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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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DNA helper oligonucleotides increase assay sensitivity independently of TINA modifications in the capture oligonucleotides.Competitive annealing of unmodified DNA and ortho- or para-TINA terminally modified oligonucleotides to denatured PCR products, with and without an unmodified DNA helper oligonucleotide. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (top), or terminally modified ortho-TINA (middle) or para-TINA (bottom) oligonucleotides targeting E. coli or P. aeruginosa rrs base pairs 446–463, with and without an unmodified DNA helper oligonucleotide (EC464; targeting E. coli rrs base pairs 464–483). Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and 300 mM monovalent cations at 52°C. Data are presented as mean raw MFI with 95% confidence intervals.
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pone-0020565-g005: DNA helper oligonucleotides increase assay sensitivity independently of TINA modifications in the capture oligonucleotides.Competitive annealing of unmodified DNA and ortho- or para-TINA terminally modified oligonucleotides to denatured PCR products, with and without an unmodified DNA helper oligonucleotide. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (top), or terminally modified ortho-TINA (middle) or para-TINA (bottom) oligonucleotides targeting E. coli or P. aeruginosa rrs base pairs 446–463, with and without an unmodified DNA helper oligonucleotide (EC464; targeting E. coli rrs base pairs 464–483). Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and 300 mM monovalent cations at 52°C. Data are presented as mean raw MFI with 95% confidence intervals.

Mentions: As shown in Figure 5, we also examined the individual effect of this DNA helper oligonucleotide on analytical sensitivity when targeting E. coli rrs gene base pairs 446–463. Addition of the helper oligonucleotide increased the analytical sensitivity of the unmodified DNA and ortho- and para-TINA modified oligonucleotides by approximately two-fold. As shown in earlier experiments (Figure 3), targeting base pair 446–463 with TINA/DNA modified oligonucleotides plus the helper nucleotide (to relieve secondary structure), gave similar levels of capture sensitivity to those obtained when targeting base pair 772–789 (no secondary structure).


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)

DNA helper oligonucleotides increase assay sensitivity independently of TINA modifications in the capture oligonucleotides.Competitive annealing of unmodified DNA and ortho- or para-TINA terminally modified oligonucleotides to denatured PCR products, with and without an unmodified DNA helper oligonucleotide. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (top), or terminally modified ortho-TINA (middle) or para-TINA (bottom) oligonucleotides targeting E. coli or P. aeruginosa rrs base pairs 446–463, with and without an unmodified DNA helper oligonucleotide (EC464; targeting E. coli rrs base pairs 464–483). Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and 300 mM monovalent cations at 52°C. Data are presented as mean raw MFI with 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020565-g005: DNA helper oligonucleotides increase assay sensitivity independently of TINA modifications in the capture oligonucleotides.Competitive annealing of unmodified DNA and ortho- or para-TINA terminally modified oligonucleotides to denatured PCR products, with and without an unmodified DNA helper oligonucleotide. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (top), or terminally modified ortho-TINA (middle) or para-TINA (bottom) oligonucleotides targeting E. coli or P. aeruginosa rrs base pairs 446–463, with and without an unmodified DNA helper oligonucleotide (EC464; targeting E. coli rrs base pairs 464–483). Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and 300 mM monovalent cations at 52°C. Data are presented as mean raw MFI with 95% confidence intervals.
Mentions: As shown in Figure 5, we also examined the individual effect of this DNA helper oligonucleotide on analytical sensitivity when targeting E. coli rrs gene base pairs 446–463. Addition of the helper oligonucleotide increased the analytical sensitivity of the unmodified DNA and ortho- and para-TINA modified oligonucleotides by approximately two-fold. As shown in earlier experiments (Figure 3), targeting base pair 446–463 with TINA/DNA modified oligonucleotides plus the helper nucleotide (to relieve secondary structure), gave similar levels of capture sensitivity to those obtained when targeting base pair 772–789 (no secondary structure).

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