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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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Related in: MedlinePlus

DTT in buffer speeds TPP probe oxidation. Without DTT (blue trace), signal is above background (black; no TPP) after 7 h. With 4 mM DTT (red), the signal levels off past 4 h of incubation. 600 nM TPP was incubated in buffer for 0–7 h, after which Q-STAR and template were added. Fluorescence intensity was measured after 1 h. This experiment was performed in duplicate; a representative trial is shown.
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gkr1313-F4: DTT in buffer speeds TPP probe oxidation. Without DTT (blue trace), signal is above background (black; no TPP) after 7 h. With 4 mM DTT (red), the signal levels off past 4 h of incubation. 600 nM TPP was incubated in buffer for 0–7 h, after which Q-STAR and template were added. Fluorescence intensity was measured after 1 h. This experiment was performed in duplicate; a representative trial is shown.

Mentions: We also observed limitations in the stability of the TPP probes under the initial conditions studied. We found that reactions reached a plateau sooner than expected for the given amount of Q-STAR probe. Moreover, we observed that TPP probes were limiting, and addition of further equivalents increased the signal (data not shown). Further investigation led to the identification of DTT in the enzyme buffer supplied by the manufacturer as a significant source of the problem. Fluorescence experiments showed that incubation of the TPP probe alone in a buffer containing 4 mM DTT before adding the probe template and Q-STAR probes caused a significant decrease in fluorescence relative to adding the Q-STAR probe and template initially, as well as relative to incubation in a buffer without DTT (Figure 4). Additionally, HPLC experiments revealed that a significant portion of the TPP probe became oxidized after incubation with the DTT-containing buffer, but the oxidized portion of TPP remained relatively constant in the buffer in which DTT was absent (Supplementary Figure S4). We hypothesized that the oxidized form of DTT (the disulfide) was acting as an oxidant for the phosphine of the TPP probe, a reaction reported previously (30). Agarose gel quantification of rolling circle products showed that the ϕ29 DNA polymerase enzyme performed comparably with and without the presence of DTT in the buffer (data not shown), so further experiments were carried out in a buffer without DTT, ameliorating the problem of insufficient reactivity of TPP probes.Figure 4.


Amplified microRNA detection by templated chemistry.

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

DTT in buffer speeds TPP probe oxidation. Without DTT (blue trace), signal is above background (black; no TPP) after 7 h. With 4 mM DTT (red), the signal levels off past 4 h of incubation. 600 nM TPP was incubated in buffer for 0–7 h, after which Q-STAR and template were added. Fluorescence intensity was measured after 1 h. This experiment was performed in duplicate; a representative trial is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1313-F4: DTT in buffer speeds TPP probe oxidation. Without DTT (blue trace), signal is above background (black; no TPP) after 7 h. With 4 mM DTT (red), the signal levels off past 4 h of incubation. 600 nM TPP was incubated in buffer for 0–7 h, after which Q-STAR and template were added. Fluorescence intensity was measured after 1 h. This experiment was performed in duplicate; a representative trial is shown.
Mentions: We also observed limitations in the stability of the TPP probes under the initial conditions studied. We found that reactions reached a plateau sooner than expected for the given amount of Q-STAR probe. Moreover, we observed that TPP probes were limiting, and addition of further equivalents increased the signal (data not shown). Further investigation led to the identification of DTT in the enzyme buffer supplied by the manufacturer as a significant source of the problem. Fluorescence experiments showed that incubation of the TPP probe alone in a buffer containing 4 mM DTT before adding the probe template and Q-STAR probes caused a significant decrease in fluorescence relative to adding the Q-STAR probe and template initially, as well as relative to incubation in a buffer without DTT (Figure 4). Additionally, HPLC experiments revealed that a significant portion of the TPP probe became oxidized after incubation with the DTT-containing buffer, but the oxidized portion of TPP remained relatively constant in the buffer in which DTT was absent (Supplementary Figure S4). We hypothesized that the oxidized form of DTT (the disulfide) was acting as an oxidant for the phosphine of the TPP probe, a reaction reported previously (30). Agarose gel quantification of rolling circle products showed that the ϕ29 DNA polymerase enzyme performed comparably with and without the presence of DTT in the buffer (data not shown), so further experiments were carried out in a buffer without DTT, ameliorating the problem of insufficient reactivity of TPP probes.Figure 4.

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