Tandem Oligonucleotide Probe Annealing and Elongation To

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Tandem oligonucleotide probe annealing and elongation to discriminate viral sequence Maria Taskova, Jesper Uhd, Laura Miotke, Matthew Kubit, John Bell, Hanlee P Ji, and Kira Astakhova Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00646 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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Analytical Chemistry

Tandem oligonucleotide probe annealing and elongation to discriminate viral sequence Maria Taskova,1 Jesper Uhd,1 Laura Miotke,2 Matthew Kubit,2 John Bell,3 Hanlee P. Ji2,3 and Kira Astakhova1,* 1

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark. * Correspondence: [email protected]; Tel.: +45-40542400 2 Division of Oncology, Department of Medicine, Stanford University, 269 Campus Drive, Stanford CA 94305 3

Stanford Genome Technology Center, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, USA

Supporting Information Placeholder

tion are in high demand in order to provide controls for existing enzymatic technologies and to create alternatives for emerging applications. In particular, there is an unmet need in rapid, reliable detection of short RNA regions which could open up new opportunities in transcriptome analysis, virology and other fields. Herein, we report for the first time a “click” chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect viral sequence. We hybridized a library of short, terminally labelled probes to Ebola virus RNA followed by click assembly and analysis of the read sequence by various techniques. As we demonstrate in this paper, using our new approach, viral RNA sequence can be detected in less than 2 hours without the need for cDNA synthesis or any other enzymatic reactions and with a sensitivity < 10 pM target RNA.

replicated DNA starting from just a few targeted molecules in order to reach their own threshold of detection. In both techniques, each enzyme used has its own inherent bias and thus the accuracy of the assay can, at best, match the accuracy of the enzymes. It is well established that polymerases introduce point errors during the synthesis of nucleic acid amplicons. This is a crucial obstacle for the detection of single-nucleotide polymorphisms in genomic DNA/RNA by 13 amplification. Additionally, over the past decade, there has been growing concern regarding the adequacy of the data 14 provided by enzymatic amplification. It was shown that 14 some DNA sequences cannot be easily amplified in vitro. Moreover, the amplification affects the sample’s stoichiome15,16 try. Therefore neither the relative nor absolute quantification of a particular sequence can be done accurately by the amplification methods. Overall, any enzymatic process introduces additional bias, complicates the performance of the 12-16 assay and compounds time and cost.

Currently, the detection of viral nucleic acid is performed by three major methods: 1) next generation sequencing (NGS), 2) quantitative polymerase chain reaction (qPCR), 3) amplification-free hybridization assays. NGS methods can then be further broken down into sequencing with fluorescent ter1-4 minators (Illumina), nanopore sequencing, and sequencing 5,6 by hybridization. The major advantage to NGS lies in its design that allows for the discovery of unknown viruses, 7,8 including long nucleic acid regions. However, this is rather expensive and unnecessary when detecting a known viral sequence. Thus, polymerase chain reaction (PCR) and its quantitative variant (qPCR) are often used for the detection of known sequences, especially in clinical settings where cost and ease of operation matter just as much as accuracy.

To address the issues of NGS and qPCR, several amplification-free hybridization assays have been developed for the detection of viral DNA/RNA. This includes Simoa and nano17-19 particle-oligonucleotide probe biosensors. Faster and less expensive than NGS, these assays limit their application to very short reads (20-25 nt), which only includes a small number of target sequences. Thus, there is a gap in existing methods for a technology that can detect longer sequence of viral RNA in small amounts, without the use of enzymes that might limit the accuracy of the results. Herein, we aim to fill that gap by combining the unique properties of locked nucleic acids (LNAs) and short hybridization probes with highly efficient copper-catalyzed click chemistry. As a proof of concept, we analyzed the RNA sequence of the Ebola virus and demonstrated the high specificity and convenience of this novel approach.

ABSTRACT: New approaches for genomic DNA/RNA detec-

Both PCR methods and nearly all NGS methods are burdened 7-11 by a dependence on enzymatic processes. In NGS, enzymatic reverse transcription is used to convert the sample RNA to cDNA in library preparation. Then, there is an enzymatic amplification step prior to sequencing to reach a 12 threshold of signal detection. PCR techniques employ synthetic templates and enzymes to create high amounts of

RESULTS AND DISCUSSION The initial assay design is shown in Figure 1. The sequence of the RNA targets was designed using the publically available viral genome assembly. For the pilot assay, 170mer and

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300mer Ebola RNA were used (Appendix S1, Supporting information).

