Polymerase-amplified release of ATP (POLARA) for detecting single nucleotide variants in RNA and DNA

ATP-releasing nucleotides are employed to detect single nucleotide polymorphisms in a novel method that is sensitive, rapid, and isothermal.


Table of Contents
LNA-containing oligonucleotides were purchased from Exiqon/Qiagen. High-performance liquid chromatography (HPLC) was performed using a system comprised of 2 Shimadzu LC-10AD pumps, SCL-10A controller, SPD-M10A photodiode array detector. The luminometer used in this experiment was Thermo Fluoroskan Ascent FL. The thermal cycler used for PCR was Eppendorf Mastercycler Gradient.

Synthesis of ARNs
ARNs were synthesized as previously reported. 1 Briefly, the sodium salt of a deoxynucleoside monophosphate (dAMP, dTMP, dGMP, dCMP) and the sodium salt of ATP were each dissolved in distilled deionized water and converted into pyridinium salts using a Dowex-50W ion exchange column (pyridinium form), stirred with 5 equivalents of tributylamine in a 25% methanol solution, and lyophilized to white powders. The resulting tributylammonium nucleotide salts were coevaporated with anhydrous DMF and stored under high vacuum overnight before the subsequent coupling reaction.
The tributylammonium salt of ATP was dissolved in 0.5 mL anhydrous DMF. 6 equivalents CDI was dissolved in 0.1 mL anhydrous DMF and added to the reaction vessel containing ATP. The mixture was stirred at room temperature for 6 hr, after which 30 equivalents of methanol was added to quench the reaction. The reaction was stirred for 10 minutes after which all solvents were removed under high vacuum overnight. The residue was redissolved in 0.5 mL anhydrous DMF. The 8-oxo-2'-deoxyguanosine monophosphate tributylammonium salt was dissolved in 0.5 mL anhydrous DMF and anhydrous MgCl2 (3-5 mg) was added. ATP-imidazolide was added to the reaction vessel containing the monophosphate, and the reaction was stirred at room temperature for 72 hours. 50 mM triethylammonium bicarbonate (TEAB) buffer was added to quench the reaction.
ARNs were purified from the reaction mixture by HPLC using a gradient of acetonitrile and TEAB. The fractions containing the ARN were pooled and lyophilized to yield a white powder.
In order to convert the ARN triethylammonium salt to a sodium salt, the lyophilized powder was redissolved in 100 µL MeOH. 400 µL 0.75M NaClO4 in acetone was added, and the mixture was centrifuged at 4°C for 2 minutes at max speed. The mixture was decanted and washed with an additional 400 µL acetone and centrifuged at 4°C for 2 minutes at max speed. The tube was then placed on high vacuum for about an hour until completely dry.
Synthesis of 500nt DNA Targets PCR was performed on two BRAF plasmids, 2 one containing a wild-type BRAF insert (a gift from Dustin Maly, Addgene plasmid #40775) and the other containing a V600E-mutated insert (Addgene plasmid #53156). The reverse PCR primer was 5′-biotinylated so that the antisense strand could be pulled down with streptavidin beads, leaving only the sense strand. The doublestranded DNA amplicons were purified using Thermo Fisher Scientific GeneJET PCR Purification Kit. Afterward streptavidin magnetic beads (New England Biolabs) were resuspended in the original container by vortexing for 10 seconds. Beads were washed with binding/washing buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, .5 M NaCl). After removing all buffer, the biotinylated PCR product was added along with binding/washing buffer and the mixture was left on the shaker at room temperature for 20 minutes. All buffer was removed and 150 mM NaOH was added to denature the double-stranded DNA. The mixture was left on the shaker at RT for 20 minutes, after which the buffer containing single-stranded DNA was pipetted into a new tube.

Synthesis of RNA Targets
For the JAK2 and BRAF genes, PCR was performed on plasmids 2-4 containing inserts with the wild-type (JAK2: Addgene plasmid #23915) or mutant (JAK2 V617F: Addgene plasmid #64610) sequence to generate 500mer double-stranded DNA templates. The forward PCR primers included the T7 promoter sequence so that the resulting DNA templates would be suitable for T7 RNA transcription. A mutant BCR-ABL1 plasmid was not available, so the mutation was introduced using a plasmid 5 (Addgene plasmid #27481) with a wild-type BCR-ABL1 insert by including a mismatch in the reverse PCR primer corresponding to the required mutation. Thus, two 484mer double-stranded DNA template were synthesized by PCR corresponding to the wildtype and mutant sequences. Lastly, since the HBB E6V mutation occurs close to the 5′ end of the sequence, two synthetic single-stranded DNAs containing 87 bases of genetic sequence were used as templates for PCR.
In all cases, T7 RNA polymerase was used to generate wild-type and mutant RNA transcripts for each allele. Reactions contained 2.5 µg double-stranded PCR amplicon containing T7 promoter sequence, 0.5 mM NTPs, 5 mM DTT, reaction buffer, T7 RNA polymerase, and nuclease-free water to 50 µL. The reaction was incubated at 37°C for 2 hours. Following transcription, DNase reaction buffer, 2 µL DNase (2 U/µL), and nuclease-free water to 100 µL were added to the same tube. After incubating for an additional hour at 37°C, the resulting RNA transcripts were   for 500mer targets and 50mer targets. Treated with 20 µM ARNs (all four) and Kf exo-for 1 hr at 37°C. Signals for 50mer matches were slightly higher than those of 500mer matches.