"Bridging the DNA divide": Understanding the interplay between replication- gaps and homologous recombination proteins RAD51 and BRCA1/2.

A key but often neglected component of genomic instability is the emergence of single-stranded DNA (ssDNA) gaps during DNA replication in the absence of functional homologous recombination (HR) proteins, such as RAD51 and BRCA1/2. Research in prokaryotes has shed light on the dual role of RAD51's bacterial ortholog, RecA, in HR and the protection of replication forks, emphasizing its essential role in preventing the formation of ssDNA gaps, which is vital for cellular viability. This phenomenon was corroborated in eukaryotic cells deficient in HR, where the formation of ssDNA gaps within newly synthesized DNA and their subsequent processing by the MRE11 nuclease were observed. Without functional HR proteins, cells employ alternative ssDNA gap-filling mechanisms to ensure survival, though this compensatory response can compromise genomic stability. A notable example is the involvement of the translesion synthesis (TLS) polymerase POLζ, along with the repair protein POLθ, in the suppression of replicative ssDNA gaps. Persistent ssDNA gaps may result in replication fork collapse, chromosomal anomalies, and cell death, which contribute to cancer progression and resistance to therapy. Elucidating the processes that avert ssDNA gaps and safeguard replication forks is critical for enhancing cancer treatment approaches by exploiting the vulnerabilities of cancer cells in these pathways.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH ∼ 4.7), at a pH ∼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.

Proof of ssDNA degraded from dsDNA for ET recombination.

The widely used ET recombination requires an ssDNA product degraded by Rac phage protein E588 from dsDNA for strand invasion. However, proof of the ssDNA product is still elusive. The study provided three levels of proof sequentially. The probable ssDNAs degraded by E588 from the fluorescent plus-, minus-, or double-stranded dsDNA pET28a-xylanase exhibited a half fluorescence intensity of the corresponding dsDNAs, equivalent to the E588 degradation nucleotides half that of the total nucleotides degraded from the corresponding dsDNA. The ssDNA product degraded by E588 from the fluorescent minus-stranded dsDNA was confirmed by gradient gel-electrophoresis and two nuclease degradation reactions. Degraded by E588 from the dsDNA pET28a-xylanase that had a phosphorothioated plus-stranded 5'-terminus, the plus-stranded ssDNA product was separated via gel electrophoresis and recovered via a DNAclean kit. The recovered ssDNA product was proven to have intact 5'- and 3'-ends by DNA sequencing analysis. This study provides a solid foundation for the mechanism of ssDNA invasion.

How to sensitize glioblastomas to temozolomide chemotherapy: a gap-centered view.

Temozolomide (TMZ) is a methylating agent used as the first-line drug in the chemotherapy of glioblastomas. However, cancer cells eventually acquire resistance, necessitating the development of TMZ-potentiating therapy agents. TMZ induces several DNA base adducts, including O 6 -meG, 3-meA, and 7-meG. TMZ cytotoxicity stems from the ability of these adducts to directly (3-meA) or indirectly (O 6 -meG) impair DNA replication. Although TMZ toxicity is generally attributed to O 6 -meG, other alkylated bases can be similarly important depending on the status of various DNA repair pathways of the treated cells. In this mini-review we emphasize the necessity to distinguish TMZ-sensitive glioblastomas, which do not express methylguanine-DNA methyltransferase (MGMT) and are killed by the futile cycle of mismatch repair (MMR) of the O 6 -meG/T pairs, vs. TMZ-resistant MGMT-positive or MMR-negative glioblastomas, which are selected in the course of the treatment and are killed only at higher TMZ doses by the replication-blocking 3-meA. These two types of cells can be TMZ-sensitized by inhibiting different DNA repair pathways. However, in both cases, the toxic intermediates appear to be ssDNA gaps, a vulnerability also seen in BRCA-deficient cancers. PARP inhibitors (PARPi), which were initially developed to treat BRCA1/2-deficient cancers by synthetic lethality, were re-purposed in clinical trials to potentiate the effects of TMZ. We discuss how the recent advances in our understanding of the genetic determinants of TMZ toxicity might lead to new approaches for the treatment of glioblastomas by inhibiting PARP1 and other enzymes involved in the repair of alkylation damage (e.g., APE1).