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and was therefore preferable for the design of mutation specific probes (Supporting Fig. 1B).

We enriched the target using a biotinylated bait hybridization probe and simultaneously annealed equimolar amounts of 12mer DNA probes (sequences are given in Appendix S1, 20 Supporting information). Each of the latter contained 3’21 azido and 5’-hexynyl groups for click chemistry. Interstrand conjugation by click chemistry has been previously demonstrated to be efficient, although in a non-templated 21-23 fashion, i.e. by a protection-deprotection strategy. Herein, we used annealing to target RNA as a key to the correct ligation of sequencing probes by click chemistry (see Figure 1). Afterwards, the target: bait probe complex was bound to streptavidin-coated magnetic beads. After washing off all unbound probes, the beads were re-suspended in a mixture of reagents for the click reaction, degassed and left to react for 1 h, followed by target degradation with RNase 24 and ligation product analysis. Figure 2. MALDI MS and HPLC analysis of short reads in the presence of complementary RNA (a,c) and without the target present (b,d).

Figure 1. Design of the re-sequencing assay. Capture probe is attached to magnetic dynabeads via biotinstreptavidin binding. Series of probes is annealed to captured RNA target followed by click chemistry, detachment and product analysis. Probe order requires sequence specific hybridization among the various k-mers. Optionally, fluorophore can be attached to one or more probes (shown as a star). That allows for the product detection by fluorometry or fluorescence microscopy. The product was detached from dynabeads at 92 °C, desalted and analyzed. We initially performed MALDI MS analysis to confirm the successful formation of the 30mer. The successful formation of products for 3 and 4 probes were additionally confirmed by gel electrophoresis and HPLC (see Figure 2). Our next goal was to demonstrate specificity of the method and to enable the detection of clinically actionable SNPs in the relatively short genomic regions. As shown in Figure 3 and in Appendix S2, we introduced affinity and specificity 25 enhancing LNA to the short probes. Moreover, a series of wild-type and mutant specific 10mers was tested to evaluate the specificity of the assay. Having LNA within hybridization probes allowed us to use warm washing upon their annealing to 300 nt long RNA, wild-type or mutant (see Appendix 20,25 5-7,17-19 S1). Advantageously to the LNA-free assays, the resulting 80mer read was indeed genotype specific which was confirmed by HPLC (see Figure 3c). To further improve probe design, we studied the influence of SNP position within RNA on probe binding. According to our data, SNP in terminal positions had highest impact on Tm

Figure 3. Principle of SNP discrimination used in the assay (a) and chemical structure of LNA, L (b). Dye is shown as a star. LNA allows for eliminating mismatched probes upon washing steps with warm buffer (38-43 °C). c-d) Read analysis by HPLC: assay carried out with mismatched nucleotides (c) and in the presence of fully matched probes (d). Ebola virus has the additional challenge of often being misdiagnosed because of sequence similarities with other virus23 es. Even though NGS has been shown to be an effective tool for Ebola sequence identification, it still fails to verify viral type, especially at low viral loads. We hypothesized that the “click” approach, owing to its enzyme-free paradigm, might increase the specificity of Ebola identification. Especially for short, species-unique reads, there is no need to sequence long parts of the genome, since the specificity of each nucleotide being read is extraordinarily high. To confirm this, we discovered and analyzed specific 50mers of control samples (influenza A) vs. Ebola RNA (Appendix S3, Supporting information). Using “clickable” tandem oligonucleotide ligation products we could easily distinguish between the sequences, with specificity reaching 99% (Table S2, Sup-

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Analytical Chemistry

porting information). Having introduced different fluorophores for terminal probes binding to wild-type (Cy5) and mutant (Cy5.5) Ebola RNA, corresponding genotype could be easily distinguished by the fluorescence read out that detected a fractional mixture where the mutation occurred at 3% (Figure S1, Supporting information). One potential application of the developed method is a confirmation or correction of the data obtained by conventional sequencing. To prove this, we analyzed extended Ebola RNA with a sequence determined during an outbreak in the 26 1970s. In this work, the RNA was prepared by in vitro transcription and confirmed by NGS (Appendix S4, Supporting information). Then, using a library of LNA/DNA probes annealed by click chemistry, we were able to read 1280 nt of Ebola RNA, taking 115-130 nt at each step (see Figure 4).

