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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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TINA modified oligonucleotides increase the analytical sensitivity in buffer of increasing ionic strength.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products in buffer of increasing ionic strength. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 772–789 and 446–463, with unmodified DNA oligonucleotide (▾), ortho-TINA () or para-TINA (⧫) modified oligonucleotides targeting P. aeruginosa base pairs 446–463 as cross-reactivity control. In experiments targeting base pairs 446–463, a conventional DNA helper oligonucleotide (base pair target 464–483) was included. Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and increasing ionic strength (100–1,000 mM) at 52°C. Data are presented as mean raw MFI with 95% confidence intervals. Full data are shown in Figure S1.
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pone-0020565-g003: TINA modified oligonucleotides increase the analytical sensitivity in buffer of increasing ionic strength.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products in buffer of increasing ionic strength. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 772–789 and 446–463, with unmodified DNA oligonucleotide (▾), ortho-TINA () or para-TINA (⧫) modified oligonucleotides targeting P. aeruginosa base pairs 446–463 as cross-reactivity control. In experiments targeting base pairs 446–463, a conventional DNA helper oligonucleotide (base pair target 464–483) was included. Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and increasing ionic strength (100–1,000 mM) at 52°C. Data are presented as mean raw MFI with 95% confidence intervals. Full data are shown in Figure S1.

Mentions: Until now, the effects of TINA molecules have only been evaluated by Tm analyses, which are good model systems, but do not provide information on how TINA-modified oligonucleotides will perform as competitive annealing probes. To address this issue, we used the Luminex® 200™ instrument to analyze the capture of denatured biotinylated E. coli rrs PCR product by magnetic microspheres coated with oligonucleotide sequences targeting base pairs 772–789 from the E. coli rrs gene. Figure 3 and Figure S1 show the capture of biotinylated rrs PCR product (in two-fold dilution series from 2.5 µL to 0.0098 µL rrs PCR product) by unmodified DNA oligonucleotides and oligonucleotides terminally modified with para- or ortho-TINA molecules in buffers of increasing ionic strength (100–1,000 mM monovalent cation). The overall level of median fluorescence intensity (MFI) was generally higher at greater ionic strength. In 150 mM buffer, the ortho-TINA modified oligonucleotide increased the analytical sensitivity 27-fold and the para-TINA modified oligonucleotide increased the analytical sensitivity seven-fold, compared with the unmodified DNA oligonucleotide. In 300 mM buffer, ortho-TINA modified oligonucleotide increased analytical sensitivity eleven-fold and para-TINA modified oligonucleotide six-fold, and even at 1,000 mM, a four-fold increase in analytical sensitivity was observed with both modified oligonucleotides compared with the unmodified equivalent.


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)

TINA modified oligonucleotides increase the analytical sensitivity in buffer of increasing ionic strength.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products in buffer of increasing ionic strength. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 772–789 and 446–463, with unmodified DNA oligonucleotide (▾), ortho-TINA () or para-TINA (⧫) modified oligonucleotides targeting P. aeruginosa base pairs 446–463 as cross-reactivity control. In experiments targeting base pairs 446–463, a conventional DNA helper oligonucleotide (base pair target 464–483) was included. Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and increasing ionic strength (100–1,000 mM) at 52°C. Data are presented as mean raw MFI with 95% confidence intervals. Full data are shown in Figure S1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108614&req=5

pone-0020565-g003: TINA modified oligonucleotides increase the analytical sensitivity in buffer of increasing ionic strength.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products in buffer of increasing ionic strength. E. coli rrs biotinylated PCR product was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 772–789 and 446–463, with unmodified DNA oligonucleotide (▾), ortho-TINA () or para-TINA (⧫) modified oligonucleotides targeting P. aeruginosa base pairs 446–463 as cross-reactivity control. In experiments targeting base pairs 446–463, a conventional DNA helper oligonucleotide (base pair target 464–483) was included. Experiments were performed in phosphate buffer, pH 7.0, with 0.03% Triton X-100 and increasing ionic strength (100–1,000 mM) at 52°C. Data are presented as mean raw MFI with 95% confidence intervals. Full data are shown in Figure S1.
Mentions: Until now, the effects of TINA molecules have only been evaluated by Tm analyses, which are good model systems, but do not provide information on how TINA-modified oligonucleotides will perform as competitive annealing probes. To address this issue, we used the Luminex® 200™ instrument to analyze the capture of denatured biotinylated E. coli rrs PCR product by magnetic microspheres coated with oligonucleotide sequences targeting base pairs 772–789 from the E. coli rrs gene. Figure 3 and Figure S1 show the capture of biotinylated rrs PCR product (in two-fold dilution series from 2.5 µL to 0.0098 µL rrs PCR product) by unmodified DNA oligonucleotides and oligonucleotides terminally modified with para- or ortho-TINA molecules in buffers of increasing ionic strength (100–1,000 mM monovalent cation). The overall level of median fluorescence intensity (MFI) was generally higher at greater ionic strength. In 150 mM buffer, the ortho-TINA modified oligonucleotide increased the analytical sensitivity 27-fold and the para-TINA modified oligonucleotide increased the analytical sensitivity seven-fold, compared with the unmodified DNA oligonucleotide. In 300 mM buffer, ortho-TINA modified oligonucleotide increased analytical sensitivity eleven-fold and para-TINA modified oligonucleotide six-fold, and even at 1,000 mM, a four-fold increase in analytical sensitivity was observed with both modified oligonucleotides compared with the unmodified equivalent.

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