Figure 2. Effects of 3′ and 5′ base mismatches on RNA templated DNA end-joining fidelity of PBCV-1 DNA ligase. For each PLP group, the contribution of.

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Figure 2. Effects of 3′ and 5′ base mismatches on RNA templated DNA end-joining fidelity of PBCV-1 DNA ligase. For each PLP group, the contribution of each PLP in the group is presented as a percentage of the average RCPs number for all combined PLPs within a group. The average RCPs number in negative controls was subtracted from all the RCPs counts. From: Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res. 2017;45(18):e161. doi:10.1093/nar/gkx708 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 1. PBCV-1 DNA ligase single nucleotide polymorphism (SNP) fidelity assay. (A) The structures of duplexes with mismatches at 3′ and 5′ sides of the nick. Eight PLPs were used to probe four identical RNA templates where a central base was one of the following: A, U, C or G (N). *: conserved bases; triangle: 3′ OH group; RNA template highlighted in blue. (B) All PLPs were subjected to PBCV-1 DNA ligase ligation and RCA. The generated RCPs were counted digitally and RCP numbers for both matching and mismatching PLPs are shown as a percentage. From: Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res. 2017;45(18):e161. doi:10.1093/nar/gkx708 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 3. iLock activation using the invader principle Figure 3. iLock activation using the invader principle. (A) The structures of iLock RNA binding modes according to traditional and alternative invader configurations. In a traditional configuration, the terminal 3′ iLock base displaces the first base in the flap from the invasive junction, and competes for hybridization to the SNP position. Alternatively, the discriminatory base is localized in the 5′ iLock arm, at a single base distance from the traditional invasive junction. Bold: SNP discriminatory bases; bases participating in an invader structure formation are highlighted with a purple rectangle; triangle: 3′ OH group. (B) The invader structure is formed if a matching iLock hybridizes with a template (iLocks with a green backbone). When Taq activates (spark) matching hybridized iLock in the traditional hybridization configuration, the flap is displaced, 5′P is exposed and a discriminatory base (N’) is positioned in the 3′ iLock terminus. In the alternative invader structure configuration, complementarity between iLock 5′ arm and RNA target allows for an/the (formation of) invader structure formation. After activation, phosphorylated discriminatory base (N’) is localized on the iLock 5′ terminus. When a mismatching iLock is annealed to the target, invader structure formation and iLock activation are compromised. A combination of an imperfect 3′ arm invasion (traditional approach) and a lack of 5′ arm complementarity renders the iLock probe non-functional. Misaligned iLock in the alternative approach might generate a single nucleotide gap or a 5′ mismatch, upon activation. From: Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res. 2017;45(18):e161. doi:10.1093/nar/gkx708 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. Effects of 3′ and 5′ base mismatches on RNA templated DNA end-joining fidelity of PBCV-1 DNA using iLocks. (A) Probe alignment schematics using an RNA template with base A in the central, polymorphic position. Triangle: 3′ OH group; dashed rectangle: bases participating in the invader structure formation. (B) In each iLock group, average number of RCPs was added and ligation fidelity for each iLock was is presented as a percentage within the group. (C) Wild-type KRAS codon 12 (GGT) was genotyped with a complementary and three mismatching (depicted as a lowercase SNP nucleotides in the figure legend) PLPs and iLocks. Average number of RCPs in negative controls was subtracted from all the RCPs counts. From: Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res. 2017;45(18):e161. doi:10.1093/nar/gkx708 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. Analysis of the RNA templated iLock probe activation and ligation by polyacrylamide gelelectrophoresis (PAGE). Match and mismatch 3′/5′ iLocks (lanes 2–10) were activated and ligated on A template (lanes 3–10). 3′/5′ T and C PLPs (lanes 11–14) were ligated on A template as a reference. Inactivated iLocks are 80-nt long (lane 2). Upon activation, flap is released (visible as a faint band <20-nt long, marked with asterisks) and iLock is shortened to 70 nt. Arrows: degradation of RNA templates; -: no Taq control; DNA Ladder (lanes 1 and 15). From: Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res. 2017;45(18):e161. doi:10.1093/nar/gkx708 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 6. miRNA detection fidelity using PLP and iLock RNA detection assay. (A) Hybridization of let-7f PLP (left) and iLock (right) on let-7f miRNA. Uppercase letters: bases in the sequence corresponding to let-7f reference; lowercase: mismatches; *: conserved bases; triangle: 3′ OH group. (B) let-7 miRNA detection fidelity using PLPs (left) and iLock (right) assay. Average number of RCPs in each group was added and each probe signal is presented as a percentage. Average RCPs number in negative controls was subtracted from all the RCPs counts. From: Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy Nucleic Acids Res. 2017;45(18):e161. doi:10.1093/nar/gkx708 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com