12 Reconstructing the RNA world

A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of self-replication as well as mutation, and hence evolution towards ever more efficient replication. As this primordial replicase appears to have been lost in time, we use synthetic biology to build modern-day “Doppelgangers” of the ancestral replicase to reconstruct and study their properties in an effort to learn more about life’s first genetic system. I will discuss our progress in the engineering and evolution of RNA polymerase ribozymes as well as the potential role that structured media such as the eutectic phase of water–ice may have played in the emergence of RNA self-replication.     

207 Nanopore immobilization of DNA polymerase enhances single-molecule sequencing

A zero-mode waveguide (ZMW) is a nanoscale optical waveguide driven at a frequency below its cut-off. In this mode, the electric field, instead of traveling down the axis of the conducting cavity, decays exponentially. By fabricating waveguides with sub-wavelength diameters and illuminating them with laser light, the electric field in the waveguide is confined enough to enable single-molecule optical detection at micromolar concentration [1]. Immobilizing single DNA polymerases in ZMWs and using special phosphate-fluorescently labeled dNTPs form the basis for single-molecule real-time DNA sequencing, one of the most promising next-generation sequencing platforms [2]. In this method, the polymerase replicates the sample DNA, and as it incorporates new bases into the product strand, the labeled dNTPs emit a burst of light before the phosphate is cleaved off. The sequence of colors corresponds to the DNA sequence (see Figure 1 below from Eid et al., 2009). Because the ZMW aperture’s diameter is sub-diffraction-limit, it is impossible to optically distinguish one polymerase in a ZMW from two. Having only one polymerase in each waveguide is critical to sequencing accuracy. In its present state, experimenters use diffusion to fill ZMWs with polymerases, resulting in a Poisson distribution for filling ZMWs, and consequently a theoretical limit of 36.8% of ZMWs having only one polymerase [2]. We achieve full polymerase occupancy of ZMWs by fabricating the structures on an ultrathin silicon nitride membrane and drilling a nanopore at the base of each waveguide with an ion beam. A short DNA fragment with biotin on either end is conjugated to a streptavidin and then drawn into the nanopore with a voltage bias. There is then a free biotin at the base of the ZMW. A polymerase–streptavidin complex can diffuse into the ZMW and bind to the exposed biotin. Because the nanopore is too small to fit more than one molecule, only one ZMW will bind to a biotin in the nanopore. Upon flushing the ZMW chamber, the biotin-bound polymerase will remain trapped in the pore, and only a single polymerase will remain at the base of each waveguide.      

187 Plucking the high hanging fruit: a systematic approach for targeting protein interfaces

Development of specific ligands for protein targets that help decode the complexities of protein–protein interaction networks is a key goal for the field of chemical biology. Despite the emergence of powerful in silico and experimental high-throughput screening strategies, the discovery of synthetic ligands that selectively modulate protein–protein interactions remains a challenge for the chemical biologists. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these domains, we can modulate the function of a particular protein with metabolically-stable synthetic molecules (Raj et al., 2013). This presentation will discuss computational approaches (Bullock et al., 2011; Jochim & Arora, 2010) to identify targetable interfaces along with synthetic methods (Patgiri et al., 2008; Tosovska & Arora, 2010) to develop protein domain mimics (PDMs) as modulators of intracellular protein–protein interactions (Henchey et al., 2010; Patgiri et al., 2011).     

152 Order from disorder: defining structure in disordered proteins

Intrinsically disordered proteins are involved in a range of functional roles in the cell, as well as being associated with a number of diverse diseases, including cancers, neurodegenerative disorders, and cardiac myopathies. We use single-molecule fluorescence approaches to characterize disordered proteins implicated in the progression of Parkinson’s and Alzheimer’s diseases. Our goal is to understand, how disease-associated modifications to these proteins alter their conformational and dynamic properties and to relate these changes to disease pathology.     

