Pair DNA Research Articles

Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery.

Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.

A conjugated aptamer and oligonucleotides-stabilized gold nanoclusters nanoplatform for targeted fluorescent imaging and efficient drug delivery

A facile approach is designed for the configuration of aptamer-gold nanoclusters nanohybrid for dual-modal of targeted fluorescent imaging and efficient drug delivery. Morphological and structural investigations indicate that Apt-C30 @AuNCs with intrinsic fluorescence consists in oligonucleotides dopped with AuNCs with an average diameter of 2–3 nm. Based on cell viability assays, Apt-C30 @AuNCs exhibit good biocompatibility. Considering that chemotherapeutic drugs such as DOX can intercalate into the base pairs of DNA, Apt-C30 @AuNCs is performed to delivery DOX. Results clearly demonstrate that Apt-C30 @AuNCs/DOX can be effectively inhibit the growth of cancer cells growth. Moreover, functionalized with aptamer, Apt-C30 @AuNCs owns the ability to bind specifically to MUC1 protein overexpressed on HeLa cells, and the viability of HeLa cells is quite low after treated with Apt-C30 @AuNCs/DOX. In vitro, fluorescent imaging results demonstrate that Apt-C30 @AuNCs present a significantly increase in the internalization and bioimaging ability, attributing to the biofunctionalization of C30 @AuNCs with aptamer. Apt-C30 @AuNCs could be successfully exploited as nanovehicles for efficient drug delivery and fluorescent agents for cellular staining, which can be used in potential application of image-guided cancer diagnosis and therapy fields.

Cryo–electron microscopy structure of the H3-H4 octasome: A nucleosome-like particle without histones H2A and H2B

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo-electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4' four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone-histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

RF19 | PSUN353 Liquid Biopsies - Detecting and Tracking Circulating Cell Free Tumor Derived DNA in Patients with Neuroendocrine Neoplasms

Abstract Introduction There is a clinical need to develop novel and better biomarkers to monitor patients with neuroendocrine neoplasms (NENs). Circulating cell free tumor derived DNA (ctDNA), a form of liquid biopsy, is finding clinical utility in an ever increasing number of malignancies however has not been widely tested in patients with NENs. Aims Our aim was to identify and track plasma ctDNA in a cohort of patients with NENs using a personalized, patient specific approach. Materials and methods A total of 35 serial plasma samples were collected from 9 patients with metastatic, well differentiated NENs (6 small intestinal and 1 lung, 1 ovarian and 1 pelvic; range 2-5 plasma samples per patient) over the space of 2-25 months. For each patient, NEN specific somatic mutations (single nucleotide variants and insertions/deletions) were identified through whole exome sequencing of paired tumor-leucocyte DNA and were used to design a bespoke multi-variant Ampliseq™ HD ctDNA panel (5-20 variants per patient) for targeted next generation sequencing. Imaging and treatment were provided as per usual clinical care. Results ctDNA was detectable in 6/9 patients and in 19/35 plasma samples. A rise in the number of ctDNA target variants and/or variant allele frequency was seen in 4/6 patients who experienced disease progression. Two of these patients received peptide receptor radionuclide therapy after which ctDNA disappeared in one patient and substantially reduced in the other, which correlated with treatment response. The 3 patients who did not have detectable ctDNA at any time point all had grade 1 small intestinal NENs with stable disease during the observation period. Discussion Our data provide exciting evidence for the feasibility of using ctDNA as a biomarker in NENs. By targeting multiple individualized variants using next generation sequencing, we have demonstrated that ctDNA can track changes in disease burden and can monitor response to treatment in patients. Of equal importance, ctDNA was not detectable in patients with quiescent disease. This could help identify patients who do not need intensive monitoring. Targeting bespoke, multiple variants per patient is a novel and powerful approach for NENs, especially given their heterogenous genetic landscape. This study provides important early evidence that ctDNA may be a clinically useful biomarker for detection and surveillance of NENs. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Sunday, June 12, 2022 1:06 p.m. - 1:11 p.m.

Somatic inactivation of breast cancer predisposition genes in tumors associated with pathogenic germline variants.

