39 research outputs found
Diverse and robust molecular algorithms using reprogrammable DNA self-assembly
Molecular biology provides an inspiring proof-of-principle that chemical systems can store and process information to direct molecular activities such as the fabrication of complex structures from molecular components. To develop information-based chemistry as a technology for programming matter to function in ways not seen in biological systems, it is necessary to understand how molecular interactions can encode and execute algorithms. The self-assembly of relatively simple units into complex products is particularly well suited for such investigations. Theory that combines mathematical tiling and statistical–mechanical models of molecular crystallization has shown that algorithmic behaviour can be embedded within molecular self-assembly processes, and this has been experimentally demonstrated using DNA nanotechnology with up to 22 tile types. However, many information technologies exhibit a complexity threshold—such as the minimum transistor count needed for a general-purpose computer—beyond which the power of a reprogrammable system increases qualitatively, and it has been unclear whether the biophysics of DNA self-assembly allows that threshold to be exceeded. Here we report the design and experimental validation of a DNA tile set that contains 355 single-stranded tiles and can, through simple tile selection, be reprogrammed to implement a wide variety of 6-bit algorithms. We use this set to construct 21 circuits that execute algorithms including copying, sorting, recognizing palindromes and multiples of 3, random walking, obtaining an unbiased choice from a biased random source, electing a leader, simulating cellular automata, generating deterministic and randomized patterns, and counting to 63, with an overall per-tile error rate of less than 1 in 3,000. These findings suggest that molecular self-assembly could be a reliable algorithmic component within programmable chemical systems. The development of molecular machines that are reprogrammable—at a high level of abstraction and thus without requiring knowledge of the underlying physics—will establish a creative space in which molecular programmers can flourish
Nucleic acid detection with CRISPR-Cas13a/C2c2
Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060)Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institutes of Health (U.S.) (Grant 1R01-MH110049
Characterization and Generation of Male Courtship Song in Cotesia congregata (Hymenoptera: Braconidae)
Background
Male parasitic wasps attract females with a courtship song produced by rapid wing fanning. Songs have been described for several parasitic wasp species; however, beyond association with wing fanning, the mechanism of sound generation has not been examined. We characterized the male courtship song of Cotesia congregata (Hymenoptera: Braconidae) and investigated the biomechanics of sound production. Methods and Principal Findings
Courtship songs were recorded using high-speed videography (2,000 fps) and audio recordings. The song consists of a long duration amplitude-modulated “buzz” followed by a series of pulsatile higher amplitude “boings,” each decaying into a terminal buzz followed by a short inter-boing pause while wings are stationary. Boings have higher amplitude and lower frequency than buzz components. The lower frequency of the boing sound is due to greater wing displacement. The power spectrum is a harmonic series dominated by wing repetition rate ~220 Hz, but the sound waveform indicates a higher frequency resonance ~5 kHz. Sound is not generated by the wings contacting each other, the substrate, or the abdomen. The abdomen is elevated during the first several wing cycles of the boing, but its position is unrelated to sound amplitude. Unlike most sounds generated by volume velocity, the boing is generated at the termination of the wing down stroke when displacement is maximal and wing velocity is zero. Calculation indicates a low Reynolds number of ~1000. Conclusions and Significance
Acoustic pressure is proportional to velocity for typical sound sources. Our finding that the boing sound was generated at maximal wing displacement coincident with cessation of wing motion indicates that it is caused by acceleration of the wing tips, consistent with a dipole source. The low Reynolds number requires a high wing flap rate for flight and predisposes wings of small insects for sound production
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Synthetic organization in vitro and in vivo
