Heteroduplex Research Articles

Anti-CRISPR proteins in Gluconobacter oxydans inactivate FnCas12a by acetylation.

Gluconobacter oxydans is an important chassis cell for one-step production of vitamin C. Previous studies reported that CRISPR/Cas12a is naturally inactivated in G. oxydans, but the specific mechanism remains unclear. Here, we identified anti-CRISPR proteins AcrVA6, AcrVA7 and AcrVA8 in G. oxydans. They functioned as acetyltransferases to inactivate FnCas12a by respectively acetylating Lys671, Lys589 and Lys823 of FnCas12a. Lys671 and Lys823 were related residues that recognise the protospacer-adjacent motif, modification of AcrVA6 and AcrVA8 untangled the interaction between FnCas12a and dsDNA, while Lys589 played an important role in binding to the crRNA-target DNA heteroduplex, AcrVA7 prevented the formation of FnCas12a-crRNA binary complexes. In addition, histone deacetylase HDAC11 was found to prevent modification of FnCas12a by AcrVA6. Quantum mechanical calculations showed that ser37 of AcrVA6, as an intermediate between acetyl group and receptor protein, achieves acetylation through ping-pong transfer mechanism. Finally, the acetyltransferase AcrVA6 and the deacetylase HDAC11 served as photoswitches by writing and erasing acetyl groups, respectively, to achieve continuous on-off of FnCas12a. Our study reveals different mechanisms by which acetyltransferase inactivates Cas12a and successfully applies reversible acetylation to the regulation of gene editing tools, providing new insights into the function and application of acetylation.

Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine learning and accelerated molecular simulations

CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.

Efficient Correction of DNA Hetero–Duplexes Formed During SELEX Procedure

Background: Aptamers, oligos-based (ssDNA or RNA) and peptide-based, are now emerging as the first-choice biomolecules in various applications in medicine and biology. DNA aptamers selection process includes repeatable steps of DNA amplification and digestion. The amplification process of the enriched DNA aptamers includes using PCR. Objective of study: In this article, we sought to describe an efficient and robust method that corrects or permanently removes the PCR by–products during the cycles of DNA aptamers selection. Materials and Methods: Our method is simple, efficient, and requires no enzymatic treatment. In this method, we applied repetitive cycles of thermal treatment to the enriched aptamers final products during the enrichment cycles. The procedure starts with loading the PCR-induced DNA on 1% agarose gel to evaluate the presence of ladder-like DNA or smearing band-like DNA. Next, we cut and purified these by–products and applied thermal treatment. The thermal treatment was by heating the DNA to 65ºC for 10 minutes and then cool them down to 20ºC for 1 minute. Results: PCR artifacts evaluation has been revealed two types of unwanted DNA sequences; the ladder-like and the smearing bands. The next–Generation illume sequencing specified the presence of long and short sequences. Upon the thermal treatment of the PCR products, the next–generation illume sequencing method results showed that the longer sequences and the shorter sequences of the DNA have been reduced considerably and effectively in the DNA pool over the cycles of the systemic evolution of ligands by exponential enrichment (SELEX) procedure. Furthermore, we could reduce the formation of the hetero–duplexes in the next cycles of (SELEX) procedure. Conclusion: This method suggests practical and routine application to prevent or reduce DNA aptamer by–products during the selection process. This treatment could reduce the mis-shaped or irrelevant non-functional aptamers in SELEX procedure

Silver Nanoclusters-Decorated Porous Microneedles Coupling Duplex-Specific Nuclease-Assisted Signal Amplification for Sampling and Detection of MicroRNA in Interstitial Fluid.

MicroRNAs (miRNAs) in dermal interstitial fluid (ISF) have recently been recognized as clinically promising biomarkers for the diagnosis and prognosis of cancer. However, the detection poses significant challenges, primarily due to the low abundance of miRNAs and the limitations of current sampling techniques. To address this issue, we develop novel porous microneedles (PMNs) array-based sensor composed of poly(vinyl alcohol) porous hydrogel and DNA-templated silver nanoclusters (AgNCs) to facilitate the enrichment and highly sensitive detection of ISF miRNA. Leveraging the capillary action facilitated by its unique porous structure and the swelling properties of the hydrogel, the PMNs array can efficiently extract 2.7 ± 0.3 mg of ISF within 5 min. Additionally, the interconnected pores within the PMNs array contribute to an increased specific surface area, thereby offering a convenient platform for the decoration of DNA-templated AgNCs. The immobilized large amount of AgNCs effectively capture the target miRNA from the extracted ISF, resulting in miRNA-induced fluorescence quenching of AgNCs. Subsequently, the introduction of the duplex-specific nuclease leads to the cleavage of DNA in DNA-RNA heteroduplexes, which release miRNA to interact with other AgNCs. This process of target recycling triggers a further reduction in fluorescence intensity, thereby enabling sensitive detection of the low-abundant miRNA down to 1.6 pM. Both in vitro and in vivo experiments validate the efficacy of the AgNCs immobilized PMNs array for the detection of miRNA biomarkers in ISF within minutes. These results indicate that the proposed PMNs array-based sensor holds great potential for the development of noninvasive personalized diagnostic strategies.

