G-ruption: The third international meeting on G-quadruplex and G-assembly

A three and a half day conference focusing on nucleic acid structures called G-quadruplexes (G4s) and other guanine-based assemblies was held in Sorrento, Italy (June 28–July 1, 2011) and featured 35 invited talks and over 89 posters. The G-quadruplex field continues to expand at an explosive rate with the emergence of new connections to biology, chemistry, physics, and nanotechnology. Following the trend established by the previous two international G4 meetings, the conference touched upon all these areas and facilitated productive exchanges of ideas between researchers from all over the world.     

On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5′-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5′- and 3′-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range.     

Biosafety in DIY‐bio laboratories: from hype to policy: Discussions about regulating DIY biology tend to ignore the extent of self‐regulation and oversight of DIY laboratories

Policymakers should treat DIY‐biology laboratories as legitimate parts of the scientific enterprise and pay attention to the role of community norms. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business, S&S: Ethics

DIY biology – very broadly construed as the practice of biological experiments outside of traditional research environments such as universities, research institutes or companies – has, during the past decade, gained much prominence. This increased attention has raised a number of questions about biosafety and biosecurity, both in the media and by policy makers who are concerned about safety and security lapses in “garage biology”. There are a number of challenges here though when it comes to policies to regulate DIY biology. For a start, the term itself escapes easy definition: synonyms or related terms abound, including garage biotechnology, bio‐hacking, self‐modification/grinding, citizen science, bio‐tinkering, bio‐punk, even transhumanism. Some accounts even use ‘DIY‐bio’ interchangeably with synthetic biology, even though these terms refer to different emerging trends in biology. Some of these terms are more charged than others but each carries its own connotations with regard to practice, norms and legality. As such, conversations about the risk, safety and regulation of DIY‐bio can be fraught.

Synonyms or related terms abound, including garage biotechnology, bio‐hacking, self‐modification/grinding, citizen science, bio‐tinkering, bio‐punk, even transhumanism.

Given the increasing policy discussions about DIY‐bio, it is crucial to consider prevailing practice thoughtfully, and accurately. Key questions that researchers, policy makers and the public need to contemplate include the following: “How do different DIY‐bio spaces exist within regulatory frameworks, and enact cultures of (bio)safety?”, “How are these influenced by norms and governance structures?”, “If something is unregulated, must it follow that it is unsafe?” and “What about the reverse: does regulatory oversight necessarily lead to safer practice?”.The DIY‐bio movement emerged from the convergence of two trends in science and technology. The first one is synthetic biology, which can broadly be defined as a conception of genetic engineering as systematic, modular and programmable. While engineering living organisms is obviously a complex endeavour, synthetic biology has sought to re‐frame it by treating genetic components as inherently modular pieces to be assembled, through rational design processes, into complex but predictable systems. This has prompted many “LEGO” metaphors and a widespread sense of democratisation, making genetic engineering accessible not only to trained geneticists, but also to anyone with an “engineering mindset”.The second, much older, trend stems from hacker‐ and makerspaces, which are – usually not‐for‐profit – community organisations that enable groups of enthusiasts to share expensive or technically complex infrastructure, such as 3D printers or woodworking tools, for their projects. These provide a model of community‐led initiatives based on the sharing of infrastructure, equipment and knowledge. Underpinning these two trends is an economic aspect. Many of the tools of synthetic biology – notably DNA sequencing and synthesis – have seen a dramatic drop in cost, and much of the necessary physical apparatus is available for purchase, often second‐hand, through auction sites.DIY‐bio labs are often set‐up under widely varying management schemes. While some present themselves as community outreach labs focusing on amateur users, others cater specifically to semi‐ or professional members with advanced degrees in the biosciences. Other such spaces act as incubators for biotech startups with an explicitly entrepreneurial culture. Membership agreements, IP arrangements, fees, access and the types of project that are encouraged in each of these spaces can have a profound effect on the science being done.     