Breast cancers (BCs) that arise in individuals heterozygous for a germline pathogenic variant in a susceptibility gene, such as BRCA1 and BRCA2, PALB2, and RAD51C, have been shown to exhibit biallelic loss in the respective genes and be associated with triple-negative breast cancer (TNBC) and distinctive somatic mutational signatures. Tumor sequencing thus presents an orthogonal approach to assess the role of candidate genes in BC development. Exome sequencing was performed on paired normal-breast tumor DNA from 124 carriers of germline loss-of-function (LoF) or missense variant carriers in 15 known and candidate BC predisposition genes identified in the BEACCON case-control study. Biallelic inactivation and association with tumor genome features including mutational signatures and homologous recombination deficiency (HRD) score were investigated. BARD1-carrying TNBC (4 of 5) displayed biallelic loss and associated high HRD scores and mutational signature 3, as did a RAD51D-carrying TNBC and ovarian cancer. Biallelic loss was less frequent in BRIP1 BCs (4 of 13) and had low HRD scores. In contrast to other established BC genes, BCs from carriers of CHEK2 LoF (6 of 17) or missense (2 of 20) variant had low rates of biallelic loss. Exploratory analysis of BC from carriers of LoF variants in candidate genes such as BLM, FANCM, PARP2, and RAD50 found little evidence of biallelic inactivation. BARD1 and RAD51D behave as classic BRCA-like predisposition genes with biallelic inactivation, but this was not observed for any of the candidate genes. However, as demonstrated for CHEK2, the absence of biallelic inactivation does not provide definitive evidence against the gene's involvement in BC predisposition.

Self-Assembled Micro-Sized Hexagons Built from Short DNA in a Crowded Environment.

DNA programmable structures of various morphologies have attracted extensive attention due to their potential for materials science and biomedical applications. Here, we report the formation of micro-sized hexagons via assembly of only one pair of short double-stranded DNA in buffer-salt poly(ethylene glycol) solution. Each DNA strand had complementary bases with a two-base overhang. The procedure of heating and subsequent cooling of blunt-ended double-stranded DNA resulted in different assemblies. These results indicated that end-to-end adhesion at the terminals induced by complementary overhangs were required to construct the hexagonal DNA assemblies. The stable formation of the hexagons was highly dependent on heating temperature. In addition, concentration adjustments of DNA and poly(ethylene glycol) were essential. Circular dichroism spectral measurements and polarization microscopy observations indicated parallel alignment of double-stranded DNA in the hexagonal platelet. Self-assembled micro-sized hexagons composed of simple building blocks may have great potential for future biomedical device development.

Cell-Free HPV DNA Kinetics Predicted by Radiographic Response in Locoregional Viral-Associated Oropharyngeal Cancer Receiving Response-Adaptive Treatment – Exploratory Analysis of a Prospective Trial

