CALF INTESTINAL ALKALINE PHOSPHATASE APTAMER BASED BIOSENSORS

Abstract

In recent years, there has been an increasing demand for newer, more accurate, technologies that can detect and identify biomolecules or biological entities related to health, agriculture or the environment. With the discovery of new properties of nucleic acids beyond the storage and transfer of genetic information, a new class of nucleic acid-based biosensors is emerging, using DNA and RNA as target recognition elements with the advantage of being simpler and more cost-effective compared to antibodies-based biosensor.

Two sequences, TrG14MC and TrG10SC, with evidence to suggest that they are capable of inhibit the metalloenzyme CIP, were isolated from a selection conducted by Dr. Razvan Nutiu. Here we study the inhibitory properties of these two aptamer candidates and measure the IC50 value, determined as 94 nM for TrG14MC and 83 nM for TrG10SC. Different bivalent constructs, designed to increase the inhibitory effect of the isolated sequences, are studied showing a pronounce influence of the linker length improving the inhibitory effect over CIP.

Modulating the interaction of the isolated sequences and the CIP is of key importance in order to develop a successful biosensor. Therefore, we try to recover CIP from the inhibition effect by using antisense sequences complementary to different segments of the construct. The maximum recovery, 75%, was achieved by an antisense sequence fully complemented to the inhibitory bivalent construct. We also study here the use of a linker in the bivalent construct that forms a secondary hairpin structure, and the effect of linearizing that structure with an antisense sequence complementary to the linker. This resulted in as 12% of the inhibitory effect.

The purpose of this investigation was to establish the first steps toward the development of a new class of biosensors capable of disinhibiting CIP upon the recognition of a specific target, taking advantage of the suggested CIP-inhibitory properties of the isolated sequences TrG14MC and TrG10SC.