DNA Meets Protein─Development, Characterization, and Application of Aptamers against Peanut Allergen.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Genome-wide characterization of single-stranded DNA in rice.

Single-stranded DNA (ssDNA) is essential for various DNA-templated processes in both eukaryotes and prokaryotes. However, comprehensive characterizations of ssDNA still lag in plants compared to non-plant systems. Here, we conducted in situ S1-seq (ISS1-seq), with starting gDNA ranging from 5 µg to 250 ng, followed by comprehensive characterizations of ssDNA in rice (Oryza sativa L.). We found that ssDNA loci were substantially associated with a subset of non-B DNA structures and functional genomic loci. Subtypes of ssDNA loci had distinct epigenetic features. Importantly, ssDNA may act alone or partly coordinate with non-B DNA structures, functional genomic loci, or epigenetic marks to actively or repressively modulate gene transcription, which is genomic-region-dependent and associated with the distinct accumulation of RNA Pol II. Moreover, distinct types of ssDNA had differential impacts on the activities and evolution of TEs (especially common or conserved TEs) in the rice genome. Our study showcases an antibody-independent technique for characterizing non-B DNA structures or functional genomic loci in plants. It lays the groundwork and fills a crucial gap for further exploration of ssDNA, non-B DNA structures, or functional genomic loci, thereby advancing our understanding of their biology in plants.

Systematic Selection of High-Affinity ssDNA Sequences to Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) have gained significant interest for their potential in biomedicine and nanoelectronics. The functionalization of SWCNTs with single-stranded DNA (ssDNA) enables the precise control of SWCNT alignment and the development of optical and electronic biosensors. This study addresses the current gaps in the field by employing high-throughput systematic selection, enriching high-affinity ssDNA sequences from a vast random library. Specific base compositions and patterns are identified that govern the binding affinity between ssDNA and SWCNTs. Molecular dynamics simulations validate the stability of ssDNA conformations on SWCNTs and reveal the pivotal role of hydrogen bonds in this interaction. Additionally, it is demonstrated that machine learning could accurately distinguish high-affinity ssDNA sequences, providing an accessible model on a dedicated webpage (http://service.k-medai.com/ssdna4cnt). These findings open new avenues for high-affinity ssDNA-SWCNT constructs for stable and sensitive molecular detection across diverse scientific disciplines.

A sensitive analytical strategy of oligonucleotide functionalized fluorescent probes for detection of nusinersen sodium in human serum.

Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disease. Nusinersen sodium (NS) is the world's first antisense oligonucleotide (ASO) drug for SMA precise targeted therapy. However, the limited half-life of oligonucleotides and their tendency to accumulate in hepatic and renal tissues presented significant challenges for clinical investigation and therapeutic drug monitoring. In this study, we proposed an analytical strategy based on the specific capture of oligonucleotide functionalized fluorescent probes by single stranded binding proteins (SSB) for ultra-sensitive and high-throughput detection of nusinersen sodium in human serum. The magnetic nanoparticles modified with single-strand binding protein (MNPs-SSB) selectively bonded to the red fluorescent quantum dots functionalized with oligonucleotides (RQDs-ssDNA) that were complementary to nusinersen sodium. Upon interaction with nusinersen sodium, RQDs-ssDNA formed a double-stranded complex (RQDs-ssDNA-NS), resulting in enhanced red fluorescence after magnetic separation as it was no longer captured by MNPs-SSB but remained in the supernatant. A quantitative analysis of nusinersen sodium in biological samples was successfully achieved by establishing a relationship between fluorescence intensity and its concentration. The detection signal F/F0 exhibited a linear correlation (R2 = 0.9871) over a wide range from 0.1 nM to 200 nM, with a limit of detection (LOD) of 0.03 nM, demonstrating the high specificity and rapid analysis time (only 30 min). This method provided a novel approach for sensitive, high-throughput, and specific analysis of nusinersen sodium and similar ASO drugs.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA.

