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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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2′-O-methyl nucleotides at the 3′-terminus protect probes from degradation. False signal from unprotected Q-STAR probes with ϕ29 DNA polymerase alone (red). Protected probes with two 2′-O-Me nucleotides at the 3′-end (blue); Q-STAR probes without polymerase (black). Experiments were carried out with 1 μM Q-STAR and 0.5 U/μl polymerase at 30°C.
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gkr1313-F3: 2′-O-methyl nucleotides at the 3′-terminus protect probes from degradation. False signal from unprotected Q-STAR probes with ϕ29 DNA polymerase alone (red). Protected probes with two 2′-O-Me nucleotides at the 3′-end (blue); Q-STAR probes without polymerase (black). Experiments were carried out with 1 μM Q-STAR and 0.5 U/μl polymerase at 30°C.

Mentions: Since Q-STAR probes had been used successfully under biological conditions in the literature (25), we did not anticipate problems in the conditions typically employed for RCA. However, initial attempts to monitor the production of rolling circle products as they were generated met with limited success using these fluorogenic probes. Control experiments revealed that unmodified Q-STAR probes yielded a signal in the presence of ϕ29 polymerase regardless of whether a target RNA or a TPP probe was present (Figure 3). Literature reports describe a 3′-exonuclease activity in this polymerase, and so we considered the possibility that full polymerase-mediated degradation of the probes to cut the fluorescein-labeled thymidine from the quencher at the extreme end might yield unwanted background signal. To address this, we incorporated two 2′-O-methyl nucleosides at the 3′-end of each of the probes (22). The modification succeeded in preventing the false signal: protected probes gave very little signal with the polymerase alone (Figure 3).Figure 3.


Amplified microRNA detection by templated chemistry.

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

2′-O-methyl nucleotides at the 3′-terminus protect probes from degradation. False signal from unprotected Q-STAR probes with ϕ29 DNA polymerase alone (red). Protected probes with two 2′-O-Me nucleotides at the 3′-end (blue); Q-STAR probes without polymerase (black). Experiments were carried out with 1 μM Q-STAR and 0.5 U/μl polymerase at 30°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1313-F3: 2′-O-methyl nucleotides at the 3′-terminus protect probes from degradation. False signal from unprotected Q-STAR probes with ϕ29 DNA polymerase alone (red). Protected probes with two 2′-O-Me nucleotides at the 3′-end (blue); Q-STAR probes without polymerase (black). Experiments were carried out with 1 μM Q-STAR and 0.5 U/μl polymerase at 30°C.
Mentions: Since Q-STAR probes had been used successfully under biological conditions in the literature (25), we did not anticipate problems in the conditions typically employed for RCA. However, initial attempts to monitor the production of rolling circle products as they were generated met with limited success using these fluorogenic probes. Control experiments revealed that unmodified Q-STAR probes yielded a signal in the presence of ϕ29 polymerase regardless of whether a target RNA or a TPP probe was present (Figure 3). Literature reports describe a 3′-exonuclease activity in this polymerase, and so we considered the possibility that full polymerase-mediated degradation of the probes to cut the fluorescein-labeled thymidine from the quencher at the extreme end might yield unwanted background signal. To address this, we incorporated two 2′-O-methyl nucleosides at the 3′-end of each of the probes (22). The modification succeeded in preventing the false signal: protected probes gave very little signal with the polymerase alone (Figure 3).Figure 3.

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