Two cationic porphyrin isomers showing different multimeric G-quadruplex recognition specificity against monomeric G-quadruplexes

Ligands that can interact specifically with telomeric multimeric G-quadruplexes could be developed as promising anticancer drugs with few side effects related to other G-quadruplex-forming regions. In this paper, a new cationic porphyrin derivative, m-TMPipEOPP, was synthesized and characterized. Its multimeric G-quadruplex recognition specificity under molecular crowding conditions was compared to its isomer p-TMPipEOPP. The slight structural difference accounts for different multimeric G-quadruplex recognition specificity for the two isomers. p-TMPipEOPP can barely discriminate between multimeric and monomeric G-quadruplexes. By contrast, m-TMPipEOPP can bind with multimeric but not with monomeric G-quadruplexes. p-TMPipEOPP might bind to multimeric G-quadruplexes by two modes: sandwich-like end-stacking mode and pocket-dependent intercalative mode. Increasing the pocket size between adjacent two G-quadruplex uints is beneficial for the latter mode. m-TMPipEOPP might bind to multimeric G-quadruplexes by a side binding mode, which confers m-TMPipEOPP with higher multimeric G-quadruplex recognition specificity compared to p-TMPipEOPP. m-TMPipEOPP increases the stability of multimeric G-quadruplex under both dilute and molecular crowding conditions but its G-quadruplex-stabilizing ability is a little weaker than p-TMPipEOPP. These results provide important information for the design of highly specific multimeric G-quadruplex ligands. Another interesting finding is that pocket size is an important factor in determining the stability of multimeric G-quadruplexes.     

Structural diversity and extreme stability of unimolecular Oxytricha nova telomeric G-quadruplex

Oxytricha nova telomeric DNA contains guanine-rich short-tandem repeat sequences (GGGGTTTT) n and terminates as a single strand at the 3'-end. This single-stranded overhang forms a novel DNA structure, namely, G-quadruplex, comprising four quartets. In this study, we investigated the structures and dynamics of unimolecular Oxytricha nova ( O. nova) telomeric G-quadruplexes by performing single molecule fluorescence resonance energy transfer (FRET) spectroscopy and bulk circular dichroism (CD) measurements. We observed that unimolecular O. nova G-quadruplexes exhibit structural polymorphism according to monovalent cations. In the presence of Na (+), only antiparallel conformation is detected, which was demonstrated in previous studies; however, in the presence of K (+), they fold into two different conformations, a parallel conformation and an antiparallel one different from that induced by Na (+). Furthermore, these G-quadruplexes show extremely high stability in their dynamics when compared with human G-quadruplexes. While human telomeric G-quadruplexes that possess three quartets display fast dynamic behavior (<100 s) at low K (+) concentrations or high temperatures, O. nova G-quadruplexes maintain their conformational state for a long time (>1000 s), even at the lowest K (+) concentration and the highest temperature investigated. This high stability is primarily due to an extra quartet that results in additional cation coordination. In addition to cation coordination, we propose that other factors such as base stacking and the size of the thymine loop may contribute to the stability of O. nova G-quadruplexes; this is based on the fact that the O. nova G-quadruplexes were observed to be more stable than the human ones in the presence of Li (+), which is known to greatly destabilize G-quadruplexes because of imprecise coordination. This extreme stability of four-quartet G-quadruplexes enables telomere protection even in the absence of protective proteins or in the case of abrupt environmental changes, although only a single G-quadruplex structure can be derived from the short single-stranded overhang.     

Engineering of interlocked DNA G-quadruplexes as a robust scaffold

Interlock is a structural element in DNA G-quadruplexes that can be compared with the commonly used complementary binding of ‘sticky ends’ in DNA duplexes. G-quadruplex interlocking can be a basis for the assembly of higher-order structures. In this study, we formulated a rule to engineer (3 + 1) interlocked dimeric G-quadruplexes and established the folding topology of the designed DNA sequences by nuclear magnetic resonance spectroscopy. These interlocked G-quadruplexes are very stable and can serve as compact robust scaffolds for various applications. Different structural elements can be engineered in these robust scaffolds. We demonstrated the anti-HIV inhibition activity of the newly designed DNA sequences.     

Verification of specific G-quadruplex structure by using a novel cyanine dye supramolecular assembly: II. The binding characterization with specific intramolecular G-quadruplex and the recognizing mechanism

The supramolecular assembly of a novel cyanine dye, 3,3′-di(3-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethyl-thiacarbocyanine triethylammonium salt (ETC) was designed to verify specific intramolecular G-quadruplexes from duplex and single-strand DNAs. Spectral results have shown that ETC presented two major distinct signatures with specific intramolecular G-quadruplexes in vitro: (i) dramatic changes in the absorption spectra (including disappearance of absorption peak around 660 nm and appearance of independent new peak around 584 nm); (ii) ∼70 times enhancement of fluorescence signal at 600 nm. Furthermore, based on ¹H-nuclear magnetic resonance and circular dichroism results, the preferring binding of ETC to specific intramolecular G-quadruplexes probably result from end-stacking, and the loop structure nearby also plays an important role.     

Structure of the Hybrid-2 type intramolecular human telomeric G-quadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence

Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K⁺ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K⁺ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.     