69 DNA-binding studies of large antiviral polyamides

Polyamides are minor groove DNA-binding agents derived from the natural product distamycin A. PA1 is a large 12 ring polyamide discovered by NanoVir LLC; it is bioactive against the HPV16 virus in cell and tissue culture (Edwards et al., 2011). To better understand the basis of this phenomenon, the interactions of PA1 with the regulatory sequence of the HPV16 genome (7662–122?bp) are being examined. Using affinity cleavage as detected by capillary electrophoresis, with PA1 attached to methyl propyl ethidium iron EDTA, 10 binding sites of PA1 were identified in this part of the HPV genome. Polyamide perfect binding sites were as predicted by recognition rules (Dervan & Edelson, 2003). Quantitative DNaseI footprinting indicates that both perfect and single mismatch sites are bound with K_ds in the low nm range. Interestingly, a wide range of K_ds are observed for double mismatch sites (1–60?nm) and are under examination. This work will permit us to build a map of PA binding to HPV sequences, thus informing mechanisms of in vivo behavior.     

102 Genome engineering nucleases derived from GIY-YIG homing endonucleases

Efficient targeted manipulation of complex genomes requires highly specific endonucleases to generate double-strand breaks at defined locations (Bibikova et al., 2003; Bogdanove and Voytas, 2011). The predominantly engineered nucleases, zinc-finger nucleases (ZFNs), and TAL effector nucleases (TALENs) use the catalytic domain of FokI as the nuclease portion. This domain, however, functions as a dimer to nonspecifically cleave DNA meaning that ZFNs and TALENs must be designed in head-to-head pairs to target a desired sequence. To overcome this limitation and expand the toolbox of genome editing reagents, we used the N-terminal catalytic domain and interdomain linker of the monomeric GIY-YIG homing endonuclease I-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), and I-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al. 2012). We also made I-TevI fusions to LAGLIDADGs homing endonucleases (I-Tev-LHEs). All the three fusions showed activity on model substrates on par with ZFNs and TALENs in yeast-based recombination assays. These proof-of-concept experiments demonstrate that the catalytic domain of GIY-YIG homing endonucleases can be targeted to relevant loci by fusing the domain to characterize DNA-binding platforms. Recent efforts have focused on improving the Tev-TAL platform by (1) understanding the spacing requirements between the nuclease cleavage site and the DNA binding site, (2) probing the DNA binding requirements of the I-TevI linker domain, and (3) demonstrating activity in mammalian systems.     

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).     

116 Deamination of both methyl- and normal-cytosine by the foreign DNA restriction enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.     

184 Small molecule identification against novel MDRA protein of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an obstinate pathogen causing tuberculosis (TB) in Homo sapiens. One third of the World population is affected by Mtb (James et al., 2008). The multidrug-resistant protein-A (MDRA) belongs to ABC transporter family. The protein MDRA and the membrane integral protein MDRB together form the efflux pump (MDRA₂B₂ complex) that confers resistance by transport of the drugs out of the cell. The MDRB protein expression depends on the expression of MDRA (Baisakhee et al., 2002). In the present study, MDRA 3-D model (Figure) was generated with the help of comparative homology modeling techniques using pair-wise sequence alignment. The predicted 3-D model was subjected to refinement and validated. The active site of the protein was predicted. The virtual screening (VS) studies were performed at MDRB binding site with an in-house library of small molecules to identify a lead molecule that can inhibits the MDRA protein. The results of VS project competitive inhibitors of MDRB, for its binding with MDRA, and its drug-resistant activity. Hence, the MDRA protein may be treated as a novel target for the development of new chemical entities for tuberculosis therapy (Bhargavi et al., 2010; Malkhed et al., 2011).     

212 Shifting interfaces: changes in protein–protein and protein–DNA interfaces probed via molecular dynamics

Over the past decade, there has been a growing interest in studying the binding of DNA to the MutSalpha protein complex. This heterodimeric protein complex, the Msh2/Msh6 complex in humans, is the initial complex that binds mismatched DNA and other DNA defects that occur during replication. This complex has also been shown to bind at least some types of damaged DNA, such as the cross-linked adducts due to the chemotherapeutics cisplatin and carboplatin, or the incorporation of the chemotherapeutic, FdU. As a result of this interest, multiple studies have contrasted the interactions of MutSalpha with its normal mismatched substrate and with the interactions of MutsSalpha with the DNA damaged by chemotherapeutic cisplatin. To complement these studies, we examine the interaction between MutSalpha and the DNA damaged by carboplatin via all-atom molecular dynamics simulations. These simulations provide evidence for subtle changes in the protein–DNA and protein–protein interfaces. The interfaces shifts found are broadly similar to those found in binding with adduct from cis-platin, but have distinct differences. These subtle differences may play a role in the way of the different damages and mismatched DNA are signaled by MutSalpha, and suggest a signaling mechanism for DNA damage that chiefly involves shifts in protein–protein interactions as opposed to changes in protein conformation.     

