Primer Extension Assay Research Articles

Linker-Mediated Inactivation of the SAM-II Domain in the Tandem SAM-II/SAM-V Riboswitch.

Tandem SAM-II/SAM-V riboswitch belongs to a class of riboswitches found in the marine bacterium 'Candidatus Pelagibacter ubique'. Previous studies have demonstrated that these riboswitches have the potential for digital modulation of gene expression at both the transcriptional and translational levels. In this study, we investigate the conformational changes in the tandem SAM-II/SAM-V riboswitch binding to S-adenosylmethionine (SAM) using selective 2'-hydroxyl acylation analyzed by the primer extension (SHAPE) assay, small-angle X-ray scattering (SAXS), and oligos depressing probing. Our findings reveal that the linker between SAM-II/SAM-V aptamers blocks the SAM response of the SAM-II domain. This result proposes a new mechanism for gene expression regulation, where the ligand-binding functions of tandem riboswitches can be selectively masked or released through a linker.

B-237 Engineered Reverse Transcriptases and Taq DNA Polymerases deliver robust yield in the presence of clinically relevant inhibitors

Abstract Background The past five years has seen explosive growth in molecular diagnostic assays that rely on sensitive and accurate detection of nucleic acids across a wide range of sample types. As the assays are required to become increasingly sensitive, the need for highly functional and purpose-built enzymatic tools has grown. Reverse transcriptase (RT) and Taq DNA Polymerase (Taq DNAP) are commonly used to detect RNA and/or DNA viruses in patient samples for point of care (POC) diagnostics. However, crude patient samples introduce inhibitors that decrease enzyme activity and increase false negative results in POC testing. In addition, there is a need for faster turn-around time for patient diagnosis which can be enabled by engineering more active and sensitive Taq variants. Herein, we present on our engineered RT and Taq DNAP enzymes that were developed to confer greater activity, thermostability, inhibitor tolerance and/or sensitivity. Methods We take a 3-pronged approach to protein engineering: rational design, computational design and directed evolution. Using these techniques, we identified and characterized many RT and Taq DNAP variants. We deeply characterized each RT variant by measuring processivity, inhibitor tolerance and thermostability. First strand synthesis was done at temperatures from 42-65°C in the presence and absence of various clinically relevant inhibitors followed by qPCR to assess cDNA yield. Next, we characterized the inhibitor tolerance of the Taq variants by performing qPCR reactions in the presence of inhibitors. To determine if the Taq variant would be an appropriate fit for fast PCR, the polymerization activity of the Taq DNAP variants were characterized using a primer extension assay. Results Reverse transcriptases termed; StellarScript, StellarScript HT and StellarScript HT+ exhibit high processivity at 42°C. StellarScript HT+ exhibited the highest inhibitor tolerance, processivity and thermostability at temperatures up to 65°C thus making StellarScript HT+ well suited to produce high yields with clinical samples. Taq Pol variants were identified that efficiently amplify DNA in the presence of inhibitors. Conclusions Based on the increased polymerase activity and inhibitor tolerance, WMG Taq variants are well suited to give robust DNA yields with clinical samples. We were successful in engineering RT and Taq DNAP variants that are highly thermostable, active and inhibitor tolerant and thus will enable breakthroughs in existing POC diagnostics.

Human T-cell leukemia virus type 1 uses a specific tRNAPro isodecoder to prime reverse transcription.

Human T-cell leukemia virus type 1 (HTLV-1) is the only oncogenic human retrovirus discovered to date. All retroviruses are believed to use a host cell tRNA to prime reverse transcription (RT). In HTLV-1, the primer-binding site (PBS) in the genomic RNA is complementary to the 3' 18 nucleotides (nt) of human tRNAPro The human genome encodes 20 cytoplasmic tRNAPro genes representing seven isodecoders, all of which share the same 3' 18 nt sequence but vary elsewhere. Whether all tRNAPro isodecoders are used to prime RT in cells is unknown. A previous study showed that a 3' 18 nt tRNAPro-derived fragment (tRFPro) is packaged into HTLV-1 particles and can serve as an RT primer in vitro. The role of this tRNA fragment in the viral life cycle is unclear. In retroviruses, N1-methylation of the tRNA primer at position A58 (m1A) is essential for successful plus-strand transfer. Using primer-extension assays performed in chronically HTLV-1-infected cells, we found that A58 of tRNAPro is m1A-modified, implying that full-length tRNAPro is capable of facilitating successful plus-strand transfer. Analysis of HTLV-1 RT primer extension products indicated that full-length tRNAPro is likely to be the primer. To determine which tRNAPro isodecoder is used as the RT primer, we sequenced the minus-strand strong-stop RT product containing the intact tRNA primer and established that HTLV-1 primes RT using a specific tRNAPro UGG isodecoder. Further studies are required to understand how this primer is annealed to the highly structured HTLV-1 PBS and to investigate the role of tRFPro in the viral life cycle.

Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps

Since its introduction in the human population, SARS-CoV-2 has evolved into multiple clades, but the events in its intrahost diversification are not well understood. Here, we compare three-dimensional (3D) self-organized neural haplotype maps (SOMs) of SARS-CoV-2 from thirty individual nasopharyngeal diagnostic samples obtained within a 19-day interval in Madrid (Spain), at the time of transition between clades 19 and 20. SOMs have been trained with the haplotype repertoire present in the mutant spectra of the nsp12- and spike (S)-coding regions. Each SOM consisted of a dominant neuron (displaying the maximum frequency), surrounded by a low-frequency neuron cloud. The sequence of the master (dominant) neuron was either identical to that of the reference Wuhan-Hu-1 genome or differed from it at one nucleotide position. Six different deviant haplotype sequences were identified among the master neurons. Some of the substitutions in the neural clouds affected critical sites of the nsp12-nsp8-nsp7 polymerase complex and resulted in altered kinetics of RNA synthesis in an in vitro primer extension assay. Thus, the analysis has identified mutations that are relevant to modification of viral RNA synthesis, present in the mutant clouds of SARS-CoV-2 quasispecies. These mutations most likely occurred during intrahost diversification in several COVID-19 patients, during an initial stage of the pandemic, and within a brief time period.

A beginner's handbook to identify and characterize i-motif DNA.

Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.

Potent Anti‐HIV Activity of Alkyl‐Modified DiPPro‐Nucleotides

Two convergent approaches for synthesizing a new class of nucleoside diphosphate prodrugs bearing different nucleoside analogs are reported herein. The DiPPro‐nucleotides comprise an acyloxybenzyl group in combination with a lipophilic alkyl residue at the β‐phosphate or β‐phosphonate group, respectively. They are selectively cleaved to form their corresponding β‐alkylated nucleoside diphosphate derivatives in chemical and biological hydrolysis studies. In contrast, there is a selective but slow cleavage observed in the hydrolysis of the DiPPro‐compounds bearing two different, nonbioreversible alkyl moieties in human CD4+ T‐lymphocyte CEM/0 cell extracts. In these studies, the delivery of nucleoside monophosphates rather than nucleoside diphosphates is being observed, most likely due to a pure chemical phosphoranhydride cleavage of the β‐phosph(on)ate moiety. The antiviral evaluation of these two types of prodrugs reveals that these compounds exhibit marked anti‐HIV efficacy in HIV‐2‐infected thymidine kinase‐deficient CD4+ CEM T‐cells (CEM/TK−), with significantly better activities (up to 6700‐fold) against HIV‐2 replication than the parent nucleosides. Primer extension assays demonstrate that the β‐dialkylphosphate‐modified nucleoside derivatives, β‐monoalkylated‐diphosphates, and nucleoside diphosphates serve as substrates for HIV reverse transcriptase for the viral DNA elongation.

