clicking here. colleagues, clients, or customers by , you can order high-quality copies for your ... more clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): April 2, 2012 www.sciencemag.org (this information is current as of The following resources related to this article are available online at
Methods for investigating the conformation and interactions of RNA and RNA/protein complexes are ... more Methods for investigating the conformation and interactions of RNA and RNA/protein complexes are currently limited. This in turn has restricted our ability to understand the dynamics and interactio...
tracked with changes in diffusion time of the larger mAb-antigen complex. With this approach, we ... more tracked with changes in diffusion time of the larger mAb-antigen complex. With this approach, we measured the binding affinities of Alexa-488 labeled streptavidin and anti-streptavidin immunoglobulin G2 (IgG2) in buffer and neat serum, and found that binding is~3-fold tighter in serum versus buffer. Through control experiments in viscosity-matched sucrose solution, we found that differences in viscosity may account for some but not all of this effect. Serum contains high concentrations of co-solutes of various sizes including albumin and g-globulins. We tested the effects of macromolecular crowding on binding using physiological concentrations of bovine serum albumin as well as polymeric crowders, and found that crowding also partially accounts for the tighter binding of the streptavidin-IgG2 pair in serum. Conversely, serum does not affect the binding affinity of anti-streptavidin IgG1 to streptavidin. Accurate characterization of therapeutic proteins in relevant conditions is integral to assessing their safety and efficacy. The implications of these results with respect to drug development are discussed.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Jun 12, 2009
Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one th... more Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here we report two complementary synthetic genetic counters in E. coli that can count up to three induction events, the first comprised of a riboregulated transcriptional cascade and the second of a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.
There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably d... more There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogenrelated RNA, it requires expensive thermocycling equipment and is comparatively slow. Isothermal methods promise procedural simplicity but have traditionally suffered from amplification artifacts that tend to preclude easy identification of target nucleic acids. Recently, the isothermal SHERLOCK system overcame this problem by using CRISPR to distinguish amplified target sequences from artifactual background signal. However, this system comes at the cost of introducing considerable enzymatic complexity and a corresponding increase in total assay time. Therefore, simpler and less expensive strategies are highly desirable. Here, we demonstrate that by nesting NASBA primers and modifying the NASBA inner primers to encode an RNA Mango aptamer sequence we can dramatically increase the sensitivity of NASBA to 1.5 RNA molecules per microliter. As this isothermal nucleic acid detection scheme directly produces a fluorescent reporter, real-time detection is intrinsic to the assay. Nested Mango NASBA is highly specific and, in contrast to existing RNA detection systems, offers a cheap, simple, and specific way to rapidly detect single-molecule amounts of pathogenic RNA.
A processive RNA replicator The RNA World Hypothesis suggests that, before modern life, there wer... more A processive RNA replicator The RNA World Hypothesis suggests that, before modern life, there were RNA molecules that were capable of carrying genetic information and driving chemical reactions, a task gradually replaced by DNA and enzymes in modern biology. Central to this theory is an RNA replicase capable of mediating general replication of RNA. Using laboratory evolution, Cojocaru et al. isolated a promoter-based RNA polymerase ribozyme that, analogous to modern-day protein polymerases, clamps onto templates to increase its processivity, making it a potential model for replication in early biology. Science , this issue p. 1225
The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA pro... more The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 complex in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and by using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for non-canonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the well conserved 6S template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate in low nutrient conditions. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions when ATP concentrations are low.
Small RNAs, defined as noncoding 20-30-nt-long RNAs, are instrumental regulators of cellular proc... more Small RNAs, defined as noncoding 20-30-nt-long RNAs, are instrumental regulators of cellular processes in most eukaryotes. In this chapter we describe three methods for extracting small RNA from cells: a general method, one plant specific and a third particular to conifers. Further, protocols are given for the analysis and quantification of small RNAs using polyacrylamide gel-based approaches. A native streptavidin gel-shift assay, useful for measuring the relative amounts of multiple small RNAs simultaneously, is presented. To further characterize small RNAs biochemically, a sodium periodate assay probing for 2', 3' hydroxyl groups on the 3' terminus of small RNAs is outlined.
The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack... more The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack of naturally fluorescent RNA molecules for RNA detection and imaging.
