Antisense Webinar Transcript

Better Antisense Design Without the Trial and Error:  Problems we Solve

Most researchers use naïve strategies to design antisense oligonucleotides.  However, to maximize assay sensitivity and specificity one must consider probe and target sequence and their respective and combined chemistries.  That is why DNA Software has built the Antisense Architect design and simulation platform for the rational design of Antisense Oligonucleotide Analogs.  Three problems that we solve are the proper evaluation of Target Accessibility and sensitivity, the minimization off-target effects that elicit immune responses and often cell death, and we have the necessary thermodynamic libraries to predict the proper use of Chemical Modifications to prevent enzymatic digestion.

Target Accessibility:  The Conventional Approach to Duplex Formation

The naïve way to design Antisense Oligonucleotides is to only consider the two-state model of probe hybridization which describes the probe and its hybridization to its complement.  Target accessibility and tail folding are not accounted for, therefore this is an incomplete model of what is really happening in the system which leads to a lack of sensitivity due to target and probe secondary structure and negates the effect of bimolecular mishybridization reactions.

Competing Secondary Structure-False-Negative Assays

The platform for each of our DNA Software products is based on the multi-state equilibrium model which accounts for the energetic cost of target and probe secondary structure through coupled equilibria, which ultimately increases assay sensitivity.  This means that the Antisense Architect will evaluate and thermodynamically score your desired design sites, and if given an entire target, it will identify all thermodynamically accessible sites.

Off-Target Effects:  Using ThermoBLAST to Predict Crosshybridization and Mishybridization

Many researchers use NCBI BLAST for their oligonucleotide design because its free, but even free has its associated costs because BLAST is the wrong tool for determining selectivity.  BLAST was meant to determine common evolutionary ancestry through sequence similarity, however sequence similarity does not equal thermodynamic stability as BLAST does not account for thermodynamic contributions of basepairing and mismatches, target bulges and dangling ends and different oligo backbone chemistries.  DNA Software’s products take all of the aforementioned thermodynamic contributions into account in both the design and simulation of Antisense oligonucleotide analogs, which results in a more accurate prediction of oligonucleotide mishybridization and crosshybridization.

Chemical Modification:   Packages are Customizable

It is well documented that the key to preventing the enzymatic digestion of Antisense oligonucleotides, or in some cases immune stimulation, lays in the chemical substitution of the probe backbone or 2’ sugar moieties. What is not well documented is the thermodynamic libraries required for rational design that takes into account the thermodynamic contributions of the modifications listed that predict probe secondary structure and the energy of hybridization and ultimately the melting temperature, or Tm.  Depending on the modifieds used the Tm could differ by as much as +/- 10 degrees celcius, which may have an unwanted and undefined effect on your assay if you are not able to simulate the use of modifieds in design. DNA Software has determined the thermodynamic effects of the listed modifieds and is prepared to build an Antisense Architect Platform that is specific to your research needs and requirements.

DNA Software’s Antisense Architect

The recurring issues of Target accessibility, Probe design, Off-target effects and the proper use of chemical modifications are solved in the Antisense Architect which uses design heuristics that allows for the rational design of Antisense oligonucleotides.  Some heuristics that are used in design include the evaluation of secondary structure and sequence composition and complexity of targets and probes.  We will discuss the design heuristics in more detail when we review the design results of our demo.

Antisense Architect: 4 easy steps

The Antisense Architect use a design wizard format to collect necessary input from the user for the design of highly sensitive and selective Antisense probes in four easy steps:

-Choose the background RefSeq of interest and the target

-Choose target design region

-Choose desired probe chemistry and hybridization conditions

-Run the job and get results.

And now…the Antisense Architect Demo!