Membrane-permeable tenofovir-di- and monophosphate analogues

The development of new antiviral agents such as nucleoside analogues or acyclic nucleotide analogues (ANPs) and prodrugs thereof is an ongoing task. We report on the synthesis of three types of lipophilic triphosphate analogues of (R)-PMPA and dialkylated diphosphate analogues of (R)-PMPA. A highly selective release of the different nucleotide analogues ((R)-PMPA-DP, (R)-PMPA-MP, and (R)-PMPA) from these compounds was achieved. All dialkylated (R)-PMPA-prodrugs proved to be very stable in PBS as well as in CEM/0 cell extracts and human plasma. In primer extension assays, both the monoalkylated and the dialkylated (R)-PMPA-DP derivatives acted as (R)-PMPA-DP as a substrate for HIV-RT. In contrast, no incorporation events were observed using human polymerase γ. The dialkylated (R)-PMPA-compounds exhibited significant anti-HIV efficacy in HIV-1/2 infected cells (CEM/0 and CEM/TK−). Remarkably, the dialkylated (R)-PMPA-MP derivative 9a showed a 326-fold improved activity as compared to (R)-PMPA in HIV-2 infected CEM/TK− cells as well as a very high SI of 14,000. We are convinced that this study may significantly contribute to advancing antiviral agents developed based on nucleotide analogues in the future.

Lipophilic Nucleoside Triphosphate Prodrugs of Anti-HIV Active Nucleoside Analogs as Potential Antiviral Compounds.

Nucleoside analogs require three phosphorylation steps catalyzed by cellular kinases to give their triphosphorylated metabolites. Herein, the synthesis of two types of triphosphate prodrugs of different nucleoside analogs is disclosed. Triphosphates comprising: i) a γ-phosphate or γ-phosphonate bearing a bioreversible acyloxybenzyl group and a long alkyl group and ii) γ-dialkyl phosphate/phosphonate modified nucleoside triphosphate analogs. Almost selective conversion of the former TriPPPro-compounds into the corresponding γ-alkylated nucleoside triphosphate derivatives is demonstrated in CEM/0 cell extracts that proved to be stable toward further hydrolysis. The latter γ-dialkylated triphosphate derivatives lead to the slow formation of the corresponding NDPs. Both types of TriPPPro-compounds are highly potent in wild-type CEM/0 cells and more importantly, they exhibit even better activities against HIV-2 replication in CEM/TK- cell cultures. A finding of major importance is that, in primer extension assays, γ-phosphate-modified-NTPs, γ-mono-alkylated-triphosphates, and NDPs prove to be substrates for HIV-RT but not for cellular DNA-polymerases α,γ.

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.

Pronucleotides of 2',3'-Dideoxy-2',3'-Didehydrothymidine as Potent Anti-HIV Compounds.

We report on the synthesis and evaluation of three different nucleotide prodrug systems: (i) nucleoside triphosphate analogues in which the γ-phosph(on)ate has two different lipophilic nonbioreversible alkyl residues with d4TDP as the released nucleotide analogue; (ii) nucleoside diphosphate analogues bearing a bioreversible and a stable β-alkyl group; or (iii) nucleoside diphosphate analogues bearing two nonhydrolysable lipophilic alkyl moieties. The delivery of d4TDP (for the triphosphate precursor) and d4TMP (for the diphosphate precursor) was demonstrated in CD4+ T-lymphocyte CEM cell extracts as well as in phosphate buffer saline (PBS). In primer extension assay, we found that γ-dialkylated d4TTP derivatives and d4TDP were accepted as substrates by HIV-RT. Several of these compounds were observed to be extremely active against HIV-1/2 replication in HIV-infected cells. A more than 45,000-fold increase in the anti-HIV activity was detected for compound 18a as compared to the parent d4T which results in a selectivity index value of 37,000.

Human Translesion Synthesis Polymerases polκ and polη Perform Error-Free Replication across N2-dG Methyleugenol and Estragole DNA Adducts.

The secondary metabolites of polypropanoids, methyleugenol (MEG), and estragole (EG), found in many herbs and spices, are commonly used as food flavoring agents and as ingredients in cosmetics. MEG and EG have been reported to cause hepatocarcinogenicity in rodents, human livers, and lung cells. The formation of N2-dG and N6-dA DNA adducts is primarily attributed to the carcinogenicity of these compounds. Therefore, these compounds have been classified as "possible human carcinogens" by the International Agency for Research on Cancer and "reasonably anticipated to be a human carcinogen" by the National Toxicology Program. Herein, we report the synthesis of the N2-MEG-dG and N2-EG-dG modified oligonucleotides to study the mutagenicity of these DNA adducts. Our studies show that N2-MEG-dG and N2-EG-dG could be bypassed by human translesion synthesis (TLS) polymerases hpolκ and hpolη in an error-free manner. The steady-state kinetics of dCTP incorporation by hpolκ across N2-MEG-dG and N2-EG-dG adducts show that the catalytic efficiencies (kcat/Km) were ∼2.5- and ∼4.4-fold higher, respectively, compared to the unmodified dG template. A full-length primer extension assay demonstrates that hpolκ exhibits better catalytic efficiency than hpolη. Molecular modeling and dynamics studies capturing pre-insertion, insertion, and post-insertion steps reveal the structural features associated with the efficient bypass of the N2-MEG-dG adduct by hpolκ and indicate the reorientation of the adduct in the active site allowing the successful insertion of the incoming nucleotide. Together, these results suggest that though hpolκ and hpolη perform error-free TLS across MEG and EG during DNA replication, the observed carcinogenicity of these adducts could be attributed to the involvement of other low fidelity polymerases.