Fast, accurate and reliable diagnostic tests are critical for controlling the spread of the 2019 ... more Fast, accurate and reliable diagnostic tests are critical for controlling the spread of the 2019 Coronavirus disease associated with SARS-CoV-2 infection. The current gold standard for testing is real time PCR (RT-PCR), however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 min), user-friendly and accurate microchip RT-PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips pre-loaded with COVID-19 primers and probes for the N gene accommodate 1.2 μl reaction volumes, lowering the required reagents by 10-fold compared to tube-based RT-PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip RT-PCR provides a significantly lower resource alternative to the CDC approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.
To further understand the transcriptome, new tools capable of measuring folding, interactions, an... more To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor–acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local substructures in a biocompatible manner. Fluorescence turn-on RNA aptamers are genetically encodable tags that appear to rigidly confine their cognate fluorophores, and thus have the potential to report angular-resolved FRET. Here, we use the fluorescent aptamers Broccoli and Mango-III as donor and acceptor, respectively, to measure the angular dependence of FRET. Joining the two fluorescent aptamers by a helix of variable length allowed systematic rotation of the acceptor fluorophore rel...
Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking tra... more Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.
Ennishi et al that lack rearrangements of MYC, BCL2, or both. Of these, 18 cases lack a matched n... more Ennishi et al that lack rearrangements of MYC, BCL2, or both. Of these, 18 cases lack a matched normal and were analyzed for somatic SNVs and indels using a combination of Strelka2 and Mutect2, using the GnomAD database to filter germline variants based on population allele fraction. Structural variants and copy number alterations were identified using Manta and Control-FREEC, respectively. Results: Our analysis identified MYC or BCL2 rearrangements that were cryptic to breakapart FISH assays, revealing that 40% of these apparently non-HGBL-DH/TH-BCL2 tumors had, in fact, a "genetic double hit" (Figure 1). These MYC rearrangements involved recurrent non-IG partner loci ZCCHC7, RFTN1, or CD96. Although the breakpoints for two of these translocations were outside of the locus interrogated by FISH, the remainder involved small insertions of either the MYC gene or enhancer regions. Three tumors had focal copy number gains of MYC, including one with a double minute, and six tumors harbored gains of MIR-17-HG, a regulator of MYC expression. Similarly, rearrangements involving BCL2 that were cryptic to breakapart FISH were identified in three tumors, including the insertion of an IGK enhancer element into the BCL2 locus. In the only case lacking both BCL2 and MYC translocations, the 11q alteration associated with MYC-negative high-grade lymphomas was found. Finally, several focal deletions were found affecting the promoter region around TSS1 of the lncRNA PVT1 gene, telomeric of MYC. This locus has recently been found to contain a boundary element that insulates MYC from the actions of downstream enhancers. Conclusions: Genetic mechanisms for the membership of apparently non-HGBL-DH/TH-BCL2 tumors to the DHITsig-pos subtype of DLBCL were revealed in 90% of tumors. These mechanisms included MYC and BCL2 rearrangements cryptic to FISH assays, copy number aberrations, and focal deletion of PVT1. This study further supports the notion that DHITsig identifies a new biological entity within DLBCL and highlights the limitations of FISH assays in the detection of genetic mechanisms of aberrant MYC and BCL2 expression.
within the 3 0 UTR of the positive strand RNA turnip crinkle virus (TCV). This T-shaped Structure... more within the 3 0 UTR of the positive strand RNA turnip crinkle virus (TCV). This T-shaped Structure (TSS) is similar in shape and size to a tRNA, despite a distinct secondary structure involving three hairpins and two coaxially stacked helix-pseudoknot motifs at the 5'-end and the 3'-end of the structure. TSS binds ribosomes and enhances translation, but upon binding to the RNA-dependent RNA-polymerase (RdRp), it can change its conformation and foster viral replication. We present a study of the unfolding pathway of the extended TCV TSS, combining steered molecular dynamics simulations (SMD) and optical tweezers (OT) experiments. Developing an SMD protocol for a structure of this size and complexity was an important and novel part of the study. Simulations and experiments indicate that the TSS unfolding starts with the 3'-side structures, followed by the long central hairpin, and ends with the 5'-side pseudoknot-hairpin motif. This order could not be deduced from the ranking of the free energies of the TSS motifs. The SMD simulations explained this sequence of unfolding based on the 3D organization of the TSS and formation of transient tertiary interactions that maintain the 5'-end pseudoknot topology and delay its full unfolding until all other structures have opened. The SMD results also explained the differences between the estimated and measured contour lengths for the TCV TSS domains, while mutational studies pinpointed the RdRp binding site. Thus, combined biochemical analysis, single molecule force spectroscopy and SMD simulations yielding complementary information at multiple levels of resolution provide a means for studying complex structures. Funded in part by HHSN261200800001E.