BACKGROUND: CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. RESULTS: Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. SIGNIFICANCE: The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

Cost-effective evaluation of Aptamer candidates in SELEX-based Aptamer isolation.

Aptamers are short, single-stranded nucleic acids with high affinity and specificity for various targets, making them valuable in diagnostics and therapeutics. Their isolation traditionally involves a time-consuming and costly process called SELEX. While SELEX methods have evolved to improve binding and amplification, the crucial step of aptamer identification from sequencing data remains expensive and often overlooked. Common identification methods require modification of aptamer candidates with labels like biotin or fluorescent dyes, which becomes costly and cumbersome for high-throughput sequencing data. This paper presents an efficient and cost-effective approach to streamline aptamer identification. It employs asymmetric polymerase chain reaction (PCR) to generate modified single-stranded DNA copies of aptamer candidates, simplifying the modification process. By using excess modified forward primers and limited reverse primers, this method reduces costs since only unmodified candidates need to be synthesized initially. The approach was demonstrated with an IgE protein aptamer and successfully applied to identify aptamers from a pool of 12 candidates against a monoclonal antibody. The validity of the results was further confirmed through the direct synthesis of fluorophore-conjugated aptamer candidates, yielding consistent outcomes while reducing the cost by threefold. This approach addresses a critical bottleneck in aptamer discovery by significantly reducing the time and cost associated with aptamer identification, facilitating aptamer-based research and making aptamers more accessible for various applications in diagnostics and therapeutics.

Unprocessed genomic uracil as a source of DNA replication stress in cancer cells.

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010  GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA).

Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe.

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.

In vivo detection of DNA secondary structures using permanganate/S1 footprinting with direct adapter ligation and sequencing (PDAL-Seq).

DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called 'non-B DNA'), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in many non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.

Improving synthesis and binding affinities of nucleic acid aptamers and their therapeutics and diagnostic applications.

Nucleic acid aptamers have captivated the attention of analytical and medicinal scientists globally due to their several advantages as recognition molecules over conventional antibodies because of their small size, simple and inexpensive synthesis, broad target range, and high stability in varied environmental conditions. These recognition molecules can be chemically modified to make them resistant to nuclease action in blood serum, reduce rapid renel clearance, improve the target affinity and selectivity, and make them amenable to chemically conjugate with a support system that facilitates their selective applications. This review focuses on the development of efficient aptamer candidates and their application in clinical diagnosis and therapeutic applications. Significant advances have been made in aptamer-based diagnosis of infectious and non-infectious diseases. Collaterally, the progress made in therapeutic applications of aptamers is encouraging, as evident from their use in diagnosing cancer, neurodegenerative diseases, microbial infection, and in imaging. This review also updates the progress on clinical trials of many aptamer-based products of commercial interests. The key development and critical issues on the subject have been summarized in the concluding remarks.

Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

The Intriguing Mystery of RPA Phosphorylation in DNA Double-Strand Break Repair.

Human Replication Protein A (RPA) was historically discovered as one of the six components needed to reconstitute simian virus 40 DNA replication from purified components. RPA is now known to be involved in all DNA metabolism pathways that involve single-stranded DNA (ssDNA). Heterotrimeric RPA comprises several domains connected by flexible linkers and is heavily regulated by post-translational modifications (PTMs). The structure of RPA has been challenging to obtain. Various structural methods have been applied, but a complete understanding of RPA's flexible structure, its function, and how it is regulated by PTMs has yet to be obtained. This review will summarize recent literature concerning how RPA is phosphorylated in the cell cycle, the structural analysis of RPA, DNA and protein interactions involving RPA, and how PTMs regulate RPA activity and complex formation in double-strand break repair. There are many holes in our understanding of this research area. We will conclude with perspectives for future research on how RPA PTMs control double-strand break repair in the cell cycle.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.