Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV‑W Family Using PNA Strand Invasion Into Double‑Stranded DNA
Total Page：16
File Type：pdf, Size：1020Kb
Molecular Biotechnology (2018) 60:124–133 https://doi.org/10.1007/s12033-017-0057-0 ORIGINAL PAPER Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV‑W Family Using PNA Strand Invasion into Double‑Stranded DNA Grzegorz Machnik1 · Estera Skudrzyk1 · Łukasz Bułdak1 · Jarosław Ruczyński2 · Agnieszka Kozłowska2 · Piotr Mucha2 · Piotr Rekowski2 · Witold Szkróbka1 · Marcin Basiak1 · Aleksandra Bołdys1 · Helena Sławska3 · Bogusław Okopień1 Published online: 8 January 2018 © The Author(s) 2018. This article is an open access publication Abstract In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fne balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the diferentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of diferent ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, fnding the linear correlation between template composition and signal intensity of fnal reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR. Keywords Peptide nucleic acid (PNA) · Human endogenous retroviruses · Multiple sclerosis-associated retrovirus (MSRV) · Quantitative methods · Polymerase chain reaction (PCR) · Strand invasion Introduction diverse modifcations of the classical process described for the frst time by Mullis et al. [1]. Modern techniques, both Detection or semi-quantitative estimation of specific qualitative and quantitative, frequently utilize fuorescent sequence type (e.g., a mutated allele) in biological speci- signal derived either from labeled oligonucleotide(s) or from mens is commonly required in genomic approaches. Until dsDNA intercalators such as SYBR Green I. These so-called now, several techniques that meet these criteria have been real-time PCR (rtPCR) methods can be used for virtually well described. These methods, basically employing the all purposes wherever a certain sequence is to be detected principles of polymerase chain reaction (PCR), represent or quantifed [2, 3]. However, some authors discussed the limitations of rtPCR techniques. They argued that their main disadvantage is the inability to detect the mutated variant * Grzegorz Machnik [email protected] if a wild-type allele occurs in a signifcant excess over the sequence of interest [4, 5]. Even the most commonly used 1 Department of Internal Medicine and Clinical Sanger’s sequencing method, which has been perceived as Pharmacology, School of Medicine in Katowice, Medical a gold standard in the mutation detection approach, displays University of Silesia, Medyków 18, 40‑752 Katowice, Poland relatively low sensitivity of detection of a minor target in mixed samples [6]. This fact implicates the possible failure 2 Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80‑308 Gdańsk, Poland of determination of rare mutants during sample processing [7]. Particularly important hazard of false-negative results 3 Department of Gynaecology, Obstetrics and Oncological Gynaecology, Medical University of Silesia, Batorego 15, obtainment can take place in tumor samples, e.g., in screen- 41‑902 Bytom, Poland ing for epidermal growth factor receptor (EGFR) mutations Vol:.(1234567890)1 3 Molecular Biotechnology (2018) 60:124–133 125 in non-small cell lung cancer (NSCLC) patients or in clini- double stranded, non-altered counterpart. Moreover, if the cal virology [8, 9]. It has been estimated that the DNA of process is efective enough, all initially double-stranded interest must comprise at least 25% of the total DNA to be DNA converts into relaxed, single-stranded form. That pro- easily detected by means of Sanger sequencing [6, 10]. To cess is well visible on the polyacrylamide gel by the appear- omit the risk of false-negative results, the addition of oligo- ance of a new band (which indicates the presence of single- nucleotides that clamp the amplifcation of wild-type DNA stranded DNA), while double-stranded DNA becomes no sequences may be utilized. This can increase the sensitivity more visible. After staining, the intensities of both ss- and to the level, when only 1% of mutated DNA is present in a dsDNA bands refect the efectiveness of a strand invasion. total DNA in the sample [11]. Some synthetic oligonucleo- In our work, we have elaborated on the relevancy between tides, such as locked nucleic acid (LNA) or peptide nucleic the strand invasion efciency