clicking here. colleagues, clients, or customers by , you can order high-quality copies for your ... more clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): April 2, 2012 www.sciencemag.org (this information is current as of The following resources related to this article are available online at
Methods for investigating the conformation and interactions of RNA and RNA/protein complexes are ... more Methods for investigating the conformation and interactions of RNA and RNA/protein complexes are currently limited. This in turn has restricted our ability to understand the dynamics and interactio...
tracked with changes in diffusion time of the larger mAb-antigen complex. With this approach, we ... more tracked with changes in diffusion time of the larger mAb-antigen complex. With this approach, we measured the binding affinities of Alexa-488 labeled streptavidin and anti-streptavidin immunoglobulin G2 (IgG2) in buffer and neat serum, and found that binding is~3-fold tighter in serum versus buffer. Through control experiments in viscosity-matched sucrose solution, we found that differences in viscosity may account for some but not all of this effect. Serum contains high concentrations of co-solutes of various sizes including albumin and g-globulins. We tested the effects of macromolecular crowding on binding using physiological concentrations of bovine serum albumin as well as polymeric crowders, and found that crowding also partially accounts for the tighter binding of the streptavidin-IgG2 pair in serum. Conversely, serum does not affect the binding affinity of anti-streptavidin IgG1 to streptavidin. Accurate characterization of therapeutic proteins in relevant conditions is integral to assessing their safety and efficacy. The implications of these results with respect to drug development are discussed.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Jun 12, 2009
Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one th... more Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here we report two complementary synthetic genetic counters in E. coli that can count up to three induction events, the first comprised of a riboregulated transcriptional cascade and the second of a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.
There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably d... more There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogenrelated RNA, it requires expensive thermocycling equipment and is comparatively slow. Isothermal methods promise procedural simplicity but have traditionally suffered from amplification artifacts that tend to preclude easy identification of target nucleic acids. Recently, the isothermal SHERLOCK system overcame this problem by using CRISPR to distinguish amplified target sequences from artifactual background signal. However, this system comes at the cost of introducing considerable enzymatic complexity and a corresponding increase in total assay time. Therefore, simpler and less expensive strategies are highly desirable. Here, we demonstrate that by nesting NASBA primers and modifying the NASBA inner primers to encode an RNA Mango aptamer sequence we can dramatically increase the sensitivity of NASBA to 1.5 RNA molecules per microliter. As this isothermal nucleic acid detection scheme directly produces a fluorescent reporter, real-time detection is intrinsic to the assay. Nested Mango NASBA is highly specific and, in contrast to existing RNA detection systems, offers a cheap, simple, and specific way to rapidly detect single-molecule amounts of pathogenic RNA.
A processive RNA replicator The RNA World Hypothesis suggests that, before modern life, there wer... more A processive RNA replicator The RNA World Hypothesis suggests that, before modern life, there were RNA molecules that were capable of carrying genetic information and driving chemical reactions, a task gradually replaced by DNA and enzymes in modern biology. Central to this theory is an RNA replicase capable of mediating general replication of RNA. Using laboratory evolution, Cojocaru et al. isolated a promoter-based RNA polymerase ribozyme that, analogous to modern-day protein polymerases, clamps onto templates to increase its processivity, making it a potential model for replication in early biology. Science , this issue p. 1225
The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA pro... more The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 complex in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and by using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for non-canonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the well conserved 6S template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate in low nutrient conditions. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions when ATP concentrations are low.
Small RNAs, defined as noncoding 20-30-nt-long RNAs, are instrumental regulators of cellular proc... more Small RNAs, defined as noncoding 20-30-nt-long RNAs, are instrumental regulators of cellular processes in most eukaryotes. In this chapter we describe three methods for extracting small RNA from cells: a general method, one plant specific and a third particular to conifers. Further, protocols are given for the analysis and quantification of small RNAs using polyacrylamide gel-based approaches. A native streptavidin gel-shift assay, useful for measuring the relative amounts of multiple small RNAs simultaneously, is presented. To further characterize small RNAs biochemically, a sodium periodate assay probing for 2', 3' hydroxyl groups on the 3' terminus of small RNAs is outlined.
The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack... more The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack of naturally fluorescent RNA molecules for RNA detection and imaging.