Galidesivir Triphosphate Promotes Stalling of Dengue-2 Virus Polymerase Immediately Prior to Incorporation.

Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 μM and 47 ± 5 μM, respectively, at an ATP concentration of 20 μM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.

Abstract 6179: Comparative study of Polθ helicase and polymerase inhibitor biochemical and cellular potency

Abstract Background: DNA Polymerase θ (Polθ) is active in Theta-mediated end joining (TMEJ), a repair pathway that utilizes resected 3’ ends and micro-homology sequences to repair double strand breaks. Polθ contains an N-terminal helicase domain and a C-terminal polymerase domain. The helicase domain facilitates stripping of RPA/RAD51 from single stranded DNA and is involved in DNA-end recognition, and micro-homology search and pairing. The polymerase domain performs DNA synthesis following micro-homology pairing. Lack of Polθ is synthetic lethal in homologous recombination deficient (HRD) cells. In this study, we utilized existing small molecule inhibitors targeting either helicase or polymerase domain of Polθ, to assess their translational potential in HRD in vitro cancer cell models. Methods: Small molecules inhibiting either the helicase or polymerase activity of Polθ were studied with biochemical and cellular assays. For the biochemical studies, recombinant helicase or polymerase domains were purified. The helicase activity of Polθ was analyzed using ADP-Glo and the polymerase activity by primer extension assay. Binding assays confirmed inhibitor binding to recombinant proteins. To study inhibitor efficacy in cellular models, we probed cell viability of BRCA2-deficient cell lines. Results: Both helicase and polymerase inhibitors showed good potency in inhibiting Polθ enzymatic activity in ATPase or primer extension assays, respectively, with IC50 values in low-nanomolar range. However, the viability of BRCA2-deficient cell lines was not significantly affected after treatment with either helicase or polymerase inhibitors. The measured compound properties (stability, efflux, solubility) did not explain the lack of efficacy in cellular viability assays. Combining Polθ inhibitor treatment with ionizing radiation led to a modest enhancement of radiation-induced decrease in cell viability. Conclusions: Recently, significant advances have been made in Polθ inhibitor development prompted by the synthetic lethal interaction between Polθ and HRD discovered in several screening efforts. Both helicase and polymerase inhibitors are highly potent in biochemical assays. However, without exogenous genotoxic stress, the potential of Polθ inhibitors as therapeutic target may not be reached in vitro. Additional studies are required to understand the translational gap between biochemical and biological read-outs. Citation Format: Johanna K. Ahlskog, Antti Pohjakallio, Ralf Paul, Martin Augustin, Elisabeth Schneider, Sakshi Johri, Tuomas Tervahauta, Hans-Georg Beisel, Lars Neumann, Julia Lindqvist, Anu Moilanen. Comparative study of Polθ helicase and polymerase inhibitor biochemical and cellular potency [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 6179.