<h3>Purpose/Objective(s)</h3> Human papillomavirus (HPV) associated oropharyngeal cancer (HPV+OPC) has a favorable prognosis. Current response-adapted strategies utilize RECIST to guide therapy. In the present study, we examined the utility of cell-free HPV DNA (cfHPV DNA) kinetic responses as an additional parameter to track therapy efficacy in de-escalation strategies. Here, we report a comparison of cfHPV DNA kinetics by RECIST and tumor volume (TV) from a prospective response-adaptive de-escalation trial. <h3>Materials/Methods</h3> Patients enrolled on the prospective phase II OPTIMA 2 (NCT03107182) de-escalation trial with available cfHPV DNA information were included for analysis. OPTIMA 2 enrolled locoregionally advanced HPV+OPC including low and high risk patients. High risk patients were defined as T4, N2c-N3 (AJCC 7^th edition), > 20 pack year smoking history, or non-HPV16 subtype. Neck CT or MRI as well as cfHPV DNA was obtained at baseline and following induction chemoimmunotherapy. Target lesions were assessed via RECIST 1.1 at baseline and post-induction therapy per protocol; patients with ≥ 50% response by RECIST received de-escalated radiation dose and volume. cfHPV DNA was detected and quantified using a CLIA-certified cfHPV sequencing assay (HPV-SEQ, Sysmex Inostics, Inc.). For this analysis, target lesions were delineated post hoc to obtain TV metrics. Linear regression was used to examine correlation between cfHPV DNA, RECIST, and TV response. Receiver Operating Characteristic (ROC) analysis was used to identify an optimal TV response to predict ≥ 95% cfHPV DNA response. <h3>Results</h3> 39 patients of which 31 had follow up cfHPV DNA information were evaluable. Median RECIST was 4.9 cm at baseline and 2.0 cm at follow up. Median TV was 29 cm³ at baseline and 6 cm³ at follow-up. 39 (100%) patients had detectable cfHPV DNA at baseline, with median 180 [IQR: 15, 966] copy number/mL. Of 31 patients with paired cfHPV DNA information at baseline, 29 (94%) had a ≥ 95% cfHPV DNA reduction. Of those, cfHPV DNA was undetectable in 21 (71%) cases. Baseline TV was associated with cfHPV DNA (p=0.04), and the median cfHPV DNA / TV ratio was 5.5 (copy number / cm³). No differences were observed between low and high-risk cohorts with respect to cfHPV DNA or radiographic metrics. RECIST was not associated with cfHPV DNA metrics. When adjusting for risk, TV response predicted cfHPV DNA response (p = 0.003). ROC analysis identified a TV response of 63% (AUC 0.88) to predict ≥ 95% cfHPV DNA response; 74% of patients had a TV response ≥ 63%. <h3>Conclusion</h3> cfHPV DNA kinetics were more pronounced than radiographic response to systemic therapy in both high and low risk groups. cfHPV DNA was predicted by TV response to chemoimmunotherapy, but was not predicted by traditional RECIST dynamics. We identified potential TV responses to predict cfHPV DNA response. This represents the first direct correlation of circulating tumor DNA levels to tumor volume and has implications for response assessment in the neoadjuvant setting as well as in other solid tumors.

‘On and Off’ intercalative binding behaviour of double chain surfactant cobalt(III) complex containing 2, 2′-bipyridyl ligand in β-Cyclodextrin: A detail approach on Host-guest inclusion of surfactant cobalt(III) complex and CT-DNA binding in micro-heterogeneous medium

The binding interaction of surfactant cobalt(III) complex, cis-[Co(bpy)2(HA)2](ClO4)3, in which bpy is 2,2-bipyridine and HA is hexadecylamine or cetylamnine with DNA was through intercalative mode via the long aliphatic chains present in the ligands. The binding was investigated by various techniques, electronic absorption, fluorescence spectroscopy, circular dichroism (CD), cyclic voltametry (CV) and viscosimetry measurements. The spectroscopic studies together with cyclic voltammetry and viscosity experiments support that the surfactant cobalt(III) complex binds to calf thymus DNA by intercalation through the aliphatic chain present in the complex into the base pairs of DNA. The presence of bipyridine ligand with larger π-frame work may also enhance intercalation. UV–vis., spectrum showed 4 nm bathochromic shift of the absorption band at 352 nm along with significant hypochromicity for the absorption band of the complex. The intrinsic binding constants(at below and above CMC are Kb = 2.41 × 105M−1, Kb = 3.12 × 106M−1 respectively) is more in keeping with intercalators and suggests this binding mode. The viscosity measurements showed that the surfactant cobalt(III) complex-DNA interaction can be hydrophobic and confirm intercalation. Moreover, the complex induced detectable changes in the CD spectrum of CT-DNA. Competitive binding study with ethidium bromide (EB) shows that the surfactant complex exhibits the ability to displace the DNA-bound EB indicating that the complex binds to DNA in strong competition with EB for the intercalative binding site. Also, CV results confirm this mode because, with increasing the CT-DNA concentration, shift to higher potential was observed. Besides the effect of binding of surfactant cobalt(III) complex to DNA in presence of β-cyclodextrin has also studied. This binding of the surfactant cobalt(III) complex in presence of β-cyclodextrin medium has been prevented (at below and above CMC are Kb = 5.45 × 104M−1, Kb = 6.92 × 105M−1 respectively) due to the incorporation of the aliphatic chains into the cavity of β-cyclodextrin. In presence of β-cyclodextrin the binding occur through surface and (or) groove binding can be attributed to the inclusion of the long aliphatic chain that is present in one of the ligands into cyclodextrin.

