Determination of T-cell clonality has an important additional value for diagnosis of T-cell lymphomas. Various molecular methods have been developed, including polymerase chain reaction (PCR) of T-cell receptor γ (TCRγ). The detection of PCR products usually relies commonly on either GeneScan (GS) analysis or heteroduplex (HD) analysis by polyacrylamide gel electrophoresis (PAGE). These techniques have their disadvantages, being relatively time-consuming and laborious or requiring expensive equipment. Here, we propose an alternative method that combines multiplex PCR and HD analysis by microcapillary electrophoresis (ME) on the Agilent 2100 Bioanalyzer. The sensitivity of the method was determined with clonal PEER T-cell line DNA dilution in polyclonal DNA and was evaluated as 1-5%. Fifty-three samples from patients with T-cell lymphoproliferative disorders were analyzed by HD analysis using ME and GS analyses. Comparison of the two techniques showed them to be highly concordant (93% similarity). The rate of clonality detection by GS analysis was higher than HD analysis by ME, but none of the discordant patients (n=5) has yet developed lymphoma. HD analysis by ME to reveal TCRγ gene rearrangements in clinical specimens was consistent with clinical data and the outcome of patients. Detection of T-cell clonality by HD analysis with ME is sensitive, practical, safe and represents a potential alternative to PAGE and GS analysis.

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

DNA/RNA heteroduplex oligonucleotide (HDO), composed of DNA/locked nucleic acid (LNA) antisense oligonucleotide (ASO) and complementary RNA, is a next-generation antisense therapeutic agent. HDO is superior to the parental ASO in delivering to target tissues, and it exerts a more potent gene-silencing effect. In this study, we aimed to elucidate the intracellular trafficking mechanism of HDO-dependent gene silencing. HDO was more preferably transferred to the nucleus after transfection compared to the parental ASO. To determine when and where HDO is separated into the antisense strand (AS) and complementary strand (CS), we performed live-cell time-lapse imaging and fluorescence resonance energy transfer (FRET) assays. These assays demonstrated that HDO had a different intracellular trafficking mechanism than ASO. After endocytosis, HDO was separated in the early endosomes, and both AS and CS were released into the cytosol. AS was more efficiently transported to the nucleus than CS. Separation, endosomal release, and initiation of nuclear transport were a series of time-locked events occurring at a median of 30 s. CS cleavage was associated with efficient nuclear distribution and gene silencing in the nucleus. Understanding the unique intracellular silencing mechanisms of HDO will help us design more efficient drugs and might also provide insight into innate DNA/RNA cellular biology.

Although techniques that permit analysis of the clonal composition of T cell populations have been used extensively to provide a better understanding of the mechanisms that influence efficacy of T cell responses in humans and mice, such methods are lacking for other animal species. In this paper we report the establishment and validation of a panel of Vbeta subfamily-specific semi-nested PCR assays, and a CDR3beta heteroduplex technique for analysing the clonal diversity of bovine alphabeta T cell responses. Development of these methods was based on available sequence data for 48 functional Vbeta genes classified within 17 subfamilies. These techniques were used to determine the clonal composition of parasite-reactive CD8(+) T cells obtained from two animals immunised with the protozoan parasite Theileria parva. Analyses of uncloned T cell lines as well as large panels of cloned T cells derived from each of these lines confirmed the specificity and sensitivity of the assays. Specific PCR products were obtained from 96% of the T cell clones examined, indicating that the currently identified Vbeta genes represent most of the functional Vbeta subfamilies in cattle. Heteroduplex analyses, coupled with sequencing of PCR products, identified over 20 clonal expansions within each of the T cell lines, distributed over a large number of Vbeta subfamilies, although a limited number of clonotypes numerically dominated the response in both animals. The development and validation of these methods provides for the first time a generic set of molecular tools that can be used to perform detailed analysis of the TCR diversity and clonal composition of bovine T cell responses.