Fast, accurate and reliable diagnostic tests are critical for controlling the spread of the 2019 ... more Fast, accurate and reliable diagnostic tests are critical for controlling the spread of the 2019 Coronavirus disease associated with SARS-CoV-2 infection. The current gold standard for testing is real time PCR (RT-PCR), however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 min), user-friendly and accurate microchip RT-PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips pre-loaded with COVID-19 primers and probes for the N gene accommodate 1.2 μl reaction volumes, lowering the required reagents by 10-fold compared to tube-based RT-PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip RT-PCR provides a significantly lower resource alternative to the CDC approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.
To further understand the transcriptome, new tools capable of measuring folding, interactions, an... more To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor–acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local substructures in a biocompatible manner. Fluorescence turn-on RNA aptamers are genetically encodable tags that appear to rigidly confine their cognate fluorophores, and thus have the potential to report angular-resolved FRET. Here, we use the fluorescent aptamers Broccoli and Mango-III as donor and acceptor, respectively, to measure the angular dependence of FRET. Joining the two fluorescent aptamers by a helix of variable length allowed systematic rotation of the acceptor fluorophore rel...
Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking tra... more Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.
Ennishi et al that lack rearrangements of MYC, BCL2, or both. Of these, 18 cases lack a matched n... more Ennishi et al that lack rearrangements of MYC, BCL2, or both. Of these, 18 cases lack a matched normal and were analyzed for somatic SNVs and indels using a combination of Strelka2 and Mutect2, using the GnomAD database to filter germline variants based on population allele fraction. Structural variants and copy number alterations were identified using Manta and Control-FREEC, respectively. Results: Our analysis identified MYC or BCL2 rearrangements that were cryptic to breakapart FISH assays, revealing that 40% of these apparently non-HGBL-DH/TH-BCL2 tumors had, in fact, a "genetic double hit" (Figure 1). These MYC rearrangements involved recurrent non-IG partner loci ZCCHC7, RFTN1, or CD96. Although the breakpoints for two of these translocations were outside of the locus interrogated by FISH, the remainder involved small insertions of either the MYC gene or enhancer regions. Three tumors had focal copy number gains of MYC, including one with a double minute, and six tumors harbored gains of MIR-17-HG, a regulator of MYC expression. Similarly, rearrangements involving BCL2 that were cryptic to breakapart FISH were identified in three tumors, including the insertion of an IGK enhancer element into the BCL2 locus. In the only case lacking both BCL2 and MYC translocations, the 11q alteration associated with MYC-negative high-grade lymphomas was found. Finally, several focal deletions were found affecting the promoter region around TSS1 of the lncRNA PVT1 gene, telomeric of MYC. This locus has recently been found to contain a boundary element that insulates MYC from the actions of downstream enhancers. Conclusions: Genetic mechanisms for the membership of apparently non-HGBL-DH/TH-BCL2 tumors to the DHITsig-pos subtype of DLBCL were revealed in 90% of tumors. These mechanisms included MYC and BCL2 rearrangements cryptic to FISH assays, copy number aberrations, and focal deletion of PVT1. This study further supports the notion that DHITsig identifies a new biological entity within DLBCL and highlights the limitations of FISH assays in the detection of genetic mechanisms of aberrant MYC and BCL2 expression.
within the 3 0 UTR of the positive strand RNA turnip crinkle virus (TCV). This T-shaped Structure... more within the 3 0 UTR of the positive strand RNA turnip crinkle virus (TCV). This T-shaped Structure (TSS) is similar in shape and size to a tRNA, despite a distinct secondary structure involving three hairpins and two coaxially stacked helix-pseudoknot motifs at the 5'-end and the 3'-end of the structure. TSS binds ribosomes and enhances translation, but upon binding to the RNA-dependent RNA-polymerase (RdRp), it can change its conformation and foster viral replication. We present a study of the unfolding pathway of the extended TCV TSS, combining steered molecular dynamics simulations (SMD) and optical tweezers (OT) experiments. Developing an SMD protocol for a structure of this size and complexity was an important and novel part of the study. Simulations and experiments indicate that the TSS unfolding starts with the 3'-side structures, followed by the long central hairpin, and ends with the 5'-side pseudoknot-hairpin motif. This order could not be deduced from the ranking of the free energies of the TSS motifs. The SMD simulations explained this sequence of unfolding based on the 3D organization of the TSS and formation of transient tertiary interactions that maintain the 5'-end pseudoknot topology and delay its full unfolding until all other structures have opened. The SMD results also explained the differences between the estimated and measured contour lengths for the TCV TSS domains, while mutational studies pinpointed the RdRp binding site. Thus, combined biochemical analysis, single molecule force spectroscopy and SMD simulations yielding complementary information at multiple levels of resolution provide a means for studying complex structures. Funded in part by HHSN261200800001E.
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