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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 over a wide range of annealing temperatures.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products at annealing temperatures from 42°C to 62°C. E. coli rrs biotinylated PCR product (at 0.16 µL and 1.25 µL concentrations) was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 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 all experiments, a conventional DNA helper oligonucleotide targeting E. coli rrs base pairs 464–483 was included. Experiments were performed in phosphate buffer, pH 7.0 with 0.03% Triton X-100, and 150 mM, 300 mM and 1000 mM ionic strengths at temperatures from 42°C to 62°C. Data are presented as mean raw MFI with 95% confidence intervals.
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pone-0020565-g004: TINA modified oligonucleotides increase the analytical sensitivity over a wide range of annealing temperatures.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products at annealing temperatures from 42°C to 62°C. E. coli rrs biotinylated PCR product (at 0.16 µL and 1.25 µL concentrations) was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 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 all experiments, a conventional DNA helper oligonucleotide targeting E. coli rrs base pairs 464–483 was included. Experiments were performed in phosphate buffer, pH 7.0 with 0.03% Triton X-100, and 150 mM, 300 mM and 1000 mM ionic strengths at temperatures from 42°C to 62°C. Data are presented as mean raw MFI with 95% confidence intervals.

Mentions: To investigate whether the modulating effect of TINA molecules was temperature specific, the DNA hybridization assay was repeated at annealing temperatures from 42–62°C at three different ionic strengths and with two different concentrations for the E. coli rrs gene 446–463 base pair target sequence. P. aeruginosa was used as a cross-reactivity control sequence. As shown in Figure 4, the relative MFI of the terminally modified ortho- and para-TINA and unmodified DNA oligonucleotides remained unchanged between 42°C and 52°C (temperature used in the ionic experiments), with the modified oligonucleotides generally providing the highest MFI. Above 52°C the difference in MFI rapidly diminished due to loss of signal. As expected, the level of cross-reactivity with the P. aeruginosa oligonucleotides rose with increasing ionic strength as the annealing temperatures decreased.


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 over a wide range of annealing temperatures.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products at annealing temperatures from 42°C to 62°C. E. coli rrs biotinylated PCR product (at 0.16 µL and 1.25 µL concentrations) was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 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 all experiments, a conventional DNA helper oligonucleotide targeting E. coli rrs base pairs 464–483 was included. Experiments were performed in phosphate buffer, pH 7.0 with 0.03% Triton X-100, and 150 mM, 300 mM and 1000 mM ionic strengths at temperatures from 42°C to 62°C. Data are presented as mean raw MFI with 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108614&req=5

pone-0020565-g004: TINA modified oligonucleotides increase the analytical sensitivity over a wide range of annealing temperatures.Competitive annealing of ortho- or para-TINA terminally modified oligonucleotides, compared with unmodified DNA oligonucleotide, to denatured PCR products at annealing temperatures from 42°C to 62°C. E. coli rrs biotinylated PCR product (at 0.16 µL and 1.25 µL concentrations) was captured by unmodified DNA oligonucleotide (▴) and ortho-TINA (•) or para-TINA (▪) modified oligonucleotides targeting E. coli rrs base pairs 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 all experiments, a conventional DNA helper oligonucleotide targeting E. coli rrs base pairs 464–483 was included. Experiments were performed in phosphate buffer, pH 7.0 with 0.03% Triton X-100, and 150 mM, 300 mM and 1000 mM ionic strengths at temperatures from 42°C to 62°C. Data are presented as mean raw MFI with 95% confidence intervals.
Mentions: To investigate whether the modulating effect of TINA molecules was temperature specific, the DNA hybridization assay was repeated at annealing temperatures from 42–62°C at three different ionic strengths and with two different concentrations for the E. coli rrs gene 446–463 base pair target sequence. P. aeruginosa was used as a cross-reactivity control sequence. As shown in Figure 4, the relative MFI of the terminally modified ortho- and para-TINA and unmodified DNA oligonucleotides remained unchanged between 42°C and 52°C (temperature used in the ionic experiments), with the modified oligonucleotides generally providing the highest MFI. Above 52°C the difference in MFI rapidly diminished due to loss of signal. As expected, the level of cross-reactivity with the P. aeruginosa oligonucleotides rose with increasing ionic strength as the annealing temperatures decreased.

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