PhD Scientific Days 2020

Molecular Sciences V. Lectures

Modified oligonucleotide library production for RSV selective aptamer generation

Előadó neve

Percze, Krisztina

Előadó munkahelye

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry

Előadó telefonszáma

+36703255856

Előadó e-mail címe

k.percze@gmail.com

Az előadás címe

Modified oligonucleotide library production for RSV selective aptamer generation

Szerző(k) neve és munkahelye

Krisztina Percze1, Tamás Mészáros1
1 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest

Szekció

Molecular Sciences V. Lectures

Language of the presentation

English

Section, first choice

Molecular Sciences

Section, second choice

Molecular Sciences

Összefoglaló szövege

The respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection during infancy and late elderhood that often leads to hospitalization. The current treatment for RSV is based on a monoclonal antibody vaccination that is accessible for only a small portion of the patients. Therefore, an affordable and safe RSV treatment is desperately needed.
Aptamers are single stranded DNA or RNA oligonucleotides that rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Aptamers are selected from a randomized oligonucleotide library by the utilization of an iterative, in vitro method. So far, only a tiny fraction of selected aptamers made their way into therapeutics due to various limiting factors including their short half-life in vivo. This shortcoming can be salvaged by application of non-natural nucleotides to increase the nuclease resistance of aptamers. Furthermore, modified nucleotides can also expand the physico-chemical properties of aptamers by introducing hydrophobic side chains.
Our long term aim is to provide hydrophobic side chain possessing aptamers with therapeutic potential of RSV infection.
Here, we present a novel method for creating a DNA aptamer library consisting of hydrophobic side chain carrying oligonucleotides. First, we studied the incorporation efficiency of a tryptamino-uracil analogue (TAdUTP) by several DNA polymerases to identify the most suitable one for modified aptamer library production. To determine if TAdUTP was successfully inserted during PCR, NGS analysis was performed. Using the most promising enzyme, the aptamer library was successfully amplified using emulsion PCR to minimize by-product formation. Melting curve analysis was also applied as a proof of incorporation of TAdUTP nucleotides. In further experimental work, this library will be used to generate RSV selective modified aptamers.
Our protocol yielded a generally applicable method for creating an aptamer library bearing tryptophan-like side chains that can further expand the possibilities of aptamer-target interactions and are also expected to increase the aptamers’ in vivo durability.

Additional Information

Tamás Mészáros
meszaros.tamas@med.semmelweis-univ.hu

Bemutatás módja

Szóbeli

Kind

Szabad

Status

elfogadva

Accepted presentation method

szóbeli

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

4788

Start

16:45

End

17:00

Authors (legacy)

Krisztina Percze1, Tamás Mészáros1
1 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest