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Amplified microRNA detection by templated chemistry.

Harcourt EM, Kool ET - Nucleic Acids Res. (2012)

Bottom Line: The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate.When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA.The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

ABSTRACT
MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate. The sequence is amplified through rolling circle amplification with 29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ∼50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ∼15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

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Time course for autoligation. Shown are yields of cyclized product as measured by denaturing gel electrophotresis of a fluorescent-labeled circle precursor, with quantification by gel imager. Ligations were carried out at 30°C with 50 nM pre-circle and RNA splint.
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gkr1313-F2: Time course for autoligation. Shown are yields of cyclized product as measured by denaturing gel electrophotresis of a fluorescent-labeled circle precursor, with quantification by gel imager. Ligations were carried out at 30°C with 50 nM pre-circle and RNA splint.

Mentions: The phosphorothioate-iodide circle ligation was investigated for efficacy at lower concentrations of precircle and template than had previously been reported (21). Products were observed by gel electrophoresis. At 50 nM concentrations, the reaction proceeded over 24 h to a total yield of 45% as measured by relative fluorescence (Figure 2). A ∼100-mer apparent dimer was also present at significant (∼20%) concentrations both in the presence and absence of RNA splint. The dimer concentration could be reduced in the presence of DTT, suggesting that it was due to disulfide formation rather than a substitution reaction (Supplementary Figure S1), but we ultimately chose not to use DTT in our reactions due to incompatibility with the TPP probe (see below). Further experiments showed that the ligation yield was not affected by the presence of ϕ29 DNA polymerase, suggesting that autoligation could be compatible with RCA for a single-tube reaction (Supplementary Figure S2).Figure 2.


Amplified microRNA detection by templated chemistry.

Harcourt EM, Kool ET - Nucleic Acids Res. (2012)

Time course for autoligation. Shown are yields of cyclized product as measured by denaturing gel electrophotresis of a fluorescent-labeled circle precursor, with quantification by gel imager. Ligations were carried out at 30°C with 50 nM pre-circle and RNA splint.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351153&req=5

gkr1313-F2: Time course for autoligation. Shown are yields of cyclized product as measured by denaturing gel electrophotresis of a fluorescent-labeled circle precursor, with quantification by gel imager. Ligations were carried out at 30°C with 50 nM pre-circle and RNA splint.
Mentions: The phosphorothioate-iodide circle ligation was investigated for efficacy at lower concentrations of precircle and template than had previously been reported (21). Products were observed by gel electrophoresis. At 50 nM concentrations, the reaction proceeded over 24 h to a total yield of 45% as measured by relative fluorescence (Figure 2). A ∼100-mer apparent dimer was also present at significant (∼20%) concentrations both in the presence and absence of RNA splint. The dimer concentration could be reduced in the presence of DTT, suggesting that it was due to disulfide formation rather than a substitution reaction (Supplementary Figure S1), but we ultimately chose not to use DTT in our reactions due to incompatibility with the TPP probe (see below). Further experiments showed that the ligation yield was not affected by the presence of ϕ29 DNA polymerase, suggesting that autoligation could be compatible with RCA for a single-tube reaction (Supplementary Figure S2).Figure 2.

Bottom Line: The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate.When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA.The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

ABSTRACT
MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate. The sequence is amplified through rolling circle amplification with 29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ∼50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ∼15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.

Show MeSH
Related in: MedlinePlus