Premium
DNA Aptamers Blocking Activity of Anthrax Lethal Toxin
Author(s) -
Ryabko Alena K,
Kozyr Arina V,
Kolesnikov Alexander V,
Marin Maxim A,
Zeninskaya Natalia A,
Lisitskaya Lidia A,
Shemyakin Igor G
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb138
Subject(s) - anthrax toxin , bacillus anthracis , aptamer , toxin , microbiology and biotechnology , biology , exotoxin , recombinant dna , fusion protein , chemistry , virology , biochemistry , bacteria , gene , genetics
Background Anthrax has been recently pinpointed as „deliberately emerging disease”. Apart from natural outbreaks occurring constantly around the world, lethality of anthrax and high resistance of B. anthracis spores to envioronment and disinfectants made it the most feared bioterrorist threat. Anthrax three‐component binary toxin consists of receptor subunit, known as protective antigen (PA), and two effectors, edema factor (EF), and lethal factor (LF). The PA‐LF complex known as lethal toxin (LeTx), causes macrophage death and, ultimately, fatal organ dysfunction. Until recently, no effective remedy existed to protect organism from LeTx action. Therapeutic antibodies (mAbs) neutralizing LeTx have been developed, however, all clinically approved mAbs are specific to PA with activity limited to the extracellular milieu, whereas LeTx already entered the cell is impune to the antibody‐mediated neutralization. Diagnostic and therapeutic potential of DNA aptamers, a new prospective class of affinity molecular tools, is actively explored in different fields, including pathogen detection and neutralization. Objective The purpose of the present study was to develop LeTx‐specific DNA aptamers capable to neutralize anthrax lethal toxin inside the mammalian cell. Results We identified DNA aptamers binding LF by employing new technique tailored to avoid carryover of target‐specific DNA molecules with aptamers binding to the solid phase. For this purpose recombinant LF was produced as fusion protein with His6 affinity tag and cleavable SUMO peptide. Random oligonucleotide library (10 14 ) was subjected to 6 rounds of panning and proteolytic separation of LF with bound aptamers from His6‐ SUMO peptide attached to IMAC magnetic beads. Selected aptamers were analyzed for capability to block proteolysis of internally quenched fluorescent LF substrate. Positively acting aptamers were tested for blocking LeTx intoxication in mice. Co‐administration of some selected aptamer species and LeTx to mice substantially increased their survival compared to the control groups injected with LeTx alone. Conclusion The data obtained outlines DNA aptamers as prospective tools for the development of the new generation of therapeutic agents, combatting anthrax. Support or Funding Information The work was supported by Russian Science Foundation research grant N o 14‐15‐00630.