Primer Extension Reaction Research Articles

Zn2+ ions improve the fidelity of metal-mediated primer extension while suppressing intrinsic and Mn2+-induced mutagenic effects by DNA polymerases.

While Mn2+ ions are well-established for reducing the fidelity of DNA polymerases, leading to the misincorporation of nucleotides, our investigation of the effects of metal ions revealed a contrasting role of Zn2+. Here, we demonstrate that Zn2+ ions enhance the fidelity of DNA polymerases (the 3' → 5' exonuclease-deficient Klenow fragment and Taq DNA polymerase) by suppressing misincorporation during primer extension reactions. Remarkably, Zn2+ ions inhibit both intrinsic misincorporation and Mn2+-induced misincorporation of nucleotides. Furthermore, Zn2+ ions also effectively suppressed misincorporation during metal-mediated primer extension reactions, which involved forming Ag+ and Hg2+ ion-mediated base pairs. These findings suggest that Zn2+ ions inhibit both intrinsic and Mn2+-induced mismatched base pair formation. Consequently, the combined use of Mn2+ and Zn2+ ions may offer a strategy for precisely regulating the fidelity of DNA polymerases. Remarkably, Zn2+ ions even suppress misincorporation in primer extension reactions that rely on metal-mediated base pairs, and conversely, this suggests that DNA polymerases recognize metal-mediated base pairs such as T-Hg2+-T, C-Ag+-A, and C-Ag+-T as relatively stable base pairs. These results imply that Zn2+ ions may also enhance the fidelity of DNA polymerases when incorporating non-canonical nucleobases, potentially paving the way for the expansion of the genetic alphabet.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Optimized Method for the Synthesis of Alkyne-Modified 2'-Deoxynucleoside Triphosphates.

A general approach is presented for synthesizing alkyne-modified nucleoside triphosphates via the Sonogashira cross-coupling reaction of unprotected halogenated 2'-deoxynucleoside, followed by monophosphorylation and the reaction of the corresponding phosphoromorpholidate with tributylammonium pyrophosphate. A highly efficient approach for the milligram-scale synthesis of base-modified nucleoside triphosphates with an amino acid-like side chain was developed. The present chemical method outweighs the other reported methods of a base-modified nucleoside triphosphates synthesis in terms of it being a protection-free strategy, the shortening of reaction steps, and increased yields (about 70%). The resulting 8-alkynylated dATP was tested as a substrate for DNA polymerases in a primer extension reaction.

DNA Replication across α-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η.

α-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' → 2' versus 5' → 3', respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3'-OH of the primer's terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

Simulations predict preferred Mg2+ coordination in a nonenzymatic primer-extension reaction center

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg2+ are required for efficient primer extension, but the mechanism by which Mg2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg2+ in facilitating the deprotonation of the primer 3′-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg2+ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion with both O3′ of the terminal primer nucleotide and the pro-Sp nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3′ and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension.

Polymerase-Based Signal Delay for Temporally Regulating DNA Involved Reactions, Programming Dynamic Molecular Systems, and Biomimetic Sensing.

Complex temporal molecular signals play a pivotal role in the intricate biological pathways of living organisms, and cells exhibit the ability to transmit and receive information by intricately managing the temporal dynamics of their signaling molecules. Although biomimetic molecular networks are successfully engineered outside of cells, the capacity to precisely manipulate temporal behaviors remains limited. In this study, the catalysis activity of isothermal DNA polymerase (DNAP) through combined use of molecular dynamics simulation analysis and fluorescence assays is first characterized. DNAP-driven delay in signal strand release ranged from 100 to 102min, which is achieved through new strategies including the introduction of primer overhangs, utilization of inhibitory reagents, and alteration of DNA template lengths. The results provide a deeper insight into the underlying mechanisms of temporal control DNAP-mediated primer extension and DNA strand displacement reactions. Then, the regulated DNAP catalysis reactions are applied in temporal modulation of downstream DNA-involved reactions, the establishment of dynamic molecular signals, and the generation of barcodes for multiplexed detection of target genes. The utility of DNAP-based signal delay as a dynamic DNA nanotechnology extends beyond theoretical concepts and achieves practical applications in the fields of cell-free synthetic biology and bionic sensing.

Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

The Impact of Activating Agents on Non-Enzymatic Nucleic Acid Extension Reactions.

Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an aqueous, protecting group free system, without the need for expensive enzymes. However, these primer extension reactions suffer from a lack of fidelity, low reaction rates, low overall yields, and short primer extension lengths. This review outlines a detailed mechanistic pathway for non-enzymatic template-directed primer extension and presents a review of the thermodynamic driving forces involved in entropic templating. Through the lens of entropic templating, the rate and fidelity of a reaction are shown to be intrinsically linked to the reactivity of the activating agent used. Thus, a strategy is discussed for the optimization of non-enzymatic template-directed primer extension, providing a path towards cost-effective in vitro synthesis of RNA and DNA.

Increasing the functional density of threose nucleic acid.

Chemical strategies that augment genetic polymers with amino acid residues that are overrepresented on the paratope surface of an antibody offer a promising route for enhancing the binding properties of nucleic acid aptamers. Here, we describe the chemical synthesis of α-l-threofuranosyl cytidine nucleoside triphosphate (tCTP) carrying either a benzyl or phenylpropyl side chain at the pyrimidine C-5 position. Polymerase recognition studies indicate that both substrates are readily incorporated into a full-length α-l-threofuranosyl nucleic acid (TNA) product by extension of a DNA primer-template duplex with an engineered TNA polymerase. Similar primer extension reactions performed using nucleoside triphosphate mixtures containing both C-5 modified tCTP and C-5 modified tUTP substrates enable the production of doubly modified TNA strands for a panel of 20 chemotype combinations. Kinetic measurements reveal faster on-rates (kon) and tighter binding affinity constants (Kd) for engineered versions of TNA aptamers carrying chemotypes at both pyrimidine positions as compared to their singly modified counterparts. These findings expand the chemical space of evolvable non-natural genetic polymers by offering a path for improving the quality of biologically stable TNA aptamers for future clinical applications.

The evolutionary loss of the Eh1 motif in FoxE1 in the lineage of placental mammals.

Forkhead box E1 (FoxE1) protein is a transcriptional regulator known to play a major role in the development of the thyroid gland. By performing sequence alignments, we detected a deletion in FoxE1, which occurred in the evolution of mammals, near the point of divergence of placental mammals. This deletion led to the loss of the majority of the Eh1 motif, which was important for interactions with transcriptional corepressors. To investigate a potential mechanism for this deletion, we analyzed replication through the deletion area in mammalian cells with two-dimensional gel electrophoresis, and in vitro, using a primer extension reaction. We demonstrated that the area of the deletion presented an obstacle for replication in both assays. The exact position of polymerization arrest in primer extension indicated that it was most likely caused by a quadruplex DNA structure. The quadruplex structure hypothesis is also consistent with the exact borders of the deletion. The exact roles of these evolutionary changes in FoxE1 family proteins are still to be determined.

Rapid Chemical Ligation of DNA and Acyclic Threoninol Nucleic Acid (aTNA) for Effective Nonenzymatic Primer Extension.

Previously, nonenzymatic primer extension reaction of acyclic l-threoninol nucleic acid (L-aTNA) was achieved in the presence of N-cyanoimidazole (CNIm) and Mn2+; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-aTNA to L-aTNA and of DNA to DNA. We found that Cd2+, Ni2+, and Co2+ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-aTNA fragment and template, resulting in unexpectedly more effective L-aTNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.

Aptamer-based regulation of DNA polymerase activity for the detection of protein-small molecule interactions

Herein, we developed a novel method for identifying protein-small molecule interactions (PSMIs) based on the switching of DNA polymerase activity. This strategy employs the small molecule-linked DNA probe (SMP) that could bind to and disrupt the DNA polymerase aptamer probe (DAP) capable of inhibiting DNA polymerase activity. Without the binding of the target protein, the SMP is readily degraded by the applied exonuclease I (exo I), preventing its binding to the DAP. The DAP then freely inhibits the polymerase activity through its specific binding to the polymerase. In the presence of the target protein, however, the binding between the target protein and the small molecule protects the SMP against the exo I-catalyzed degradation. The intact SMP now binds to the DAP and disrupts its active aptameric structure, preventing the DAP from inhibiting the polymerase activity. The uninhibited DNA polymerase catalyzes the primer extension reaction in conjugation with the TaqMan probe (TP), leading to a significant fluorescence enhancement. Taking advantage of this elaborate design principle, we successfully identified the two model PSMIs, streptavidin/biotin (SA/BTN) and anti-digoxigenin/digoxigenin (ADIG/DIG), as low as 4.01 nM and 6.72 nM, respectively, with excellent specificity. We further validated its practical applicability by reliably identifying the SA/BTN interaction in heterogeneous human serum specimens.