Homologous pairing in short double-stranded DNA-grafted colloidal microspheres

Homologous pairing (HP), i.e., the pairing of similar or identical double-stranded DNA, is an insufficiently understood fundamental biological process. HP is now understood to also occur without protein mediation, but crucial mechanistic details remain poorly established. Unfortunately, systematic studies of sequence dependence are not practical due to the enormous number of nucleotide permutations and multiple possible conformations involved in existing biophysical strategies even when using as few as 150 basepairs. Here, we show that HP can occur in DNA as short as 18 basepairs in a colloidal microparticle-based system. Exemplary systematic studies include resolving opposing reports of the impact of % AT composition, validating the impact of nucleotide order and triplet framework and revealing isotropic bendability to be crucial for HP. These studies are enabled by statistical analysis of crystal size and fraction within coexisting fluid-crystal phases of double-stranded DNA-grafted colloidal microspheres, where crystallization is predicated by HP.

A highly divergent Wolbachia with a tiny genome in an insect-parasitic tylenchid nematode.

Wolbachia symbionts are the most successful host-associated microbes on the planet, infecting arthropods and nematodes. Their role in nematodes is particularly enigmatic, with filarial nematode species either 100% infected and dependent on symbionts for reproduction and development, or not at all infected. We have discovered a highly divergent strain of Wolbachia in an insect-parasitic tylenchid nematode, Howardula sp., in a nematode clade that has not previously been known to harbour Wolbachia. While this nematode is 100% infected with Wolbachia, we did not detect it in related species. We sequenced the Howardula symbiont (wHow) genome and found that it is highly reduced, comprising only 550 kilobase pairs of DNA, approximately 35% smaller than the smallest Wolbachia nematode symbiont genomes. The wHow genome is a subset of all other Wolbachia genomes and has not acquired any new genetic information. While it has lost many genes, including genes involved in cell wall synthesis and cell division, it has retained the entire haem biosynthesis pathway, suggesting that haem supplementation is critical. wHow provides key insights into our understanding of what are the lower limits of Wolbachia cells, as well as the role of Wolbachia symbionts in the biology and convergent evolution of diverse parasitic nematodes.

Purine-Purine Base Pairs in Parallel DNA: β-D Anomeric 8-Aza-7-deazaisoguanine and 7-Functionalized Conjugates Form Stable Base Pairs with α-D 5-Aza-7-deaza-2'-deoxyguanosine.

Anomeric purine-purine DNA represents a new recognition system with strands in parallel orientation. This work investigates the new heterochiral system and the positional impact of nucleobase functionalization. Tracts of anomeric isoguanine/8-aza-7-deazaisoguanine base pairs with 5-aza-7-deazaguanine were embedded in anomeric Watson-Crick DNA. It was discovered that stable purine-purine base pairs are formed in anomeric DNA. Nucleobase functionalization of the novel base pair system with short ethynyl and bulky octadiynyl chains showed that the position of functionalization is critical. From Tm values and thermodynamic data, it is disclosed that side chains at 7-position of the β-D 8-aza-7-deaza-2'-deoxyisoguanosine-α-D 5-aza-7-deaza-2'-deoxyguanosine purine-purine pair are well accommodated in this new heterochiral DNA, whereas functionalization at 8-position of isoguanine hinders base pair formation. The new DNA base pair system has the potential to be applied in chemical biology, bioconjugation, and nanobiotechnology.

Sputum cell-free DNA for detection of alterations of multiple driver genes in lung adenocarcinoma.

