MAGNETIC-ASSISTED RAPID APTAMER SELECTION METHOD FOR GENERATING HIGH AFFINITY DNA APTAMER

摘要

A magnetic-assisted rapid aptamer selection (MARAS) protocol for screening DNA aptamer is proposed. The MARAS protocol is able to efficiently generate aptamers with high affinity and specificity. A rotating magnetic field or alternating magnetic field was used in combination with target-bound magnetic micro-particles or nanoparticles to select DNA aptamers having desirable affinity and specificity to the target.

主权项

1. An aptamer selection protocol, comprising:
a) providing a sample comprising at least a random sequence library having a plurality of oligonucleotide sequences and a plurality of magnetic nanoparticles or micro-particles having target molecules joined thereon, wherein a first portion of the plurality of oligonucleotide sequences is bound to the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon; b) performing a first round of magnetic separation by using a magnetic stand to collect the first portion of the plurality of oligonucleotide sequences bound to the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon; c) dispersing the first portion of the plurality of oligonucleotide sequences in a buffer; d) performing a magnetic-assisted screening by applying an oscillation magnetic field to the first portion of the plurality of oligonucleotide sequences bound to the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon, so that a portion of the first portion of the plurality of oligonucleotide sequences is detached from the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon; e) performing a second round of magnetic separation by using the magnetic stand to collect a second portion of the first portion of the plurality of oligonucleotide sequences bound to the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon, so that the portion of the first portion of the plurality of oligonucleotide sequences detached from the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon is removed; f) dispersing the second portion of the first portion of the plurality of oligonucleotide sequences bound to the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon in the buffer; g) eluting the second portion of the first portion of the plurality of oligonucleotide sequences from the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon; h) performing a third round of magnetic separation by using the magnetic stand to remove the plurality of magnetic nanoparticles or micro-particles having the target molecules joined thereon; and i) performing a negative selection to the second portion of the first portion of the plurality of oligonucleotide sequences to select aptamers specific to the target molecules, wherein the aptamers specific to the target molecules are not bound to the plurality of magnetic nanoparticles or micro-particles without having the target molecules joined thereon.