Limits...
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.

Show MeSH

Related in: MedlinePlus

Synthesis of ortho-TINA phosphoramidite.i–iii = synthesis of starting compound; iv–v = two-step synthesis of ortho-TINA phosphoramidite [Reagents and conditions: i) KOH, toluene, reflux; ii) 80% aq. CH3COOH, room temperature (RT); iii) DMTCl, pyridine, RT; iv) Et3N, N2, Pd(PPh3)2Cl2, CuI, 1-Ethynylpyrene, RT; v) NC(CH2)2OP(NiPr2)2, diisopropylammonium tetrazolide, CH2Cl2, 0°C to RT.] 1 = S-(+)-2,2-dimethyl-1,3-dioxalane-4-methanol; 2 = 2-iodobenzylbromide; 3 =  (S)-1-O-(4,4′-dimethoxytriphenylmethyloxy)-3-O-(2-iodobenzyloxy)propan-2-ol; 4 = DMT-protected ortho-TINA; 5 = ortho-TINA phosphoramidite.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3108614&req=5

pone-0020565-g002: Synthesis of ortho-TINA phosphoramidite.i–iii = synthesis of starting compound; iv–v = two-step synthesis of ortho-TINA phosphoramidite [Reagents and conditions: i) KOH, toluene, reflux; ii) 80% aq. CH3COOH, room temperature (RT); iii) DMTCl, pyridine, RT; iv) Et3N, N2, Pd(PPh3)2Cl2, CuI, 1-Ethynylpyrene, RT; v) NC(CH2)2OP(NiPr2)2, diisopropylammonium tetrazolide, CH2Cl2, 0°C to RT.] 1 = S-(+)-2,2-dimethyl-1,3-dioxalane-4-methanol; 2 = 2-iodobenzylbromide; 3 =  (S)-1-O-(4,4′-dimethoxytriphenylmethyloxy)-3-O-(2-iodobenzyloxy)propan-2-ol; 4 = DMT-protected ortho-TINA; 5 = ortho-TINA phosphoramidite.

Mentions: Full details of the synthesis procedure are provided in the materials and methods section, and in Supplementary Data S1. In brief (Figure 2), the starting compound (3) was prepared (80% overall yield) in three steps from commercially available compounds S-(+)−2,2-dimethyl-1,3-dioxalane-4-methanol (1) and 2-iodobenzylbromide (2). In the first step of the ortho-TINA phosphoramidite synthesis, 1-ethynylpyrene was coupled to compound 3 using the Sonogashira coupling mixture [14]. To eliminate oxygen, the reaction mixture was degassed with nitrogen prior to the addition of tritylated compound 3; when the reaction mixture was not degassed, the product yield decreased significantly. DMT-protected ortho-TINA (4) was obtained as yellow foam (85% yield), and its structure was confirmed by NMR spectrometry. Finally, the secondary hydroxyl group was phosphatized. Signals in the 31P NMR spectrum with chemical shifts of 148.9 and 149.3 ppm, respectively, confirmed the formation of the phosphoramidite (5).


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)

Synthesis of ortho-TINA phosphoramidite.i–iii = synthesis of starting compound; iv–v = two-step synthesis of ortho-TINA phosphoramidite [Reagents and conditions: i) KOH, toluene, reflux; ii) 80% aq. CH3COOH, room temperature (RT); iii) DMTCl, pyridine, RT; iv) Et3N, N2, Pd(PPh3)2Cl2, CuI, 1-Ethynylpyrene, RT; v) NC(CH2)2OP(NiPr2)2, diisopropylammonium tetrazolide, CH2Cl2, 0°C to RT.] 1 = S-(+)-2,2-dimethyl-1,3-dioxalane-4-methanol; 2 = 2-iodobenzylbromide; 3 =  (S)-1-O-(4,4′-dimethoxytriphenylmethyloxy)-3-O-(2-iodobenzyloxy)propan-2-ol; 4 = DMT-protected ortho-TINA; 5 = ortho-TINA phosphoramidite.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020565-g002: Synthesis of ortho-TINA phosphoramidite.i–iii = synthesis of starting compound; iv–v = two-step synthesis of ortho-TINA phosphoramidite [Reagents and conditions: i) KOH, toluene, reflux; ii) 80% aq. CH3COOH, room temperature (RT); iii) DMTCl, pyridine, RT; iv) Et3N, N2, Pd(PPh3)2Cl2, CuI, 1-Ethynylpyrene, RT; v) NC(CH2)2OP(NiPr2)2, diisopropylammonium tetrazolide, CH2Cl2, 0°C to RT.] 1 = S-(+)-2,2-dimethyl-1,3-dioxalane-4-methanol; 2 = 2-iodobenzylbromide; 3 =  (S)-1-O-(4,4′-dimethoxytriphenylmethyloxy)-3-O-(2-iodobenzyloxy)propan-2-ol; 4 = DMT-protected ortho-TINA; 5 = ortho-TINA phosphoramidite.
Mentions: Full details of the synthesis procedure are provided in the materials and methods section, and in Supplementary Data S1. In brief (Figure 2), the starting compound (3) was prepared (80% overall yield) in three steps from commercially available compounds S-(+)−2,2-dimethyl-1,3-dioxalane-4-methanol (1) and 2-iodobenzylbromide (2). In the first step of the ortho-TINA phosphoramidite synthesis, 1-ethynylpyrene was coupled to compound 3 using the Sonogashira coupling mixture [14]. To eliminate oxygen, the reaction mixture was degassed with nitrogen prior to the addition of tritylated compound 3; when the reaction mixture was not degassed, the product yield decreased significantly. DMT-protected ortho-TINA (4) was obtained as yellow foam (85% yield), and its structure was confirmed by NMR spectrometry. Finally, the secondary hydroxyl group was phosphatized. Signals in the 31P NMR spectrum with chemical shifts of 148.9 and 149.3 ppm, respectively, confirmed the formation of the phosphoramidite (5).

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