quantification of the ligated read at the concentration of < 10 pM (see Figure 4c). We compared the genome reference sequence against the click approach (Appendix S4, Supporting information). Nucleotides were identical in 99.1% positions, and 11 nucleotide positions (0.9%) differed. This confirms our hypothesis that this method affects the data on the target sequence, In the present version, the assay needs information on the sequence. In that way it cannot be used as a primary source of completely unknown genomic fragments. However, in the case of Ebola, sequence identification is sometimes needed in time and cost effective way, and using a relatively short se26 quence unique to the virus. A similar approach can be used in oncology and to assess other pathogens, and click chemistry probes combined with bioinformatics may in the future provide an opportunity to decode unknown sequences. Being compared to NGS and nanopore sequencing, the new method has a lower performance regarding sensitivity (Sup26 porting Table S4). However, this is compensated by other advantageous features including that it is very simple, accurate, time and cost effective. In turn, in comparison to already developed hybridization assays such as Simoa and nanoparticle-oligonucleotide sensors, the new method has advantage of high specificity, owing to LNA, and of extended read, while the sensitivity is comparable to other hybridiza26,27 tion based methods (Supporting Table S4).

Figure 4. Re-sequencing of 1280 nt long Ebola RNA by “clickable” probes. Length is 130 nt for 5’-terminal and 115 nt for other 10 reads. The latter is due to 15 nt sequence recognition termini. Full length read is synthesized upon complementarity of each probe to the target (A); when a mismatch is present in the target, a break occurs in the ligation product (B). Quantification of 3’-terminal ligation product by fluorescence (C). Sequences of all reads are given in the Supporting information, Appendix S4. In this work we selected a simple approach where every n+1 read starts with the same sequence as the terminal region of the n-read. Length of these overlapping identification sequences for the ligation products was chosen to be 15 nucleo20 tides which is a unique length for the genome. Resulting reads and their assignment are shown in Table S3, Appendix S4. Notably, the click reactions for each read were run in parallel, and the overall sequencing time was < 2 h. To the best of our knowledge, this sequencing chemistry is more time and cost effective compared to currently used approaches. Finally, a simple attachment of an organic fluorophore to the “top” sequence allowed for the detection and

In conclusion, we applied double-labelled oligonucleotide probes which, after binding the target, were cross-linked by convenient click chemistry. Following this simple strategy, short reads confirmed unique types of Ebola virus, whereas overlapping multiple reads allowed us to extend the sequenced region to 1280 nt. The main advantage of our approach is its enzyme-free principle, resulting in considerable time and cost effectiveness while maintaining extraordinary specificity and sensitivity, as well as target quantification. Altogether, this resulted in target RNA detection without the need for reverse transcription, PCR or any other method relying on enzymes. This work may in several potential ways impact the field of nucleic acid sequencing and diagnostics. First, as demonstrated in the paper, taking NGS data as a starting point, this approach can be used to proofread the obtained sequence. Second, very short reads specific to species, for example Ebola 50mers, can be detected at high specificity and resolu28 tion. Finally, existing diagnostics of oligonucleotides and genomic DNA/RNA can strongly benefit from the time and cost effectiveness of this enzyme-free approach that also allows for target quantification and opens up an opportunity to study SNP dynamics directly in live cells. This includes, but is not limited to, the detection of cancer-related SNPs, chimeric splicing, bacteria and viruses of clinical signifi29-32 cance.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website and includes: Probe design and sequences; RNA targets, protocols and characterization of

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ligation products; performance metrics of novel assay, NGS, qPCR and nanoparticle-probe hybridization methods.

AUTHOR INFORMATION Corresponding Author

[email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT Lumiprobe GmbH is acknowledged for providing oligonucleotide probes and reagents for click chemistry. Prof. Dr. E. Gilboa and Dr. B. Schrand, University of Miami, USA; are acknowledged for providing Ebola RNA targets. MT, JU and KA acknowledge financial support from the Villum Foundation, grant no 73516. This work was also supported by the following grant from the NIH: NHGRI P01HG000205 to JMB and HPJ.

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