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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

Test of selectivity among closely related RNAs. Shown are initial rates of Q-STAR signaling after incubation under ligation/RCA conditions for the times indicated.
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gkr1313-F7: Test of selectivity among closely related RNAs. Shown are initial rates of Q-STAR signaling after incubation under ligation/RCA conditions for the times indicated.

Mentions: Autoligation via nucleophilic displacement of iodide was previously shown to have some sensitivity to single-nucleotide mismatches in the template strand, particularly toward mismatches in the center of the binding arm regions (21). Sequence selectivity is an important goal in miRNA detection, as miRNA sequences within families often vary by one or two nucleotides. The ligation/RCA/Q-STAR method was tested for sensitivity toward let-7a against two closely related miRNA sequences, let-7d and let-7f. Let-7f places a mismatch close to the ligation site, while let-7d has a mismatch several bases from the ligation site and an additional mismatch at the 3′-end of the target (Table 1). The mismatch strands were incubated under the ligation-RCA conditions for 3–24 h, after which Q-STAR probes were added. The results showed no selectivity for let-7a over let-7d, but 5-fold or greater sensitivity against let-7f at incubation times up to 18 h (Figure 7). As an additional test, a random miRNA sequence was added to a let-7a sample in equal or greater concentration. This RNA had no effect on the ultimate Q-STAR signal, indicating no interference from a random sample (data not shown).Figure 7.


Amplified microRNA detection by templated chemistry.

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

Test of selectivity among closely related RNAs. Shown are initial rates of Q-STAR signaling after incubation under ligation/RCA conditions for the times indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1313-F7: Test of selectivity among closely related RNAs. Shown are initial rates of Q-STAR signaling after incubation under ligation/RCA conditions for the times indicated.
Mentions: Autoligation via nucleophilic displacement of iodide was previously shown to have some sensitivity to single-nucleotide mismatches in the template strand, particularly toward mismatches in the center of the binding arm regions (21). Sequence selectivity is an important goal in miRNA detection, as miRNA sequences within families often vary by one or two nucleotides. The ligation/RCA/Q-STAR method was tested for sensitivity toward let-7a against two closely related miRNA sequences, let-7d and let-7f. Let-7f places a mismatch close to the ligation site, while let-7d has a mismatch several bases from the ligation site and an additional mismatch at the 3′-end of the target (Table 1). The mismatch strands were incubated under the ligation-RCA conditions for 3–24 h, after which Q-STAR probes were added. The results showed no selectivity for let-7a over let-7d, but 5-fold or greater sensitivity against let-7f at incubation times up to 18 h (Figure 7). As an additional test, a random miRNA sequence was added to a let-7a sample in equal or greater concentration. This RNA had no effect on the ultimate Q-STAR signal, indicating no interference from a random sample (data not shown).Figure 7.

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