Sputum cell-free DNA (cfDNA) has been confirmed to be a valued surrogate sample for detection of EGFR mutations in patients with lung adenocarcinoma (LAC). Whether it is suitable for detection of mutations of multiple driver genes has not been reported. A total of 83 patients with LAC were enrolled and their sputum and paired tumor samples were collected. A next-generation sequencing (NGS)-based 10-gene panel was used to test sputum supernatant-derived cfDNA and paired tumor DNA. The sputum sediments were used for cytological evaluation. The total positive rates of hotspot mutations of the 10 driver genes in sputum cfDNA and matched tissue samples were 65.1% and 77.1%, respectively. The overall detection sensitivity of variants in sputum cfDNA was 81.3% (95% confidence interval [CI], 69.2%, 89.5%) and the specificity was 100% (95% CI, 79.1%, 100%). The sensitivities of testing sputum cfDNA from patients with stage IIIB-IV was 87.0% (95% CI, 74.5%, 94.1%); the sensitivities of testing sputum cfDNA from patients with malignant sputum was 92.3% (95% CI, 78.0%, 98.0%); and the sensitivity of testing sputum cfDNA from patients with malignant sputum in stage IIIB-IV were 94.1% (95% CI, 78.9%, 99.0%). This study demonstrated that sputum cfDNA were successfully used for the detection of multiple driver genes by NGS. Sputum cfDNA could be a valuable surrogate clinical sample for all-in-one test of mutations to guide target therapies, especially for patients with advanced LAC and malignant sputum.

Building integrative functional maps of gene regulation.

Every cell in the human body inherits a copy of the same genetic information. The three billion base pairs of DNA in the human genome, and the roughly 50 000 coding and non-coding genes they contain, must thus encode all the complexity of human development and cell and tissue type diversity. Differences in gene regulation, or the modulation of gene expression, enable individual cells to interpret the genome differently to carry out their specific functions. Here we discuss recent and ongoing efforts to build gene regulatory maps, which aim to characterize the regulatory roles of all sequences in a genome. Many researchers and consortia have identified such regulatory elements using functional assays and evolutionary analyses; we discuss the results, strengths and shortcomings of their approaches. We also discuss new techniques the field can leverage and emerging challenges it will face while striving to build gene regulatory maps of ever-increasing resolution and comprehensiveness.

P11.46.A Whole exome sequencing identifies novel SLIT2 mutations in primary CNS lymphoma

Abstract Background Whole exome sequencing (WES) data on primary CNS lymphoma (PCNSL) is scarce and correlation with clinical observations is needed to facilitate personalized care. Here we present WES data of six PCNSL and correlate genetic findings with clinical outcome. Material and Methods WES was performed on paired blood and tumor DNA samples from six PCNSL patients to a median coverage of 100x (range: 73-135x) and 194x (range: 127-232x), respectively. Selected, novel mutations were validated with Sanger sequencing. Epstein-Barr virus (EBV) status was assessed in tumor tissue. Progression free survival (PFS) and overall survival (OS) were analyzed in our cohort and in an extension cohort from the cBio Cancer Genomics Portal using Kaplan-Meier method and log-rank tests. P values &amp;lt; 0.05 were considered significant. Results We found a median of 22 (range: 11-68) somatic copy number variations, 171 (range: 82-298) somatic single-nucleotide variants and 11 (range: 0-22) somatic insertions/deletions per PCNSL. CDKN2A loss, identified in five samples, was the most commonly detected copy number alteration. PCNSL carried mutations in previously described PIM1, MYD88, CD79B or BTG genes. In three PCNSL, we identified novel SLIT2 mutations (p.N63S, p.T590M, p.T732S) clustered around the leucine-rich repeats (LRR) domains. WNT-reporter and NF-kB reporter luciferase assays suggested alterations were loss-of-function variants. SLIT2 mutations were associated with shorter PFS in our cohort and with shorter OS in an extension cohort of 1856 lymphoid malignancies from the cBio Cancer Genomics Portal. Conclusion PCNSL harbored previously unknown SLIT2 mutations clustered around domains that are critical for interaction with ROBO proteins. SLIT2 mutations may be predictive of less favorable outcome in PCNSL. Additional clinical and molecular evaluation of these mutations is warranted.