7 Imperfect RNA synthesis via model prebiotic reactions and consequences for functional RNAs

Contemporary life synthesizes RNA of homogeneous length and regioisomer composition via sophisticated enzymatic catalysis. Before such catalysts existed, RNA could have been produced only via simpler, non-enzymatic means, which model prebiotic systems have shown produce pools of products that are similar, but varied (e.g. in regioisomer composition). Recently, we have demonstrated that functional RNAs (ribozymes and aptamers) containing mixed-regioisomer backbones (i.e. 2′–5′ vs. 3′–5′ linkages) retain function. This observation, coupled with the well-known fact that mixed-regioisomer RNAs exhibit depressed melting temperatures relative to native RNA, suggests that mixed-regioisomer backbones could actually be adaptive in an RNA (or pre-RNA) world. In this poster, we will show our recent work with functional RNAs representative of those produced in non-enzymatic polymerization reactions and their behaviours as catalysts and receptors.     

164 Extracting dynamics information from multiple structures

Meaningful dynamics information can be extracted from multiple experimental structures of the same, or closely related, proteins or RNAs. The covariance matrix of atom positions is decomposable into its principal components, and in this way, it is possible to rank-order the changes in the set of structures, and to determine what the most significant changes are. Usually, only a few principal components dominate the motions of the structures, and these usually relate to the functional dynamics. This dynamics information provides strong evidence for the plasticity of protein and RNA structures, and also suggests that these structures almost always have a highly limited repertoire of motions. In some cases, such as HIV protease, the dominant motions are opening and closing over the active site. For myoglobin, the changes are much smaller, reflecting in part the small changes in sequence, but nonetheless they show characteristic details that depend on the species. Sets of structures can also be used to derive the effective microscopic forces that are forcing a given conformational transition.     

101 Exploring the 3D spatial organization of the genome

Knowledge about the 3D organization of the genome will offer great insights into how cells retrieve and process the genetic information. Knowing the spatial probability distributions of individual genes will provide insights into gene regulatory and replication processes, and fill in the missing links between epigenomics, functional genomics, and structural biology. We will discuss an approach to determine 3D genome structures and structure–function maps of genomes by integrating divers types of data. To address the challenge of modeling highly variable genome structures, we discuss a population-based modeling approach, where we construct a large population of 3D genome structures that together are entirely consistent with all available experimental data including data from genome-wide chromosome conformation capture and imaging experiments. We interpret the result in terms of probabilities of a sample drawn from a population of heterogeneous structures. We will discuss results on the 3D spatial organization of genomes in human lymphoblastoid cells and budding yeast.     

123 Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology

Deregulation of mini-chromosome maintenance (MCM) proteins are associated with genomic instability and cellular abnormality. MCM complexes are recruited to replication origins for S phase genome duplication. Paradoxically, MCM proteins are expressed in large number of origins and are associated with unreplicated chromatin regions away from the origins during G1 and S phases. We report an unusually wide left-handed filament structure for an archeal MCM, as determined by X-ray and electron microscopy. The crystal structure reveals that an α-helix bundle formed between two neighboring subunits plays a critical role in filament formation which we show through mutation and electron microscopy. The filament interior has a remarkably strong electro-positive surface spiraling along the inner filament channel which we establish as the binding site for double-stranded DNA. We find that MCM filament binding to DNA causes dramatic topological changes which negatively supercoil circular DNA through inducing loosening of the double helix. This newly identified biochemical activity of MCM may imply a wider functional role for MCM in DNA metabolism beyond helicase function, for the non-origin bound MCMs. Finally, using yeast genetics, we show that the inter–subunit interactions important for MCM filament formation play a role for cell growth and survival.     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.     

