Amplified microRNA detection by templated chemistry

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 ϕ29 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.

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

“…We envisioned driving the cyclization by means of a nucleophilic sulfur-halide substitution. This type of chemistry has been successfully used by Letsinger, [28] Kool, [29,30,53] and Abe and Ito [33] to ligate DNA conjugates and prepare DNA macrocycles. We assumed that short cyclization linkers would be required to obtain cyclic PNA with low DNA affinity.…”

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

“…[19][20][21][22][23][24][25][26] The let-7 family of miRNAs in particular has been shown to play significant roles in ovarian, prostate, liver and pancreatic cancer. [27][28][29][30] Since miRNAs are short, polymerase chain reaction (PCR) cannot be carried out on the unmodified target.…”

“…The strategy is versatile, detecting DNA or RNA, and short or long targets can be sensitively detected with judicious design of primer The sensitivity of the ARN/luciferase method compares well to literature methods for isothermal detection of nucleic acids, such as RCA detection of miRNAs. 25,26 Compared to PCR-based approaches to miRNA detection, 19 the current method is simpler, requiring fewer primers and enzymes, fewer steps, and no thermal cycling equipment. One possible limitation of the current approach is that it is difficult to detect single-nucleotide variants in a miRNA target if the polymorphism occurs near the center or 5′ end, since only a complementary 3′ terminus is needed to prime synthesis.…”

“…In this strategy, RCA is carried out, primed by the miRNA on the circular DNA. [21][22][23][24][25][26] This can in principle be extended yet further with hyperbranched RCA. 11 Isothermal detection of miRNAs via rolling circle templates has been reported previously, using templated fluorogenic chemistry 10,31 or DNA-binding fluorescent dyes [22][23][24][25] to report on the products.…”

“…25,26 Compared to PCR-based approaches to miRNA detection, 19 the current method is simpler, requiring fewer primers and enzymes, fewer steps, and no thermal cycling equipment. One possible limitation of the current approach is that it is difficult to detect single-nucleotide variants in a miRNA target if the polymorphism occurs near the center or 5′ end, since only a complementary 3′ terminus is needed to prime synthesis.…”