Organized complexity is a hallmark of biology in general, and eukaryotes in particular. This phenomenon abounds across many size scales ranging from tissues to organelles to protein complexes. Scaffold molecules, which facilitate the assembly of protein complexes, serve as guides for organization. These scaffolds encompass a wide variety of materials, including DNA, RNA, and proteins, and are used for engineering metabolic reactions and signaling pathways. However, the structures which have been produced to date are fairly simple in geometry, and in many cases are not compatible with in vivo assembly. Here, I aim to recapitulate biological organization synthetically using nucleic acids and lipids as scaffolds for proteins used as markers and as reaction catalysts, both in vitro and in vivo. In Chapter 1, I discuss a new method for assaying the assembly of DNA nanostructures using next-generation DNA sequencing. In Chapter 2, I explore a new set of DNA nanostructures capable of self-assembly across a wide range of temperatures and conditions. In Chapter 3, I develop a method called distributed cell division counting (DCDC) for counting bacterial cell divisions that utilizes the segregation of self-assembling fluorescent particles, and apply DCDC to measure the growth rate of a native gut microbe in the mammalian gut. In Chapter 4, I discuss a new set of lipid-based scaffolds that can co-localize enzymes in vivo and apply this technique to enhance indigo biosynthesis. Together, these results indicate that self-assembly can be designed to occur under a wide range of conditions, and demonstrate several practical applications of self-assembled nanostructures.Systems Biolog
Synthetic Lipid-Containing Scaffolds Enhance Production by Colocalizing Enzymes
Subcellular organization
is critical for isolating, concentrating,
and protecting biological activities. Natural subcellular organization
is often achieved using colocalization of proteins on scaffold molecules,
thereby enhancing metabolic fluxes and enabling coregulation. Synthetic
scaffolds extend these benefits to new biological processes and are
typically constructed from proteins or nucleic acids. To expand the
range of available building materials, we use a minimal set of components
from the lipid-encapsulated bacteriophage Ď•6 to form synthetic
lipid-containing scaffolds (SLSs) in <i>E. coli</i>. Analysis
of diffusive behavior by particle tracking in live cells indicates
that SLSs are >20 nm in diameter; furthermore, density measurements
demonstrate that SLSs contain a mixture of lipids and proteins. The
fluorescent proteins mCitrine and mCerulean can be colocalized to
SLSs. To test for effects on enzymatic production, we localized two
enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent
increase in indigo production, showing that SLSs can enhance the production
of a commercially relevant metabolite
Synthetic Lipid-Containing Scaffolds Enhance Production by Colocalizing Enzymes
Subcellular organization
is critical for isolating, concentrating,
and protecting biological activities. Natural subcellular organization
is often achieved using colocalization of proteins on scaffold molecules,
thereby enhancing metabolic fluxes and enabling coregulation. Synthetic
scaffolds extend these benefits to new biological processes and are
typically constructed from proteins or nucleic acids. To expand the
range of available building materials, we use a minimal set of components
from the lipid-encapsulated bacteriophage Ď•6 to form synthetic
lipid-containing scaffolds (SLSs) in <i>E. coli</i>. Analysis
of diffusive behavior by particle tracking in live cells indicates
that SLSs are >20 nm in diameter; furthermore, density measurements
demonstrate that SLSs contain a mixture of lipids and proteins. The
fluorescent proteins mCitrine and mCerulean can be colocalized to
SLSs. To test for effects on enzymatic production, we localized two
enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent
increase in indigo production, showing that SLSs can enhance the production
of a commercially relevant metabolite
Synthetic Lipid-Containing Scaffolds Enhance Production by Colocalizing Enzymes
Subcellular organization
is critical for isolating, concentrating,
and protecting biological activities. Natural subcellular organization
is often achieved using colocalization of proteins on scaffold molecules,
thereby enhancing metabolic fluxes and enabling coregulation. Synthetic
scaffolds extend these benefits to new biological processes and are
typically constructed from proteins or nucleic acids. To expand the
range of available building materials, we use a minimal set of components
from the lipid-encapsulated bacteriophage Ď•6 to form synthetic
lipid-containing scaffolds (SLSs) in <i>E. coli</i>. Analysis
of diffusive behavior by particle tracking in live cells indicates
that SLSs are >20 nm in diameter; furthermore, density measurements
demonstrate that SLSs contain a mixture of lipids and proteins. The
fluorescent proteins mCitrine and mCerulean can be colocalized to
SLSs. To test for effects on enzymatic production, we localized two
enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent
increase in indigo production, showing that SLSs can enhance the production
of a commercially relevant metabolite
Inertial guidance systems in insects: from the neurobiology to the structural mechanics of biological gyroscopes
Flying insects employ a vast array of sensory modalities to coordinate complex aerial maneuvers with incredible speed and acuity. One central feature underlying this is their ability to rapidly acquire and process information about rotational motion. In Diptera (flies), gyroscopic sensing is accomplished with halteres, organs that are derived from hindwings. In Lepidoptera (moths and butterflies), a recent study has suggested that antennae serve this critical function. Here, we review the biomechanical and sensory aspects of these biological gyroscopes. We focus on past and ongoing research to understand how the physical and physiological aspects of these inertial guidance units interact to determine their functional performance