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Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA.

Schneider UV, Géci I, Jøhnk N, Mikkelsen ND, Pedersen EB, Lisby G - PLoS ONE (2011)

Bottom Line: This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators.Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation.At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength.

View Article: PubMed Central - PubMed

Affiliation: QuantiBact Inc, Hvidovre, Denmark. uvs@quantibact.com

ABSTRACT
The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

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Related in: MedlinePlus

Structure of Twisted Intercalating Nucleic Acids showing the para-TINA (X) and ortho-TINA (Y) molecules.
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pone-0020565-g001: Structure of Twisted Intercalating Nucleic Acids showing the para-TINA (X) and ortho-TINA (Y) molecules.

Mentions: To increase the stability of dsDNA, a number of DNA stabilizing molecules have been developed [1]–[13]. DNA stabilizing molecules comprising intercalators, except TINA and AMANY were developed to increase the stability of Watson-Crick based antiparallel duplex formation[8], [10]–[12], [14], [15]. TINA and AMANY molecules were designed to stabilize Hoogsteen based triplex and parallel duplex formation[14], [15]. Surprisingly, a recent thermal stability study of TINA molecule design demonstrated that changing the attachment of the ethynylpyrene functional group from para to ortho (Figure 1) produced significant changes in antiparallel duplex stability [12], [16]. Internal insertion of ortho-TINA ((R)-1-O-[2-(1-pyrenylethynyl)phenylmethyl]glycerol) molecule stabilized the Watson-Crick based antiparallel duplex formation, in contrast to the original para-TINA ((R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) molecule which was only shown to stabilize antiparallel DNA duplex formation when placed at the 5′ terminal [14]. Here, we investigate the optimal placement of para- and ortho-TINA molecules in antiparallel DNA duplex formation, and report the first evaluation of para- and ortho-TINA molecules in antiparallel DNA duplex based hybridization capture assays.


Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA.

Schneider UV, Géci I, Jøhnk N, Mikkelsen ND, Pedersen EB, Lisby G - PLoS ONE (2011)

Structure of Twisted Intercalating Nucleic Acids showing the para-TINA (X) and ortho-TINA (Y) molecules.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108614&req=5

pone-0020565-g001: Structure of Twisted Intercalating Nucleic Acids showing the para-TINA (X) and ortho-TINA (Y) molecules.
Mentions: To increase the stability of dsDNA, a number of DNA stabilizing molecules have been developed [1]–[13]. DNA stabilizing molecules comprising intercalators, except TINA and AMANY were developed to increase the stability of Watson-Crick based antiparallel duplex formation[8], [10]–[12], [14], [15]. TINA and AMANY molecules were designed to stabilize Hoogsteen based triplex and parallel duplex formation[14], [15]. Surprisingly, a recent thermal stability study of TINA molecule design demonstrated that changing the attachment of the ethynylpyrene functional group from para to ortho (Figure 1) produced significant changes in antiparallel duplex stability [12], [16]. Internal insertion of ortho-TINA ((R)-1-O-[2-(1-pyrenylethynyl)phenylmethyl]glycerol) molecule stabilized the Watson-Crick based antiparallel duplex formation, in contrast to the original para-TINA ((R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) molecule which was only shown to stabilize antiparallel DNA duplex formation when placed at the 5′ terminal [14]. Here, we investigate the optimal placement of para- and ortho-TINA molecules in antiparallel DNA duplex formation, and report the first evaluation of para- and ortho-TINA molecules in antiparallel DNA duplex based hybridization capture assays.

Bottom Line: This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators.Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation.At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength.

View Article: PubMed Central - PubMed

Affiliation: QuantiBact Inc, Hvidovre, Denmark. uvs@quantibact.com

ABSTRACT
The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

Show MeSH
Related in: MedlinePlus