The role of IGFBP2 in pre‐symptomatic Alzheimer's disease: a multimodal longitudinal study of cognitively unaffected individuals with a family history of sporadic Alzheimer’s disease

AbstractBackgroundInsulin‐like growth factors (IGF) play pivotal roles in neurogenesis, neuronal survival, synaptogenesis and compensatory repair mechanisms following injury. Insulin‐like growth factor binding proteins (IGFBP) bind to IGFs, and regulate interactions between IGFs and their cell surface receptors. In the brain, IGFBP2 is the most abundant IGFBP. Several studies have observed altered IGFBP2 levels in Alzheimer’s disease (AD) patients. However, the role of IGFBP2 during pre‐symptomatic AD remains unknown. Thus, we used longitudinal data acquired from the pre‐symptomatic PREVENT‐AD cohort to examine its possible contributions to AD pathophysiology.MethodFor initial cross‐sectional analyses, the Mini‐Mental State Examination was used to stage the participants (PREVENT‐AD, CCNA and Douglas cohorts) across the AD spectrum. Olink’s primer extension assay was used to measure cerebrospinal fluid (CSF) IGFBP2 levels. PREVENT‐AD participants were followed longitudinally for 6‐8 years. Annual visits included cognitive performance assessments, lumbar punctures and neuroimaging. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) evaluated five cognitive domains (immediate memory, delayed memory, attention, language and visuospatial abilities) and included a total summary score.ResultCSF IGFBP2 levels were significantly reduced in MCI (p < 0.001) and AD participants (p = 0.027 and p < 0.001 respectively), relative to controls (Figure 1). In the PREVENT‐AD cohort, baseline CSF IGFBP2 levels were negatively correlated with RBANS cognitive performance trajectory slopes estimated over the course of 6 to 8 years, for the delayed memory (p = 0.003), visuospatial (p = 0.008) and total scale indices (p = 0.021) (Figure 2). However, baseline CSF IGFBP2 levels were not associated with cognitive decline for the immediate memory (p = 0.162), attention (p = 0.890) and language (p = 0.841) indices (Figure 3). Finally, in the PREVENT‐AD cohort, baseline CSF IGFBP2 levels were negatively correlated with baseline left entorhinal cortex volume (p = 0.011), but were not correlated with right entorhinal cortex volume (p = 0.868)(Figure 4).ConclusionOur results suggest that IGFBP2 may exhibit a bi‐directional association with AD stage pathology. Our findings revealed that IGFBP2 is strongly correlated with early cognitive decline in the delayed memory and visuospatial domains, in pre‐symptomatic individuals.

Macrophage Scavenger Receptor 1 in the CSF pre‐symptomatic with a parental history of AD and in the cortex of autopsy‐confirmed Alzheimer’s Disease cases.

AbstractBackgroundThe macrophage scavenger receptor 1 (MSR1) also known as SCARA1, is a gene that encodes for a macrophage/microglia cell surface receptor. Its primary function is to mediate the endocytosis of modified (acetylated or oxidized) low density lipoproteins (LDLs)(Frenkel, Wilkinson et al. 2013). Interestingly, MSR1 has been found to bind to soluble β‐amyloid (Aβ) and it is thought that it participates to its clearance via a microglia‐mediated process (PrabhuDas, Baldwin et al. 2017). Using multiple cohorts covering a wide range of brain health stages, MSR1 gene expression and protein level alterations were investigated to map the associations between MSR1 and Alzheimer’s disease (AD) pathophysiology.MethodMSR1 CSF protein levels were assessed in the PREVENT‐AD (PRe‐symptomatic EValuation of Experimental or Novel Treatments for Alzheimer’s Disease) cohort which is composed of subjects with parental history of AD. MSR1 has been measured with a primer extension assay using Olink technology (J. Alz. Dis., In press). ELISA has been used to quantify the MSR1 protein levels in frontal cortex homogenates of 86 brains from the Douglas Bell Canada Brain Bank (DBCBB) whereas MSR1 mRNA prevalence was measured using Gene Chip Clarium D human microarray. All data compilation and statistical analysis has been made using JMP pro 15.1 software.ResultStrong correlations between MSR1 CSF protein levels and CSF AD biomarkers, notably phosphorylated Tau (R2=0.108; p=0.0007) and Aβ42 (R2=0.055; p=0.0175) have been found in the PREVENT‐AD cohort. An APOEε4‐independent correlation was observed between CSF MSR1 and APOE (R2=0.156; p˂0.0001) in these cognitively unaffected subjects. In autopsy‐confirmed cortical homogenates, MSR1 protein levels display negative correlations with both Aβ plaques (R2=0.035; p=0.1768) and neurofibrillary tangles (R2=0.136; p=0.0067). On the other hand, MSR1 mRNA levels of the same samples are not correlated with plaques (R2=0.008; p=0.5255) and tangles (R2=0.009; p=0.4971) density. Studies are underway to contrast MSR1 levels with amyloid‐42, p(181)tau and total tau in the same tissues.ConclusionMSR1 plays a significant role in the pathophysiology of AD and recent evidence suggests that elevated MSR1 protein observed in the early stages of AD are followed by a progressive decline that coincides with the emergence of Tau pathology.