We describe a method to identify and recover minor human immunodeficiency virus type 1 (HIV-1) sequence variants from a complex population. The original heteroduplex tracking assay (HTA) was modified by incorporating a biotin tag into the probe to allow for direct sequence determination of the query strand. We used this approach to recover sequences from minor HIV-1 variants in the V3 region of the env gene, and to identify minor drug-resistant variants in pro. The biotin-HTA targeting of the V3 region of env allowed us to detect minor V3 variants, of which 45% were classified as CXCR4-using viruses. In addition, the biotin-protease HTA was able to detect mixtures of wild-type sequence and drug-resistance mutations in four subjects that were not detected by bulk sequence analysis. The biotin-HTA is a robust assay that first separates genetic variants then allows direct sequence analysis of major and minor variants.

The accurate measurement of T-cell receptor (TCR) repertoire changes requires the analysis of a representative sampling of complex T-cell populations. The number and frequency of clonally expanded TCR beta-chain transcripts bearing distinct CDR3 sequences were accurately determined using a simple DNA heteroduplex tracking assay. This method allowed major and minor clonal expansions (> or = 1% of a Vbeta subfamily's transcripts) to be rapidly and reproducibly quantified. Oligoclonal CD8 + cell expansions were detected in all young adults tested, while CD4 + cells generally expressed more polyclonal beta-chain repertoires. The same pattern of CD8 + cells oligoclonality and CD4 + cells polyclonality was observed in asymptomatic HIV-1 infected individuals with high CD4 + cell counts. CD8 + CD45RA + and CD8 + CD45RO + cell fractions both displayed oligoclonal, although distinct, TCR beta chain repertoires while CD8 + cells from umbilical cord blood were generally polyclonal. Oligoclonal CD8 + cell repertoires from young adults were generally stable over a period of weeks, although minor, transient, clonal expansions could also be detected in the absence of symptomatic infections. DNA heteroduplex tracking analysis provided a higher level of sensitivity for the detection of TCR beta chain transcript expansions than CDR3 length (spectrotyping/immunoscope) analysis. DNA heteroduplex tracking of TCR beta-chain transcripts is therefore a simple and sensitive method for assessing the level of clonality and for measuring changes in the TCR beta chain repertoire of different T-cell populations.

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

Antisense oligonucleotide (ASO)-based therapy is one of the next-generation therapy, especially targeting neurological disorders. Many cases of ASO-dependent gene expression suppression have been reported. Recently, we developed a tocopherol conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO) as a new type of drug. Toc-HDO is more potent, stable, and efficiently taken up by the target tissues compared to the parental ASO. However, the detailed mechanisms of Toc-HDO, including its binding proteins, are unknown. Here, we developed native gel shift assays with fluorescence-labeled nucleic acids samples extracted from mice livers. These assays revealed two Toc-HDO binding proteins, annexin A5 (ANXA5) and carbonic anhydrase 8 (CA8). Later, we identified two more proteins, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and flap structure-specific endonuclease 1 (FEN1) by data mining. shRNA knockdown studies demonstrated that all four proteins regulated Toc-HDO activity in Hepa1-6, mouse hepatocellular cells. In vitro binding assays and fluorescence polarization assays with purified recombinant proteins characterized the identified proteins and pull-down assays with cell lysates demonstrated the protein binding to the Toc-HDO and ASO in a biological environment. Taken together, our findings provide a brand new molecular biological insight as well as future directions for HDO-based disease therapy.

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails.

The activin type II receptor (ACVR2) contains two identical microsatellites in exons 3 and 10, but only the exon 10 microsatellite is frameshifted in mismatch repair (MMR)-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exons 3 or 10 of ACVR2 were cloned +1 bp out of frame of enhanced green fluorescent protein (EGFP), allowing -1 bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells, and subsequent non-fluorescent cells were sorted, cultured and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A(7)/T(8)) in hMLH1(-/-) cells, but only exon 10 microsatellites fully mutated (A(7)/T(7)) in both hMLH1(-/-) and hMSH6(-/-) backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1(-/-) cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.

Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.

Genomic imprinting is an epigenetic mechanism that restricts gene expression to one inherited allele. Improper maintenance of imprinting has been implicated in a number of human diseases and developmental syndromes. Assays are needed that can quantify the contribution of each paternal allele to a gene expression profile. We have developed a rapid, sensitive quantitative assay for the measurement of individual allelic ratios termed Pyrosequencing for Imprinted Expression (PIE). Advantages of PIE over other approaches include shorter experimental time, decreased labor, avoiding the need for restriction endonuclease enzymes at polymorphic sites, and prevent heteroduplex formation which is problematic in quantitative PCR-based methods. We demonstrate the improved sensitivity of PIE including the ability to detect differences in allelic expression down to 1%. The assay is capable of measuring genomic heterozygosity as well as imprinting in a single run. PIE is applied to determine the status of Insulin-like Growth Factor-2 (IGF2) imprinting in human and mouse tissues.

High resolution melting is a new method of genotyping and variant scanning that can be seamlessly appended to PCR amplification. Limitations of genotyping by amplicon melting can be addressed by unlabeled probe or snapback primer analysis, all performed without labeled probes. High resolution melting can also be used to scan for rare sequence variants in large genes with multiple exons and is the focus of this article. With the simple addition of a heteroduplex-detecting dye before PCR, high resolution melting is performed without any additions, processing or separation steps. Heterozygous variants are identified by atypical melting curves of a different shape compared to wild-type homozygotes. Homozygous or hemizygous variants are detected by prior mixing with wild-type DNA. Design, optimization, and performance considerations for high resolution scanning assays are presented for rapid turnaround of gene scanning. Design concerns include primer selection and predicting melting profiles in silico. Optimization includes temperature gradient selection of the annealing temperature, random population screening for common variants, and batch preparation of primer plates with robotically deposited and dried primer pairs. Performance includes rapid DNA preparation, PCR, and scanning by high resolution melting that require, in total, only 3h when no variants are present. When variants are detected, they can be identified in an additional 3h by rapid cycle sequencing and capillary electrophoresis. For each step in the protocol, a general overview of principles is provided, followed by an in depth analysis of one example, scanning of CYBB, the gene that is mutated in X-linked chronic granulomatous disease.

Haplo-insufficiency at the paired box gene 6 (PAX6) locus causes aniridia,which is characterized by iris hypoplasia and other anterior and posterior eye defects leading to poor vision. This study aimed to identify novel PAX6 mutations that lead to familial and sporadic aniridia in northeastern China.

microRNAs (miRNAs) associate with Ago proteins to post-transcriptionally silence gene expression by targeting mRNAs. To characterize the modes of miRNA-binding, we developed a novel computational framework, called optiCLIP, which considers the reproducibility of the identified peaks among replicates based on the peak overlap. We identified 98 999 binding sites for mouse and human miRNAs, from eleven Ago2 CLIP-seq datasets. Clustering the binding preferences, we found heterogeneity of the mode of binding for different miRNAs. Finally, we set up a quantitative model, named miRgame, based on an adaptation of the game theory. We have developed a new algorithm to translate the miRgame into a score that corresponds to a miRNA degree of occupancy for each Ago2 peak. The degree of occupancy summarizes the number of miRNA-binding sites and miRNAs targeting each binding site, and binding energy of each miRNA::RNA heteroduplex in each peak. Ago peaks were stratified accordingly to the degree of occupancy. Target repression correlates with higher score of degree of occupancy and number of miRNA-binding sites within each Ago peak. We validated the biological performance of our new method on miR-155-5p. In conclusion, our data demonstrate that miRNA-binding sites within each Ago2 CLIP-seq peak synergistically interplay to enhance target repression.

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.