix descriptions of transcription-factor binding sites to locate the matches in DNA sequences. This software is also suitable for designing an mutated ODN as control or to ensure that the scrambled ODN affects no other transcription factor (off-target effect). The consensus sequence typically comprises six to twenty bases and is targeting to the specific transcription factor. The exceptions is found when there is an overlapping binding sites between different transcription factors. For example, the cAMP response element (CRE) binds not only to the CRE binding protein (CREB), and also the members of the activator protein-1 (AP-1) family. Another exception is the common bindings sites that interact with kinds of proteins and thus causes a competitive manner. For example, the STRE-binding transcription factors early growth response protein-1 (Egr-1) and Sp-1 are verified to compete for the similar sequence. To solve this problem, it is logical to derive the binding-sequence from the binding site in the promoter of the primary target gene, instead of adopting the consensus binding site. Another issue is observed in the large

transcription factors families. Like the AP-1 and the STAT family, each member may binds to the same or similar sequence. That is, the competition binding between each might occurred among the family. Therefore, the consensus sequence derived dODNs of transcription factor in the large family might have low discrimination.

Several experimental methods have been reported for verifying the specificity of the dODN. The most common used methods are electrophoretic mobility shift analyses (EMSA) and enzyme-linked immunosorbent assay (ELISA), and down stream gene expression. In a typical EMSA, the cell extracts are mixed with the radiation-labelled dsDNA probe. The probe comprise a binding sequence for the transcription factor. The tested dODN displaces the probe that binds to the transcription factor in the extract, which results in the reduction of the probe–protein complex. Followed by the antibody-based super-shift analyses, the identification of the targeted transcription factor can then be achieved by. ELISA, which also allows the researchers to estimate the binding provide higher efficacy. In addition, it can be used determine to clarify how chemical modifications affects the sODN. For example, we can use ELISA to evaluate the how phosphorothioation affect the affinity and specificity toward the transcription factor.

The third common method is to determine the variation of the gene expression that governed by the transcription factor. This method is feasible only in the cellular model and can be achieved by using reverse transcription quantitative polymerase chain

reaction (RT-qPCR). The extracted mRNAs are reverse transcript to the cDNA and amplified with the PCR approach to quantify the level of the certain gene expression.

This method provides the cellular evidence of dODN effect on the transcription factor and hence is widely used.

• Means to enhance the transfection efficiency

In the last section, the importance of sequence design on the efficacy is mentioned.

Besides that, the transfection efficiency of dODN also dominates the efficacy. That is, a dODN that is easily and fast uptake by the cell is expected to benefit the efficacy. The common way used to determine the transfection efficiency is counting the fluorescent dODN-transfected cell with the flow cytometry. The dODN is labelled with fluorescent dye, such as Texas Red or Alexa Fluor 594. Besides the flow cytometry, fluorescence microscopy or any fluorescence reader are also the possible instrument to carry out the determination. Furthermore, the tested sample is not limited in cultured cells. The tissues or organs ex vivo are also suitable for the fluorescence determination (Shangguan D, et al. 2008)(figure 1.2). Besides the fluorescent dye, radiation labelling is also widely employed. For example, the phosphorothioated dODNs can be labeled with the ³⁵S to track its location in vitro. Notably, the ³²P or ³³P-labelling of the nucleic acids has been verified to be less suitable, due to the fast decay in the cell.

According to the past researches, four major parameters govern the transfection efficiency of dODN into the cell. They are concentration, length, nucleotide sequence, and nucleotide composition. The optimal condition for DNA transfection is 10 mM sequence with at least one hour of pre-incubation. In most cases, this condition provides a satisfactory outcome, which is a maximised inhibition of target gene expression. A 60-80% higher inhibition can be obtained compared to the control. Secondly, the optimal

length of the dODN is less than 30 base pairs. On the other hand, the transfection efficiency of a 25 base pair dODN is higher than a 10 base pair one. That is why most of the dODNs reported recently is in the range of 15-25 base pair. Although the previous literatures have pointed out an optimal condition of concentration and length, the transection efficiency of each dODN is not consistent. This phenomenon is related to the involved transport system during crossing the cell membrane. The transport pathway is highly dependent on the sequence and composition of the nucleotide.

Therefore, the characteristics of these transport systems is worth to be explored. There are many method fro determining the transport system that is involved.

Figure 1.2 Fluorescence microscopic image that demonstrates the uptake of a Texas Red-labelled Stat-1 decoy ODN into the human skin cells (red). The nucleus was stained with DAPI (blue). (Shangguan D, et al. 2008)

• Stability of decoy oligodeoxynucleotide in cell

The dissolved dODN in aqueous solution of neutral pH and ambient temperature is stable. Many stability analysis reports have been published to support the use of the dODNs in clinical application. For the long-term storage, the freeze-dried dODNs under 5℃ is recommended. The stability of the transfected dODN in cell is usually estimated according to the duration of their efficacy. For example, continuously monitoring the mRNA expression of the cells to see whether the transcription factor is still regulated by the dODN or not. According to some animal experiments, the dODN (with phosphorothioation at the 3’- and 5’-terminal three bases) remains its function until 48 hours after transfection (Buchwald et al., 2002).

To precisely determine the stability in cell, the ³⁵S-labelled dODN has been transfected into the human umbilical vein endothelial cells. With the aids of the native polyacrylamide gel electrophoresis and autoradiography, it is found that there is no degradation of the dODNN within 24 hours after the transfection. The results suggest that the dODNs that transfected into the cells remains stable and activity for quite a long period. The chemical modifications enhance the stability in a further extent. With the incubation of rat serum at 37℃, the calculated half-life of phosphorothioated dODNs is 16 hours, which is much longer than the unmodified one.