A CRISPRi-based investigation reveals that multiple promoter elements drive gene expression from the genome of mycobacteriophage D29.

A unique feature found in the genomes of mycobacteriophages such as L5 belonging to the A cluster is the presence of multiple dispersed repeated elements known as stoperators. The phage repressor binds these repeat elements, shutting off transcription globally and thereby promoting lysogeny. Interestingly, the sequence of these stoperators closely matches that of the consensus -35 region of prokaryotic promoters, leading us to propose that they may have a role to play in the initiation of transcription by serving as RNA polymerase binding sites. Mycobacteriophage D29 is closely related to phage L5, and their genome organizations are very similar. As in L5, there are multiple stoperators in the genome of D29. The positions occupied by the stoperators in the two genomes are almost identical. The significant difference between the two phages is that D29 lacks the gene encoding the equivalent of the L5 repressor. Since phage D29 does not produce a repressor, we considered it to be a suitable model for testing our hypothesis that the stoperators function as promoters in the absence of the repressor. To prove our point, we targeted CRISPR guide RNAs against six stoperators. In the case of five out of the six, we found a significant reduction in downstream gene expression and phage growth. Based on this observation and primer extension assays, we conclude that promoting gene expression is likely to be the primary function of stoperators.

Biochemical analysis of DNA synthesis blockage by G-quadruplex structure and bypass facilitated by a G4-resolving helicase.

G-quadruplex (G4) DNA poses a unique obstacle to DNA synthesis during replication or DNA repair due to its unusual structure which deviates significantly from the conventional DNA double helix. A mechanism to overcome the G4 roadblock is provided by the action of a G4-resolving helicase that collaborates with the DNA polymerase to smoothly catalyze polynucleotide synthesis past the unwound G4. In this technique-focused paper, we describe the experimental approaches of the primer extension assay using a G4 DNA template to measure the extent and fidelity of DNA synthesis by a DNA polymerase acting in concert with a G4-resolving DNA helicase. Important parameters pertaining to reaction conditions and controls are discussed to aid in the design of experiments and interpretation of the data obtained. This methodology can be applied in multiple capacities that may depend on the DNA substrate, DNA polymerase, or DNA helicase under investigation.

Insight into RNA-DNA primer length counting by human primosome.

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA–DNA primers of a limited length for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA–DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (∼35-mer): (i) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5′-end, and (ii) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allow us to conclude that p58C is a key regulator of all steps of RNA–DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.

Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA.

Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson–Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.

The Second Class of Tetrahydrofolate (THF-II) Riboswitches Recognizes the Tetrahydrofolic Acid Ligand via Local Conformation Changes.

Riboswitches are regulatory noncoding RNAs found in bacteria, fungi and plants, that modulate gene expressions through structural changes in response to ligand binding. Understanding how ligands interact with riboswitches in solution can shed light on the molecular mechanisms of this ancient regulators. Previous studies showed that riboswitches undergo global conformation changes in response to ligand binding to relay information. Here, we report conformation switching models of the recently discovered tetrahydrofolic acid-responsive second class of tetrahydrofolate (THF-II) riboswitches in response to ligand binding. Using a combination of selective 2′-hydroxyl acylation, analyzed by primer extension (SHAPE) assay, 3D modeling and small-angle X-ray scattering (SAXS), we found that the ligand specifically recognizes and reshapes the THF-II riboswitch loop regions, but does not affect the stability of the P3 helix. Our results show that the THF-II riboswitch undergoes only local conformation changes in response to ligand binding, rearranging the Loop1-P3-Loop2 region and rotating Loop1 from a ~120° angle to a ~75° angle. This distinct conformation changes suggest a unique regulatory mechanism of the THF-II riboswitch, previously unseen in other riboswitches. Our findings may contribute to the fields of RNA sensors and drug design.