Structures of LIG1 uncover the mechanism of sugar discrimination against 5′-RNA-DNA junctions during ribonucleotide excision repair

Ribonucleotides in DNA cause several types of genome instability and can be removed by ribonucleotide excision repair (RER) that is finalized by DNA ligase 1 (LIG1). However, the mechanism by which LIG1 discriminates the RER intermediate containing a 5'-RNA–DNA lesion generated by RNase H2-mediated cleavage of ribonucleotides remains unknown. Here, we determine X-ray structures of LIG1/5'-rG:C at the initial step of ligation where AMP is bound to the active site of the ligase, and uncover a large conformational change downstream the nick resulting in a shift at Arg(R)871 residue in the Adenylation domain of the ligase. Furthermore, we demonstrate a diminished ligation of the nick DNA substrate with a 5'-ribonucleotide in comparison to an efficient end joining of the nick substrate with a 3'-ribonucleotide by LIG1. Finally, our results demonstrate that mutations at the active site residues of the ligase and LIG1 disease-associated variants significantly impact the ligation efficiency of RNA–DNA heteroduplexes harboring “wrong” sugar at 3'- or 5'-end of nick. Collectively, our findings provide a novel atomic insight into proficient sugar discrimination by LIG1 during processing of the most abundant form of DNA damage in cells, genomic ribonucleotides, during initial step of the RER pathway.

Photo-cross-linking reactions in RNA–DNA heteroduplexes using photoreactive DNA incorporated with diazirine and aryl azide warheads

Photo-cross-linking reactions in RNA–DNA heteroduplexes using photoreactive DNA incorporated with diazirine and aryl azide warheads

Structural basis for pegRNA-guided reverse transcription by a prime editor

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9–M-MLV RTΔRNaseH–pegRNA–target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA–synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Biophysical Investigation of RNA ⋅ DNA : DNA Triple Helix and RNA : DNA Heteroduplex Formation by the lncRNAs MEG3 and Fendrr.

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.

Smartphone-based device for rapid and single-step detection of piRNA–651 using anti-DNA:RNA hybrid antibody and enzymatic signal amplification

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs/piRs) are a class of small noncoding RNAs that play a crucial role in regulating various biological processes, including carcinogenesis. One specific piRNA, piR–651, has been reported to be overexpressed in both human blood serum and solid cancer tissues, that can be used a viable biomarker in cancer diagnosis. Early diagnosis of cancer can help reduce the burden of the disease and improve survival rates. In the present work, we report for the first time a smartphone-based colorimetric biosensor for highly sensitive and specific detection of piR–651 thanks to an enzymatic signal amplification, which yielded high colorimetric intensities. Indeed, a heteroduplex DNA:RNA was formed in the presence of piR–651 with the capture DNA probe immobilized on the magnetic beads for easy magnetic separation. Then, a HRP tethered to anti-DNA:RNA (S9.6) was used to reveal the DNA-RNA heteroduplex formed by catalyzing the oxidation of TMB substrate into colorimetric TMBox, which absorbs at 630 nm. The absorbance is positively proportional to the piR–651 concentrations. On the other hand, the colorimetric product of the assay can be photographed with a smartphone camera and analyzed using ImageJ software. Using a smartphone and under optimal conditions, the biosensor responded linearly to the logarithm of piRNA–651 from 8 fM to 100 pM with a detection limit of 2.3 fM and discriminates against other piRNAs. It was also successfully applied to the determination of piRNA–651 levels in spiked human serum.

Structure-specific DNA endonuclease T7 endonuclease I cleaves DNA containing UV-induced DNA lesions.

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication and repair in Escherichia coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analysed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.

Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.

The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.

Heterozygosity alters Msh5 binding to meiotic chromosomes in the baker's yeast.