and initial template DNA acid (PNA) with improved properties compared to the natu- amount in the investigated sample. Moreover, as PNA oli- rally occurring DNA, displayed high discriminatory poten- gomers ofer the capability for the detection of an allele- tial [12]. PNA molecule is also prone to modifcations that specifc DNA template, we used the PNA-directed strand can further improve its DNA-binding abilities [13]. invasion assay for the determination of certain sequences in Peptide nucleic acid (PNA), a fully synthetic oligonucleo- the biological samples. For the experimental purposes, we tide analogue, exhibits higher afnity for complementary have chosen specifc sequences belonging to human endog- DNA, RNA or PNA sequences than natural nucleic acids enous retroviruses (HERVs) as endogenous retroviruses have [14, 15]. On the other hand, if a base-pair mismatch occurs been of special interests for our research group for several in the template DNA at any position, the binding strength years [25–28]. of such DNA/PNA complex decreases dramatically. This feature makes PNA very suitable for single-nucleotide poly- morphism (SNP) analysis [16]. Materials and Methods Another unique feature of PNA, which clearly distin- guishes it from any other synthetic DNA-binding molecules, Sequence Selection for Analysis is its ability to invade the target sites in double-stranded DNA (dsDNA). This phenomenon, termed strand invasion, Sequences belonging to the human endogenous retroviral if directed only by sequence-specifc PNA, is intrinsically HERV-W family were obtained from GenBank database poorly efcient and occurs only under certain conditions, and analyzed using EMBOSS software package [29] These e.g., in the presence of purine- and pyrimidine-rich targets sequences encode for envelope (env) proteins that have been [17]. In order to gain fexibility in clinical conditions, more implicated to play the most signifcant role in a number of universal procedure must be employed to overcome above- medical conditions. Four sequences from diferent loci were mentioned limitation. Takumi Ishizuka and co-workers, in taken into consideration. The comparison of data was car- a number of publications, proposed the addition of a single- ried out using Emma software, a Multiple Sequence Align- stranded DNA-binding protein (SSB) to the PNA-directed ment ClustalW wrapper from EMBOSS package. The most strand invasion assay to improve the reaction efciency appropriate target site for the hybridization of PNA probe [18–20]. Due to PNA-to-DNA-binding characteristics, a was designed within a highly conserved region. strand invasion phenomenon would allow an analysis of Polymerase chain reaction (PCR) was performed using genetic polymorphisms in a variety of assays and studies oligonucleotide primer pair (HERV_F and HERV_R) that [16, 21, 22]. The possible recognition mechanisms and some fanks the PNA target regions giving a PCR product of 270 applications of peptide nucleic acids that target double- base pair (ERVWE1270), Table 1. The length of this dsDNA stranded DNA have been comprehensively elaborated by Li molecule corresponds to the fragment that has been previ- et al. [23]. ously reported to be optimal in PNA-mediated strand inva- In PNA-directed, SSB-assisted strand invasion assay, the sion approaches [19]. efciency of strand displacement relies on several factors including: (1) PNA-to-DNA ratio; (2) SSB amount that is Polymerase Chain Reactions, Mutagenesis used to facilitate the double-strand invasion; (3) The length and Cloning of PNA oligomer; (4) The complementarity between PNA and DNA template to be invaded [19]. The visualization For cloning procedures, human genomic DNA was extracted of a PNA strand invasion phenomenon was achieved by from placental tissue specimens obtained from the Depart- polyacrylamide gel mobility shift assay, as described previ- ment of Gynaecology, Obstetrics and Oncological Gynae- ously [21, 24]. The principle of this assay is based on the cology, Medical University of Silesia, Bytom, Poland. The observation that double-stranded DNA, being relaxed in the ERVWE1 DNA fragment amplifcation, mutagenesis, and presence of complementary PNA, migrates slower than its subsequent cloning of DNA fragments into pSC-B-amp/kan 1 3 126 Molecular Biotechnology (2018) 60:124–133 Table 1 Oligonucleotides used in this work Oligonucleotide/ Oligonucleotide/PNA sequence Product length Remarks