ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

Abstract

We report a new strategy to produce luminescence signals from DNA synthesis by designing chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. We describe the synthesis of ATP-releasing nucleotides (ARNs) as derivatives of the four canonical nucleotides. We find that the four are good substrates for DNA polymerase, with K m values averaging 13-fold higher than those of natural dNTPs, and k cat values within 1.5-fold of those of native nucleotides. Importantly, ARNs are found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. Using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs. TOC imageChimeric dinucleotides were designed to release ATP during polymerase synthesis of DNA. This ATP can be used to generate a signal with luciferase, allowing the sensitive isothermal detection of both large (phage DNA) and small (miRNA) nucleic acids.* kool@stanford.edu.Supporting information for this article is given via a link at the end of the document. The use of luciferase signaling can also provide sensitive reporting of DNA synthesis. This enzyme is employed in the "pyrosequencing" methodology developed for high-throughput DNA sequencing. 8 In this technology, four enzymes are employed, two of them to recycle the pyrophosphate product of the DNA polymerase reaction, generating modified ATP. This method is sensitive, but is also relatively complex, and as a result, the method is not used broadly beyond its application in pyrosequencing instruments. HHS Public AccessFurther improvements in methods for general reporting of polymerase synthesis may be useful in amplified detection of native nucleic acids, in reporting on isothermal amplification methods such as rolling circle amplification (RCA), 9-12 and in future-generation approaches to DNA sequencing. To this end, it would be desirable to take advantage of the high sensitivity and specificity of luciferase in detecting DNA synthesis.Here we describe the design and application of ATP-releasing nucleotides (ARNs, Figure 1) as reporters of DNA synthesis. These tetraphosphate-bridged chimeric RNA-DNA dinucleotides are employed sequentially as substrates for DNA polymerases and for luciferase. In this design, DNA polymerase uses the ARNs to copy a target strand, releasing one equivalent of ATP for every deoxynucleotide incorporated. In a subsequent reaction, luciferase processes the ATP products to generate light signals in the presence of luciferin. In principle, the longer the target nucleic acid molecule, the more signals are generated, thus giving the possibility of high sensitivity.Although dimeric polyphosphate-linked nucleotides are known in the literature, 13-15 the ATP-rele...

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“…34 Our lab used preformed circular probes and miRNAs as primers to generate long repeating products by RCA, which were subsequently detected by a second isothermal amplification, 41 using templated fluorogenic probes, or by luciferase signaling with specialized nucleotides. 42 …”

Luminescence method for detection of aflatoxin B1 using ATP-releasing nucleotides

“…In the former paper, ARNs was prepared using ATP as the starting material. 30 However, even aer several separation, tiny amount of ATP still introduces to the nal product. The tiny amount of ATP will increase the background signal and decrease the detection sensitivity.…”

“…To generate illuminescence signal from nucleic acid ampli-cation, one way is using the ATP-releasing nucleotides (ARNs). 30 ARNs are chimeric nucleoside tetraphosphate dimers in which ATP rather than pyrophosphate, is the leaving group during the DNA amplication. The generated ATP can react with luciferase and produce luminescence signal with high sensitivity and specicity.…”

“…However, the former synthesis method using ATP as the starting material can introduce some tiny amount of ATP to the nal product ARNs. 30 The tiny ATP will increase the detection background. To meet this challenge, we plan to prepare ARNs by coupling of adenosine monophosphate (AMP) with deoxyribonucleoside triphosphates (dNTPs).…”