RNA aptamer inhibitors of a restriction endonuclease

Estefaniá Mondragón, L James Maher III

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (K<inf>d</inf> ∼12-30 nM) selective competitive inhibitors (IC<inf>50</inf> ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.

Original languageEnglish (US)
Pages (from-to)7544-7555
Number of pages12
JournalNucleic Acids Research
Volume43
Issue number15
DOIs
StatePublished - Jun 29 2015

Fingerprint

Nucleotide Aptamers
DNA Restriction Enzymes
DNA-Binding Proteins
RNA
Deoxyribonuclease BamHI
SELEX Aptamer Technique
Molecular Mimicry
DNA
Bacteriophages
Inhibitory Concentration 50
Transcription Factors
Binding Sites
Infection

ASJC Scopus subject areas

  • Genetics

Cite this

RNA aptamer inhibitors of a restriction endonuclease. / Mondragón, Estefaniá; Maher III, L James.

In: Nucleic Acids Research, Vol. 43, No. 15, 29.06.2015, p. 7544-7555.

Research output: Contribution to journalArticle

Mondragón, Estefaniá ; Maher III, L James. / RNA aptamer inhibitors of a restriction endonuclease. In: Nucleic Acids Research. 2015 ; Vol. 43, No. 15. pp. 7544-7555.
@article{1d93d87f0e5740d49086489b8d6e4a91,
title = "RNA aptamer inhibitors of a restriction endonuclease",
abstract = "Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.",
author = "Estefani{\'a} Mondrag{\'o}n and {Maher III}, {L James}",
year = "2015",
month = "6",
day = "29",
doi = "10.1093/nar/gkv702",
language = "English (US)",
volume = "43",
pages = "7544--7555",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "15",

}

TY - JOUR

T1 - RNA aptamer inhibitors of a restriction endonuclease

AU - Mondragón, Estefaniá

AU - Maher III, L James

PY - 2015/6/29

Y1 - 2015/6/29

N2 - Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.

AB - Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.

UR - http://www.scopus.com/inward/record.url?scp=84941060041&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84941060041&partnerID=8YFLogxK

U2 - 10.1093/nar/gkv702

DO - 10.1093/nar/gkv702

M3 - Article

C2 - 26184872

AN - SCOPUS:84941060041

VL - 43

SP - 7544

EP - 7555

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 15

ER -