Meiotic crossovers are initiated from programmed DNA double-strand breaks. The Msh4-Msh5 heterodimer is an evolutionarily conserved mismatch repair-related protein complex that promotes meiotic crossovers by stabilizing strand invasion intermediates and joint molecule structures such as Holliday junctions. In vivo studies using homozygous strains of the baker's yeast Saccharomyces cerevisiae (SK1) show that the Msh4-Msh5 complex associates with double-strand break hotspots, chromosome axes, and centromeres. Many organisms have heterozygous genomes that can affect the stability of strand invasion intermediates through heteroduplex rejection of mismatch-containing sequences. To examine Msh4-Msh5 function in a heterozygous context, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) analysis in a rapidly sporulating hybrid S. cerevisiae strain (S288c-sp/YJM789, containing sporulation-enhancing QTLs from SK1), using SNP information to distinguish reads from homologous chromosomes. Overall, Msh5 localization in this hybrid strain was similar to that determined in the homozygous strain (SK1). However, relative Msh5 levels were reduced in regions of high heterozygosity, suggesting that high mismatch densities reduce levels of recombination intermediates to which Msh4-Msh5 binds. Msh5 peaks were also wider in the hybrid background compared to the homozygous strain (SK1). We determined regions containing heteroduplex DNA by detecting chimeric sequence reads with SNPs from both parents. Msh5-bound double-strand break hotspots overlap with regions that have chimeric DNA, consistent with Msh5 binding to heteroduplex-containing recombination intermediates.

AcrIIC4 inhibits type II-C Cas9 by preventing R-loop formation

Anti-CRISPR (Acr) proteins are encoded by phages and other mobile genetic elements and inhibit host CRISPR-Cas immunity using versatile strategies. AcrIIC4 is a broad-spectrum Acr that inhibits the type II-C CRISPR-Cas9 system in several species by an unknown mechanism. Here, we determined a series of structures of Haemophilus parainfluenzae Cas9 (HpaCas9)-sgRNA in complex with AcrIIC4 and/or target DNA, as well as the crystal structure of AcrIIC4 alone. We found that AcrIIC4 resides in the crevice between the REC1 and REC2 domains of HpaCas9, where its extensive interactions restrict the mobility of the REC2 domain and prevent the unwinding of target double-stranded (ds) DNA at the PAM-distal end. Therefore, the full-length guide RNA:target DNA heteroduplex fails to form in the presence of AcrIIC4, preventing Cas9 nuclease activation. Altogether, our structural and biochemical studies illuminate a unique Acr mechanism that allows DNA binding to the Cas9 effector complex but blocks its cleavage by preventing R-loop formation, a key step supporting DNA cleavage by Cas9.

Mechanistic and evolutionary insights into a type V-M CRISPR-Cas effector enzyme.

RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.

Predicting and Improving Insertions by Prime Editing

Predicting and Improving Insertions by Prime Editing

Cryo-EM structure of the transposon-associated TnpB enzyme

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR–Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA–target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR–Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR–Cas12 effectors.

Hominoid SVA-lncRNA AK057321 targets human-specific SVA retrotransposons in SCN8A and CDK5RAP2 to initiate neuronal maturation

SINE-VNTR-Alu (SVA) retrotransposons arose and expanded in the genome of hominoid primates concurrent with the slowing of brain maturation. We report genes with intronic SVA transposons are enriched for neurodevelopmental disease and transcribed into long non-coding SVA-lncRNAs. Human-specific SVAs in microcephaly CDK5RAP2 and epilepsy SCN8A gene introns repress their expression via transcription factor ZNF91 to delay neuronal maturation. Deleting the SVA in CDK5RAP2 initiates multi-dimensional and in SCN8A selective sodium current neuronal maturation by upregulating these genes. SVA-lncRNA AK057321 forms RNA:DNA heteroduplexes with the genomic SVAs and upregulates these genes to initiate neuronal maturation. SVA-lncRNA AK057321 also promotes species-specific cortex and cerebellum-enriched expression upregulating human genes with intronic SVAs (e.g., HTT, CHAF1B and KCNJ6) but not mouse orthologs. The diversity of neuronal genes with intronic SVAs suggest this hominoid-specific SVA transposon-based gene regulatory mechanism may act at multiple steps to specialize and achieve neoteny of the human brain.

Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.

Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection).

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2-4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.

Serial enrichment of heteroduplex DNA using a MutS-magnetic bead system.

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing.