Tuesday, April 9, 2013

A look at Antisense Technologies

One of the greatest technologies to determine how to specifically treat diseases in humans, animals and plants is with antisense technologies.  These technologies, which manipulate DNA or RNA, end up interrupting the normal cellular processing to a gene and we can therefore discover what the gene functions as.  Integrated Gene Technologies recently released a white paper examining this issue.

Here is an excerpt:
Antisense Oligonucleotides
Oligonucleotide-based antisense techniques represent the most common and, to date, the most successful approach to achieving suppression or elimination of a genetic message. The antisense effect of a synthetic oligonucleotide sequence was first demonstrated in the late 1970s by Zamecnik and Stephenson [1]. Using nucleotide sequences from the 5’ and 3’ ends of the 35S RNA of Rous sarcoma virus (RSV), Zamecnik and Stephenson identified a repeated sequence of 21 nucleotides (nt) that appeared to be crucial to viral integration. They synthesized a 13-mer oligonucleotide, d(AATGGTAAAATGG), complement to a portion of this viral sequence. When this synthetic oligonucleotide sequence was introduced into cultured fibroblast cells infected with RSV, viral production was significantly inhibited. They correctly concluded that the oligonucleotide was inhibiting viral integration by hybridizing to the crucial sequences and blocking them. The term they introduced to describe such oligonucleotides was “hybridon.”

At the same time as this work was being done, other groups, notably Tennant et al. [2] and Miller et al. [3], were reporting similar effects for synthetic oligonucleotides in other systems. These results stimulated a rash of studies focusing on the ability of synthetic oligonucleotides to interfere with genetic processes. Many of theses studies failed to achieve the desired effect and it quickly became clear that there were a number of issues that needed to be addressed if synthetic oligonucleotides were to become generally useful reagents for these studies. The most immediately important of these issues was what can be called “persistence.” Synthetic oligonucleotides are foreign to the cells into which they are introduced and they immediately become prey for endogenous nucleases. If synthetic oligonucleotides were to attain the level of persistence in the cell that would be needed for them to accomplish their tasks, they would have to be protected from those endogenous nucleases. Following Kurreck [4], there are three possible sites on a nucleotide where protective modifications could be introduced (Figure 1). In both DNA and RNA nucleotides the base can be altered or changes can be effectedin the phosphate backbone. In RNA nucleotides the 2’ hydroxyl group, missing in DNA nucleotides, can also be modified. The “trick” involved in protective modifications of nucleotides is to introduce an alteration that is protective against nuclease degradation that does not, at the same time, eliminate the desired effect of the oligonucleotide sequence by blocking complementary hybridization or harming the cell. 

Read the full white paper here.

This May at the TIDES Event,we will be looking at antisense technologies in a number of presentations including Antisense Development Portfolio Advances with Richard S. Geary, Ph.D., Senior Vice President, Development, Isis Pharmaceuticals, Inc. and Characterization of Oligonucleotide Biotherapeutics by LC/MS/MS with Pfizer. For more information on these sessions, download the agenda. If you'd like to join us at the TIDES event taking place May 12-15, 2013 in Boston, as a reader of this blog when you register to join us and mention code TIDES12JP, you'll save 20% off the standard rate!

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