Rapid DNA Chemical Ligation for Amplification of RNA and DNA Signal

Abe, Hiroshi; Kondô, Yûkô; Jinmei, Hiroshi; Abe, Naoko; Uchiyama, Atsushi; Tsuneda, Satoshi; Aikawa, Kyoko; Matsumoto, Isamu; Ito, Yoshihiro

doi:10.1021/bc700244s

Cited by 31 publications

(27 citation statements)

References 36 publications

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“…These features support the use of templated reactions in nucleic acid detection in which product formation indicates the presence of the target structure. [2,3] Several chemical reactions have been used in a DNA-or RNA-templated fashion, including organic reactions such as ligation, [4][5][6][7][8][9][10][11] extension, [12,13] cleavage, [14][15][16][17][18] and transfer [19][20][21] reactions as well as organometal approaches. [22][23][24] The work in the field of nucleic acid diagnostics addresses two major issues, selectivity and sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Grossmann

Seitz

2009

Chemistry A European J

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DNA- and RNA-templated chemical reactions can serve as a diagnostic means for the detection of nucleic acids. Reaction schemes that allow amplified detection are of high interest for polymerase chain reaction (PCR)-free DNA and RNA diagnosis. These reactions typically draw upon the catalytic activity of the template, which is able to trigger the conversion of many signaling molecules per template molecule. However, the design of reactive probes that allow both sensitive and selective nucleic acid detection is a challenge and requires insight into three major parameters: a) reactivity of functional groups involved, b) affinity of probes for the template, and c) the readout system. In this study we used peptide nucleic acid (PNA)-based probes to investigate in detail the signaling power and the selectivity of a transfer reaction derived from a native chemical ligation. We show that subtle variations of the thioesters involved had a tremendous impact on the sensitivity and selectivity of the reaction system. The results suggest that reactions at turnover conditions require low rates of non-templated reaction pathways to provide high target selectivity and sensitivity. On the other hand, very high rates of templated reactions should be avoided to allow mismatched probe-template complexes to dissociate prior to bond formation. Furthermore, the temperature dependence of the DNA-catalyzed transfer reaction was studied and provided insight into crucial strand-exchange processes. Further improvements of selective signaling were achieved through a new readout based on pyrene-transfer reactions. This method reduces background signals and enables significant increases in the signaling rates compared with previous fluorescence-based methods.

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Section: Introductionmentioning

confidence: 99%

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Grossmann

Seitz

2009

Chemistry A European J

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“…We envisioned driving the cyclization by means of a nucleophilic sulfur-halide substitution. This type of chemistry has been successfully used by Letsinger, [28] Kool, [29,30,53] and Abe and Ito [33] to ligate DNA conjugates and prepare DNA macrocycles. We assumed that short cyclization linkers would be required to obtain cyclic PNA with low DNA affinity.…”

Section: Resultsmentioning

confidence: 99%

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Roloff¹,

Seitz²

2013

ChemBioChem

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Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA-triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid-programmed ligation reactions. A DNA-triggered ligation-cyclization sequence ("cycligation") of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA-thioesters and isocysteinyl-PNA-cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product-template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur-halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2-3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25-0.01 equiv). We believe that cyclization of products from DNA-templated reactions could ultimately afford systems that completely overcome product inhibition.

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“…A limitation is that the efficiency shifts greatly among different RNA systems, and the optimal efficiency for a single ligation by using DNA ligase or RNA ligase may vary between 80 and 40% [39,45,49], which is the bottleneck for incorporating discrete labels into a RNA by multiple ligations. Ligations can alternatively be achieved by chemical reactions, for example, the linkage between two RNA substrates is achieved by using reactive imidazolide or cyanogen bromide (BrCN) [33,47,[50][51][52][53]. This method has been more widely used for DNAs than RNAs.…”

Section: Splint Ligation Methodsmentioning

confidence: 99%

Specific labeling: An effective tool to explore the RNA world

2015

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Our knowledge about the functional diversity and importance of RNA in biology has grown enormously over the past three decades and has driven efforts to develop better tools to characterize RNAs. Amongst these tools are methods for preparing specifically labeled or chemically modified RNAs, which are essential for basic research, biomedical, and clinical applications. Understanding the potential and limits of these different RNA synthesis and labeling strategies is important in deciding how to approach the preparation of a particular RNA molecule. Here, we review these various labeling methods and future directions of the field.

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Rapid DNA Chemical Ligation for Amplification of RNA and DNA Signal

Cited by 31 publications

References 36 publications

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Specific labeling: An effective tool to explore the RNA world

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