169 Polymorphic assembly of computationally designed hydrophobic-rich collagen peptides

We seek to understand how the position and length of hydrophobic content within a collagen peptide sequence dictates morphology of self-assembly. We modeled collagen assembly using diffusion limited aggregation¹ (DLA) (Parkinson et al. 1995). of discretized, rigid rods composed of hydrophilic and hydrophobic spheres. Simulations predicted that the inclusion of short hydrophobic domains should direct the assembly of lamellar structures. We designed a set of collagen peptide sequences with six, five and four contiguous nonpolar residues. Electron microscopy of aggregates revealed the peptide with six nonpolar residues self-assembled into uniform fibrils and the peptide with five residues assembled into both fibrils and plates, while including four hydrophobic residues that formed only plates. This polymorphic behavior can be explained by packing models of rod vs. screw-like-particles.     

82 Targeting intramolecular DNA structures with complementary strands

Antisense, antigene, and siRNA strategies are currently used to control the expression of genes. To this end, our laboratory is mimicking the targeting of mRNA by reacting DNA stem-loop motifs with their partially complementary strands. Specifically, we used a combination of isothermal titration (ITC), differential scanning calorimetry (DSC), and temperature-dependent UV spectroscopy to investigate: (1) the unfolding of a pseudoknot and a complex containing joined triplex-duplex motifs (shown below); and (2) the reaction of these compact structures with single strands that are complementary to the bases in the loops and to a portion of their stem.     

24 Evolutionary dynamics of inteins in bacteria

Inteins are protein sequences that autocatalytically splice themselves out of protein precursors – analogous to introns – and ligate the flanking regions into a functional protein. Inteins are present in all three kingdoms of life, but have a sporadic distribution. They are found predominantly in proteins involved in DNA replication and repair such as helicases. The distribution of inteins suggests an adaptive function. The evolutionary forces which shaped the observed distribution of inteins are generally unknown. Some authors view inteins only as the selfish elements and argue that frequent horizontal transfer is behind inteins sporadic dissemination (Gogarten et al., 2002). On the other hand, the ancient nature of the inteins and the process of gain/loss could lead to the scattered distribution of inteins among species (Pietrokovski, 2001). It is necessary to note that the exclusively selfish nature of inteins is questionable; recent findings support the hypothesis of possible functional roles of inteins in protein regulation (Callahan et al., 2011). Moreover, both hypotheses were built on a limited number of the intein representatives. The amount of genomic data available for bacteria is enormous and in silico analysis for diverse inteins is warranted. We decided to take advantage of these microbial genomic data and performed comprehensive mining for the inteins using a bioinformatic pipeline. Altogether, 1757 species were analysed from 19 major phyla yielding more than 4500 intein-like sequences. The majority of these bacterial inteins were not described previously. Approximately 55% of the inteins were found in polymerases, helicases, or recombinases (Figure 1). Phylogenetic analysis indicated the complex evolutionary dynamics of inteins which includes horizontal transfers, high evolutionary rates coupled with recurrent gains, and losses. The preponderance of inteins in helicases and reductases is being investigated in terms of functional relevance.     

23 The phylogenomic roots of modern biochemistry,translation, and the genetic code

The origin and evolution of modern biochemistry is a complex problem that has puzzled scientists for almost a century. In my laboratory, we have dissected the emergence of the very early macromolecules that populated primordial cells using ideographic (historical, retrodictive) approaches. Deep evolutionary signals were retrieved from a census of molecular structures and functions in thousands of nucleic acids and millions of proteins using powerful phylogenomic methods. These clock-like signals revealed that modern biochemistry resulted from gradual coevolution and accretion of molecular parts and molecules. This was made evident in the study of aminoacyl-tRNA synthetase (aaRS) enzymes and the ribosomal ensemble. aaRSs coevolved with tRNAs, as catalytic aaRS domains and acceptor arm tRNAs accreted domains, and RNA substructures. Similarly, the ribosome originated in its central ratchet mechanism and expanded by coevolving rRNA–protein interactions (Figure 1). Remarkably, while the first biochemical functions were metabolic, the translation, the genetic code, and the ribosome appeared quite late as ‘exacting’ mechanisms that enhanced protein folding speed and flexibility, benefiting the search for new molecular functions. Our timelines reveal that translation unfolded only after the rise of viruses but prior to the appearance of diversified archaeal microbes. Remarkably, its debut coincided with the rise of nucleotide and amino acid biosynthetic pathways .