DNA architecture: from G to Z

G-quadruplexes and Z-DNA are two important non-B forms of DNA architecture. Results on novel structural elements, folding and unfolding kinetics, and interactions with small molecules and proteins have been reported recently for these forms. These results will enhance our understanding of the biology of these structures and provide a platform for drug design.     

G-quadruplexes and their regulatory roles in biology

‘If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo’ (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future.     

Bioenergetics of early life: Coupling of reaction networks and compartments may have sparked the first life forms

Living cells are powered by intricate networks of chemical reactions of thousands of molecules. Understanding how living systems emerged through the assembly of chemical processes is one of the biggest challenges in science. Subject Categories: Biotechnology & Synthetic Biology, Evolution & Ecology, Metabolism

How can chemistry turn into biology? How can living cells be built from molecules? These are fundamental questions in biology and, despite much research efforts, remain unanswered. Yet, the past two decades have seen considerable advances in our knowledge of how and which (bio)physical and (bio)chemical processes could have driven the emergence of the first living cells. These achievements have led not only to a better understanding of the molecular origins of life, but also spurred significant developments in synthetic biology, biophysics and supramolecular chemistry. Although the exact events that sparked life on Earth will quite likely remain a mystery, at least partially, exploring the chemical origins of life offers clues about our primordial past and could contribute to shaping our future.

Although the exact events that sparked life on Earth will quite likely remain a mystery […] exploring the chemical origins of life offers clues about our primordial past and could contribute to shaping our future.

     

Ten simple rules for designing and running a computing minor for bio/chem students

Science students increasingly need programming and data science skills to be competitive in the modern workforce. However, at our university (San Francisco State University), until recently, almost no biology, biochemistry, and chemistry students (from here bio/chem students) completed a minor in computer science. To change this, a new minor in computing applications, which is informally known as the Promoting Inclusivity in Computing (PINC) minor, was established in 2016. Here, we present the lessons we learned from our experience in a set of 10 rules. The first 3 rules focus on setting up the program so that it interests students in biology, chemistry, and biochemistry. Rules 4 through 8 focus on how the classes of the program are taught to make them interesting for our students and to provide the students with the support they need. The last 2 rules are about what happens “behind the scenes” of running a program with many people from several departments involved.     

Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases,Revealing their Potential to Lead to Genetic Change

Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.     

Benzo(h)quinoline derivatives as G-quadruplex binding agents

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity. This compound contains a 4-pyridone group which has not been previously utilized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that these compounds are capable of binding G-quadruplexes with a K_a in the range of 3 × 10⁵ M^?1 and with a 10-fold selectivity for quadruplex over duplex DNA.     

G-quadruplexes with (4n?- 1) guanines in the G-tetrad core: formation of a G-triad·water complex and implication for small-molecule binding

G-quadruplexes are non-canonical structures of nucleic acids, in which guanine bases form planar G-tetrads (G·G·G·G) that stack on each other in the core of the structure. G-quadruplexes generally contain multiple times of four (4n) guanines in the core. Here, we study the structure of G-quadruplexes with only (4n - 1) guanines in the core. The solution structure of a DNA sequence containing 11 guanines showed the formation of a parallel G-quadruplex involving two G-tetrads and one G-triad with a vacant site. Molecular dynamics simulation established the formation of a stable G-triad·water complex, where water molecules mimic the position of the missing guanine in the vacant site. The concept of forming G-quadruplexes with missing guanines in the core broadens the current definition of G-quadruplex-forming sequences. The potential ability of such structures to bind different metabolites, including guanine, guanosine and GTP, in the vacant site, could have biological implications in regulatory functions. Formation of this unique binding pocket in the G-triad could be used as a specific target in drug design.     

A stabilizing and denaturing dual-effect for natural polyamines interacting with G-quadruplexes depending on concentration

Both G-quadruplexes and natural polyamines are intimately associated with tumor growth and proliferation. The effect of the natural polyamines on telomeric and some oncogenic G-quadruplexes including bcl-2, c-kit, and c-myc G-quadruplexes has been studied by using absorption, fluorescence, CD, and NMR methods. The results exhibited an interesting dual-effect depending on polyamine? concentration. Polyamines promote and stabilize G-quadruplexes under a lower concentration (less than 1 mM) but denature G-quadruplexes under a higher concentration (more than 1 mM). Probably the electrostatic and hydrophobic effect of polyamines and the hydrogen-bonding interaction between guanines and polyamines were respectively responsible for the stabilizing and denaturing effect.