Nonenzymatic Template-Directed Primer Extension Using 2'-3' Cyclic Nucleotides Under Wet-Dry Cycles.

RNA World Hypothesis is centred around the idea of a period in the early history of life's origin, wherein nonenzymatic oligomerization and replication of RNA resulted in functional ribozymes. Previous studies in this endeavour have demonstrated template-directed primer extension using chemically modified nucleotides and primers. Nonetheless, similar studies that used non-activated nucleotides led to the formation of RNA only with abasic sites. In this study, we report template-directed primer extension with prebiotically relevant cyclic nucleotides, under dehydration-rehydration (DH-RH) cycles occurring at high temperature (90 °C) and alkaline conditions (pH 8). 2'-3' cyclic nucleoside monophosphates (cNMP) resulted in primer extension, while 3'-5' cNMP failed to do so. Intact extension of up to two nucleotide additions was observed with both canonical hydroxy-terminated (OH-primer) and activated amino-terminated (NH2-primer) primers. We demonstrate primer extension reactions using both purine and pyrimidine 2'-3' cNMPs, with higher product yield observed during cAMP additions. Further, the presence of lipid was observed to significantly enhance the extended product in cCMP reactions. In all, our study provides a proof-of-concept for nonenzymatic primer extension of RNA, using intrinsically activated prebiotically relevant cyclic nucleotides as monomers.

Abstract 6530: Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs

Abstract Background: The presence of circulating tumor DNA (ctDNA) can be used for minimal residual disease (MRD) monitoring and guide adjuvant therapy decisions. However, even when present, total ctDNA could be less than 0.01% of cell free DNA (cfDNA). Sequencing such low mutation frequency can be costly due to high sequencing read depth and accuracy requirements. The ability to enrich for low-abundance variants could improve detection performance and greatly lower sequencing cost by reducing the sequencing depth required. Purpose: The KAPA HyperPETE (Primer Extension Target Enrichment) workflow employs primer extension reactions to specifically capture and release target library molecules for sequencing, detects all major somatic variant types (SNV, InDel, CNV, MSI status, and novel fusion transcript partners) and could be leveraged to specifically target certain alleles. In this study, we assessed the variant enrichment capabilities of the KAPA HyperPETE workflow. Methods: Variant specific primers were designed to 8 low allele frequency single nucleotide variants (SNVs) in mixed cell line samples (NA12878 and NA24143, two Epstein-Barr virus transformed B cell female cell lines, with variant allele frequency (VAF) 1%, 0.5%, and 0.1%). Variant enrichment primers were used alongside a 37kb panel in the KAPA HyperPETE workflow. Additional optimizations and a study enriching all low allele frequency SNVs* in SeraCare ctDNA Mutation Mix v2 reference samples (VAF 1%, 0.5%, and 0.125%) are planned. Results: The preliminary results exhibited a median of 249✕, 159✕, and 86.5✕ enrichment of the targeted variants compared to the expected VAFs of 0.1%, 0.5%, and 1%, respectively. All 8 targeted variants were detected at 100K, 150K, and 550K read pairs per variant for VAF 1%, 0.5%, and 0.1%, respectively. Results from further optimizations and the study using SeraCare ctDNA samples will be shared at the AACR meeting. Conclusion: Preferential enrichment of specific variants down to 0.1% can be achieved using the KAPA HyperPETE Workflow. Utilizing primers designed for variant enrichment, we simultaneously enriched multiple variants and demonstrated the potential to minimize required sequencing. The KAPA HyperPETE workflow can be a powerful, cost-effective tool for detecting low frequency mutations in MRD research applications. KAPA HYPERPETE is For Research Use Only. Not for use in diagnostic procedures. KAPA HYPERPETE is a trademark of Roche. *Except GNA11 (COSM52969) due to stretches of polybase. Seraseq ctDNA Mutation Mix v2 gene list available on SeraCare website. Citation Format: Florence K. Crary-Dooley, Liu Xi, Ruben van der Merwe, Nitya M. Furtado, Jingchuan Li, Junyan Lin, Manuel Ochoa, Brian Godwin. Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6530.

DNA Polymerase ζ without the C-Terminus of Catalytic Subunit Rev3 Retains Characteristic Activity, but Alters Mutation Specificity of Ultraviolet Radiation in Yeast.

