Glowing locked nucleic acids

Brightly fluorescent probes for detection of nucleic acids in cells

Michael E. Østergaard, Pallavi Cheguru, Madhusudhan R. Papasani, Rodney A. Hill, Patrick J. Hrdlicka

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2′-N-(pyren-1-yl)carbonyl-2′-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (εF = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.
Original languageEnglish
Pages (from-to)14221-14228
Number of pages8
JournalJournal of the American Chemical Society
Volume132
Issue number40
DOIs
Publication statusPublished - 13 Oct 2010

Cite this

Østergaard, Michael E. ; Cheguru, Pallavi ; Papasani, Madhusudhan R. ; Hill, Rodney A. ; Hrdlicka, Patrick J. / Glowing locked nucleic acids : Brightly fluorescent probes for detection of nucleic acids in cells. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 40. pp. 14221-14228.
@article{68e27961df4d47498027b1f96f12302f,
title = "Glowing locked nucleic acids: Brightly fluorescent probes for detection of nucleic acids in cells",
abstract = "Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2′-N-(pyren-1-yl)carbonyl-2′-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (εF = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.",
author = "{\O}stergaard, {Michael E.} and Pallavi Cheguru and Papasani, {Madhusudhan R.} and Hill, {Rodney A.} and Hrdlicka, {Patrick J.}",
year = "2010",
month = "10",
day = "13",
doi = "10.1021/ja1057295",
language = "English",
volume = "132",
pages = "14221--14228",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "40",

}

Glowing locked nucleic acids : Brightly fluorescent probes for detection of nucleic acids in cells. / Østergaard, Michael E.; Cheguru, Pallavi; Papasani, Madhusudhan R.; Hill, Rodney A.; Hrdlicka, Patrick J.

In: Journal of the American Chemical Society, Vol. 132, No. 40, 13.10.2010, p. 14221-14228.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Glowing locked nucleic acids

T2 - Brightly fluorescent probes for detection of nucleic acids in cells

AU - Østergaard, Michael E.

AU - Cheguru, Pallavi

AU - Papasani, Madhusudhan R.

AU - Hill, Rodney A.

AU - Hrdlicka, Patrick J.

PY - 2010/10/13

Y1 - 2010/10/13

N2 - Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2′-N-(pyren-1-yl)carbonyl-2′-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (εF = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

AB - Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2′-N-(pyren-1-yl)carbonyl-2′-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (εF = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

UR - http://www.scopus.com/inward/record.url?scp=77957699357&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77957699357&partnerID=8YFLogxK

U2 - 10.1021/ja1057295

DO - 10.1021/ja1057295

M3 - Article

VL - 132

SP - 14221

EP - 14228

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 40

ER -