Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

Afonina, Irina; Kutyavin, Igor V.; Lukhtanov, Eugeny A.; Meyer, Rich B.; Gamper, Howard

doi:10.1073/pnas.93.8.3199

Cited by 33 publications

(20 citation statements)

References 28 publications

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“…8 and S12). 29 Minor groove binders conjugated to ONs are well known to increase the strength and specificity of hybridization 45,46. By analogy, major groove binding of the pyrene moiety of monomer Z may explain the observed increased mismatch discrimination of Z2 (Table 3).…”

Section: Resultsmentioning

confidence: 99%

Functionalized 2′-Amino-α-L-LNA: Directed Positioning of Intercalators for DNA Targeting

et al. 2008

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Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (Locked Nucleic Acid) and its diastereomer α-L-LNA are two promising examples hereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small non-aromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases of circular dichroism signals, and molecular modeling studies suggest that pyrene functionalized 2′-amino-α-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety, allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development probes for emerging therapeutic and diagnostic applications focusing on DNA-targeting.

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Section: Resultsmentioning

confidence: 99%

Functionalized 2′-Amino-α-L-LNA: Directed Positioning of Intercalators for DNA Targeting

et al. 2008

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“…These conjugates manifest an enhanced site specificity, which is provided by both oligonucleotide and binder moieties of conjugate [9][10][11][12][13][14]. The use of MGBs with oligonucleotide primers was successful, since it allowed an improved strength and specificity of the primer binding to the target DNA [15].…”

Section: Introductionmentioning

confidence: 99%

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove binders

et al. 2005

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“…When such MGBs are conjugated with oligodeoxynucleotides, the conjugates form very stable hybrids with complementary DNA (Kumar et al 1998). Commercial MGB probes have the MGB at their 3 end, since they are easier to synthesise but the MGB can be placed either at the 5 or 3 ends (Afonina et al 1996). Fluorescence quenching is more efficient, giving increased sensitivity (Kutyavin et al 2000) and the higher melting temperatures (T m ) allows the design of significantly shorter probes that are also more specific, especially if there is a mismatch in the MGB region of the duplex.…”

Section: Minor Groove Binding Probesmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,221

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.

Abstract: A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo [3,2-e]

Cited by 33 publications

References 28 publications

Functionalized 2′-Amino-α-L-LNA: Directed Positioning of Intercalators for DNA Targeting

Functionalized 2′-Amino-α-L-LNA: Directed Positioning of Intercalators for DNA Targeting

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove binders

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

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