DNA polymerase ζ (pol ζ) plays a central role in replicating damaged genomic DNA. When DNA synthesis stalls at a lesion, it participates in translesion DNA synthesis (TLS), which helps replication proceed. TLS prevents cell death at the expense of new mutations. The current model indicates that pol ζ-dependent TLS events are mediated by Pol31/Pol32 pol ζ subunits, which are shared with replicative polymerase pol δ. Surprisingly, we found that the mutant rev3-ΔC in yeast, which lacks the C-terminal domain (CTD) of the catalytic subunit of pol ζ and, thus, the platform for interaction with Pol31/Pol32, retains most pol ζ functions. To understand the underlying mechanisms, we studied TLS in normal templates or templates with abasic sites in vitro in primer extension reactions with purified four-subunit pol ζ versus pol ζ with Rev3-ΔC. We also examined the specificity of ultraviolet radiation (UVR)-induced mutagenesis in the rev3-ΔC strains. We found that the absence of Rev3 CTD reduces activity levels, but does not alter the basic biochemical properties of pol ζ, and alters the mutation spectrum only at high doses of UVR, alluding to the existence of mechanisms of recruitment of pol ζ to UVR-damaged sites independent of the interaction of Pol31/Pol32 with the CTD of Rev3.

Selective Unnatural Base Pairing and Recognition of 2-Hydroxy-2'-deoxyadenosine in DNA Using Pseudo-dC Derivatives.

The formation of unnatural base pairs within duplex DNA would facilitate DNA nanotechnology and biotechnology. Iso-2'-deoxyguanosine (iso-dG) forms base pairs with iso-2'-deoxycytidine, and its use as an unnatural base pair was investigated. Iso-dG is one of the tautomers of 2-hydroxy-2'-deoxyadenosine (2-OH-dA), known as an oxidatively damaged nucleobase, and its selective recognition in DNA plays an important role in the diagnosis and pathogenesis of disease. Therefore, we focused on pseudo-dC (ψdC) as a suitable molecule that recognizes 2-OH-dA in DNA. Since 2-OH-dA shows tautomeric structures in DNA, we designed and used ψdC, which also has a tautomeric structure. We successfully synthesized a ψdC phosphoramidite compound for the synthesis of oligonucleotides (ODNs) as well as its triphosphate derivative (ψdCTP). Tm measurements revealed that ODNs including ψdC showed stable base pair formation with ODNs having 2-OH-dA. In contrast, low Tm values were observed for other bases (dG, dA, dC, and T). The results obtained for the single-nucleotide primer extension reaction revealed that ψdCTP was incorporated into the complementary position of 2-OH-dA in template DNA with high selectivity. In addition, the primer elongation reaction was confirmed to proceed in the presence of dNTPs. The present study reports an artificial nucleic acid that selectively and stably forms unnatural base pairs with 2-OH-dA in DNA.

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57–60% GC-content at a rate of 50–100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

Efficient Incorporation and Template‐Dependent Polymerase Inhibition are Major Determinants for the Broad‐Spectrum Antiviral Activity of Remdesivir

Remdesivir (RDV) is a direct‐acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). RDV exhibits broad‐spectrum antiviral activity against positive‐sense RNA viruses, e.g. SARS‐CoV‐2 and hepatitis C virus (HCV) and non‐segmented negative‐sense RNA viruses, e.g. Nipah virus (NiV), while segmented negative‐sense RNA viruses such as influenza (Flu) virus or Crimean‐Congo hemorrhagic fever virus (CCHFV) are not sensitive to the drug. The reasons for this apparent pattern are unknown. Here, we expressed and purified representative RNA‐dependent RNA polymerases (RdRp) and studied three biochemical parameters that have been associated with the inhibitory effects of RDV‐triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV‐TP, (ii) the effect of the incorporated RDV‐monophosphate (MP) on primer extension, and (iii) the effect of RDV‐MP in the template during incorporation of the complementary UTP. The results of this study revealed a strong correlation between antiviral effects and efficient incorporation of RDV‐TP. Inhibition in primer extension reactions is heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template‐dependent inhibition of UTP incorporation opposite the embedded RDV‐MP is seen with all polymerases. Molecular modeling suggests a steric conflict between the 1’‐cyano group of RDV‐MP and conserved residues of RdRp motif F. We conclude that future efforts in the development of nucleotide analogues with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1’‐modification.C.J.G, H.W.L, and E.P.T contributed equally to this work

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications.

In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.