Biophysical characterization of DNA and RNA aptamer interactions with hen egg lysozyme

Potty, Ajish S. R.; Kourentzi, Katerina; Han, Fang; Schuck, Peter; Willson, Richard C.

doi:10.1016/j.ijbiomac.2010.12.007

Cited by 49 publications

(47 citation statements)

References 40 publications

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“…Despite these differences, the net result is the same, a high disturbance of lysozyme interaction with its specific aptamer. The effect of salt as observed by SPRreduced binding of lysozyme by the aptamer with increase in ionic strength -was previously documented by us and others [8,32].…”

Section: Circular Dichroismsupporting

confidence: 55%

Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection

Mihai¹,

Vezeanu²,

Polonschii³

et al. 2015

Sensors and Actuators B: Chemical

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Section: Circular Dichroismsupporting

confidence: 55%

Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection

Mihai¹,

Vezeanu²,

Polonschii³

et al. 2015

Sensors and Actuators B: Chemical

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“…Kanamycin A [30] and B [31], neomycin [32], tetracycline [33], chloramphenicol [34], as well as for various bacterial pathogens, including Salmonella typhimurium [35], Escherichia coli O157:H7 [36] Listeria monocytogenes [37] and Staphylococcus aureus [38]. Lysozyme is the only example of a food allergen that aptamers have been selected for and both RNA [39] and DNA [40] aptamers have been selected, with equilibrium dissociation constants of 2.8±0.3 nM and 0.8±2.0 nM [41], as measured by fluorescence anistropy at 25°C.…”

Section: Introductionmentioning

confidence: 99%

DNA Aptamers against the Lup an 1 Food Allergen

et al. 2012

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Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.

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“…These data suggest that two reactions occur during formation of the Lys1.2minE-lysozyme complex: (1) canonical displacement of cations from the phosphodiester backbone at the protein-RNA interface; and (2) cation uptake by one of the components of the protein-RNA complex. We note that another aptamer whose interactions with lysozyme are characterized elsewhere is unrelated to Lys1 (Potty et al 2011).…”

Section: +mentioning

confidence: 89%

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

Padlan

Malashkevich

Almo

et al. 2014

RNA

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RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å 2 of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na + stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

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Biophysical characterization of DNA and RNA aptamer interactions with hen egg lysozyme

Cited by 49 publications

References 40 publications

Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection

Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection

DNA Aptamers against the Lup an 1 Food Allergen

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

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