Mutational and splicing landscape in a cohort of 43,000 patients tested for hereditary cancer

DNA germline genetic testing can identify individuals with cancer susceptibility. However, DNA sequencing alone is limited in its detection and classification of mRNA splicing variants, particularly those located far from coding sequences. Here we address the limitations of splicing variant identification and interpretation by pairing DNA and RNA sequencing and describe the mutational and splicing landscape in a clinical cohort of 43,524 individuals undergoing genetic testing for hereditary cancer predisposition.

Visualizing "Alternative Isoinformational Engineered" DNA in A- and B-Forms at High Resolution.

A fundamental property of DNA built from four informational nucleotide units (GCAT) is its ability to adopt different helical forms within the context of the Watson-Crick pair. Well-characterized examples include A-, B-, and Z-DNA. For this study, we created an isoinformational biomimetic polymer, built (like standard DNA) from four informational "letters", but with the building blocks being artificial. This ALternative Isoinformational ENgineered (ALIEN) DNA was hypothesized to support two nucleobase pairs, the P:Z pair matching 2-amino-imidazo-[1,2a]-1,3,5-triazin-[8H]-4-one with 6-amino-3-5-nitro-1H-pyridin-2-one and the B:S pair matching 6-amino-4-hydroxy-5-1H-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one. We report two structures of ALIEN DNA duplexes at 1.2 Å resolution and a third at 1.65 Å. All of these are built from a single self-complementary sequence (5'-CTSZZPBSBSZPPBAG) that includes 12 consecutive ALIEN nucleotides. We characterized the helical, nucleobase pair, and dinucleotide step parameters of ALIEN DNA in these structures. In addition to showing that ALIEN pairs retain basic Watson-Crick pairing geometry, two of the ALIEN DNA structures are characterized as A-form DNA and one as B-form DNA. We identified parameters that map differences effecting the transition between the two helical forms; these same parameters distinguish helical forms of isoinformational natural DNA. Collectively, our analyses suggest that ALIEN DNA retains essential structural features of natural DNA, not only its information density and Watson-Crick pairing but also its ability to adopt two canonical forms.

Chromatin structure meets cryo-EM: Dynamic building blocks of the functional architecture

Chromatin is a dynamic molecular complex composed of DNA and proteins that package the DNA in the nucleus of eukaryotic cells. The basic structural unit of chromatin is the nucleosome core particle, composed of ~150 base pairs of genomic DNA wrapped around a histone octamer containing two copies each of four histones, H2A, H2B, H3, and H4. Individual nucleosome core particles are connected by short linker DNAs, forming a nucleosome array known as a beads-on-a-string fiber. Higher-order structures of chromatin are closely linked to nuclear events such as replication, transcription, recombination, and repair. Recently, a variety of chromatin structures have been determined by single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), and their structural details have provided clues about the chromatin architecture functions in the cell. In this review, we highlight recent cryo-EM structural studies of a fundamental chromatin unit to clarify the functions of chromatin.

Designing and executing prime editing experiments in mammalian cells.

Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3' ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (&gt;5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2-4 weeks.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

PAX Genes in Cardiovascular Development.

The mammalian heart is a four-chambered organ with systemic and pulmonary circulations to deliver oxygenated blood to the body, and a tightly regulated genetic network exists to shape normal development of the heart and its associated major arteries. A key process during cardiovascular morphogenesis is the septation of the outflow tract which initially forms as a single vessel before separating into the aorta and pulmonary trunk. The outflow tract connects to the aortic arch arteries which are derived from the pharyngeal arch arteries. Congenital heart defects are a major cause of death and morbidity and are frequently associated with a failure to deliver oxygenated blood to the body. The Pax transcription factor family is characterised through their highly conserved paired box and DNA binding domains and are crucial in organogenesis, regulating the development of a wide range of cells, organs and tissues including the cardiovascular system. Studies altering the expression of these genes in murine models, notably Pax3 and Pax9, have found a range of cardiovascular patterning abnormalities such as interruption of the aortic arch and common arterial trunk. This suggests that these Pax genes play a crucial role in the regulatory networks governing cardiovascular development.