Direct Sequence Analysis Method for Rapid Discovery of DNA Ligand Binding Sequences for Biosensor Technologies

Primary Partner: Syracuse University
Project Type: CARTI I
Web Address: http://chemistry.syr.edu/faculty/borer.html

Technical Description:

This material is based upon work supported by the United States Environmental Protection Agency under Award Number EPA 05 X-83232501-0.

Nucleic acid constructs have been found that have a high affinity and specificity to biological targets, making them candidates for capture-based biosensor technologies. However, this area of biosensor development is currently unmet due to:

1. the complexity of the existing nucleic acid discovery method (called SELEX) which is prone to errors and difficult to automate and
2. the expensive upfront cost and terms for licensing the SELEX platform. Here we propose a novel alternative to SELEX called the Direct Sequence Analysis (DSA) method. The DSA method will use a structurally defined library of short, variable DNA oligomers that can be rapidly screened, cloned and sequenced in a single cycle to identify DNA constructs that bind biological targets with high affinity, called ligand binding sequences (LBS). Sequences generated from these screens will be easier to characterize, cheaper to synthesize and very simple to engineer into bistable molecular sensors, termed OrthoSwitches or AlloSwitches (OrthoSystem, Inc.).

Expected Outcomes:

In this project we aim to develop and optimize the DSA technique using polyvalent biological targets. If successful the method will be automated to allow us to create a screening platform to rapidly screen a collection of biological targets in parallel.

Accomplishments:

In this project we focused on:

1. optimizing the novel blunt-end ligation reaction,
2. assess-ing the quality of cloned sequences using control probe sets, and
3. investigated partitioning (screening) protocols such that high affinity probes can be subsequently cloned directly in plasmids.

The project concluded by successfully isolating a unique DNA probe sequence for thrombin protein that resembles a published high affinity DNA aptamers probe termed the Thrombin Binding Aptamer (TBA).

Benefits:

These DNA probes can be used in biosensor applications, cell staining, Orthosystems' AlloSwitch technology, and for antibody replacement.

For more information: http://chemistry.syr.edu/faculty/borer.html

DSA Scheme.

Image Credit: Philip Borer, Syracuse University.

Researcher Information:



http://www-che.syr.edu/faculty/borer.html