Tiny tweezers allow precision control of enzymes


July 3, 2013

Tweezers are a handy instrument when it comes to removing a splinter or plucking an eyebrow.

In new research, Hao Yan and his colleagues at Arizona State University’s Biodesign Institute describe a pair of tweezers shrunk down to an astonishingly tiny scale. When the jaws of these tools are in the open position, the distance between the two arms is about 16 nanometers – over 30,000 times smaller than a single grain of sand. Download Full Image

The group demonstrated that the nanotweezers, fabricated by means of the base-pairing properties of DNA, could be used to keep biological molecules spatially separated, or to bring them together as chemical reactants, depending on the open or closed state of the tweezers.

In a series of experiments, regulatory enzymes – central components in a host of living processes – are tightly controlled with the tweezers, which can switch reactions on or off depending on their open or closed condition.

“The work has important implications for regulating enzymatic function and may help usher in a new generation of nanoscale diagnostic devices as well as aid in the synthesis of valuable chemicals and smart materials,” Yan said.

Results of the new research appear in the current issue of the journal Nature Communications. Minghui Liu, a researcher in Biodesign’s Center for Single Molecule Biophysics and the Department of Chemistry and Biochemistry at ASU is the paper’s lead author. Other authors include Jinglin Fu, Yan Liu, Neal Woodbury from ASU, and Christian Hejesen and Kurt Gothelf from Aarhus University, Denmark.

Enzymes are large molecules responsible for thousands of chemical interactions essential to life. A primary role for enzymes is to accelerate or catalyze myriad chemical reactions involved in processes ranging from digestion to DNA synthesis. To do this, enzymes lower the activation energy – the minimum energy needed for chemical reactions to occur – thereby speeding up the rate of such reactions. Enzymes are critical factors for health and disease, helping cells maintain their delicate homeostasis. When mutations lead to over- or under-production in certain key enzymes, severe genetic diseases – some of them, lethal – can result.

Because of the central importance of enzymes for biological systems, researchers want to gain a better understanding of how normal enzymatic reactions occur and how they may go awry. Such knowledge may encourage the development of techniques to mimic cellular processes involved in enzyme regulation.

In the current study, the authors create a nanoscale tool designed to manipulate enzymatic reactions with fine-grained control. The group dubs their device a tweezer-actuated enzyme nanoreactor.

The clever design separates an enzyme and a cofactor essential for successful reactions on separate arms of the tweezer-like instrument. Enzyme function is inhibited when the tweezers are in their open position and the two molecules are held apart. Enzyme activation takes place when the tweezer prongs close, bringing enzyme and cofactor in contact. (The closing of the tweezers occurs when a specific DNA sequence is added, altering the thermodynamics of the system and causing a conformational change in the structure.)

The current study explores reactions in regulatory enzymes – multitasking entities that are important for modulating biochemical pathways. Regulatory enzymes, which can catalyze reactions over and over again, accomplish their feats by binding with biomolecular cofactors. (Hormone production and regulation are just one example of regulatory enzyme activity.)

In a series of experiments, the group was able to externally control the inhibition and activation of the enzyme through successive cycles. The authors stress that the nanoreactor tweezers could be used to regulate other types of enzymes and their control could be further refined by means of feedback and feed-forward loops.

Engineering nanostructures from the bottom up, using DNA as a construction material, affords researchers exacting control over the resulting geometry. Previously, Yan has created nanostructures in two- and three-dimensions, ranging from flat shapes to bowls, baskets, cages, Möbius strips and a spider-like autonomous walker.

In the tweezer design, a pair of 14 nm arms is connected at their ends by means of a 25 nucleotide single strand of DNA. This strand controls the opening and pinching of the tweezers, much the way a spring acts in a pair of gardening shears.

Two types of complementary sequence strands interact with this component, either forming a rigid DNA double helix, which supports the tweezers in their open position (set strands), or disabling the structural support and closing the tweezers (fuel strands).

Two techniques were used to measure and analyze the resulting structures with nanoscale precision: Fluorescence Resonance Energy Transfer (FRET) and Atomic Force Microscopy. Experiments demonstrated a high yield for enzyme-bound tweezers and successful switching between open and closed states was observed. The use of FRET allowed the process to be monitored in real time.

Lengthening the cofactor linker dangling from one of the tweezer’s arms enhanced successful opening and closing of the enzyme tweezers. Analysis revealed a 5-fold increase in enzymatic activity in the closed state, compared with the open state. The study also demonstrated durability in the tweezers, which were able to cycle between the open and closed positions nine times without losing structural integrity. The process was only limited by the accumulation of set strands and fuel strands.

Future work will explore similar responsive enzyme nanodevices capable of selective chemical amplification, with potentially broad impacts for medical diagnostics. Nanoreactors may also be applied as precision biocatalysts for the production of useful chemicals and smart materials.

In addition to his position in Biodesign’s Center for Single Molecule Biophysics, Hao Yan holds the Milton D. Glick Distinguished Chair in Chemistry and Biochemistry in ASU's College of LIberal Arts and Sciences.

Richard Harth

Science writer, Biodesign Institute at ASU

480-727-0378

Clues about autism may come from the gut


July 3, 2013

Bacterial flora inhabiting the human gut have become one of the hottest topics in biological research. Implicated in a range of important activities – including digestion, fine-tuning body weight, regulating immune response and producing neurotransmitters that affect brain and behavior – these tiny workers form diverse communities. Hundreds of species inhabit the gut, and although most are beneficial, some can be very dangerous.

In new research appearing in the journal PLOS ONE, a team led by Rosa Krajmalnik-Brown, a researcher at Arizona State University’s Biodesign Institute, presents the first comprehensive bacterial analysis focusing on commensal or beneficial bacteria in children with autism spectrum disorder (ASD). Download Full Image

After publishing earlier research exploring crucial links between intestinal microflora and gastric bypass, Krajmlanik-Brown convinced James Adams, director of the ASU Autism/Asperger’s Research Program, that similar high throughput techniques could be used to mine the microbiome of patients with autism. Previously, Adams had been studying the relationship between the gut microbiome and autism using traditional culturing techniques.

“One of the reasons we started addressing this topic is the fact that autistic children have a lot of GI problems that can last into adulthood,” Krajmalnik-Brown  says. “Studies have shown that when we manage these problems, their behavior improves dramatically.”

Following up on these tantalizing hints, the group hypothesized the existence of distinctive features in the intestinal microflora found in autistic subjects compared to typical children. The current study confirmed these suspicions and found that children with autism had significantly fewer types of gut bacteria, probably making them more vulnerable to pathogenic bacteria. Autistic subjects also had significantly lower amounts of three critical bacteria: Prevotella, Coprococcus and Veillonellaceae.

Krajmalnik-Brown, along with the paper’s lead authors Dae-Wook Kang and Jin Gyoon Park, suggest that knowledge gleaned through such research may ultimately be used both as a quantitative e diagnostic tool to pinpoint autism and as a guide to developing effective treatments for ASD-associated gastrointestinal (GI) problems. The work also offers hope for new prevention and treatment methods for ASD itself, which has been on a mysterious and rapid ascent around the world.

A disquieting puzzle

Autism is defined as a spectrum disorder due to the broad range of symptoms involved and the influence of both genetic and environmental factors; features that often confound efforts at an accurate diagnosis. The disease’s prevalence in children exceeds juvenile diabetes, childhood cancer and pediatric AIDS, combined.

Controversy surrounds the apparent explosive rise in autism cases. Heightened awareness of autism spectrum disorders and more diligent efforts at diagnosis must account for some of the increase, yet many researchers believe a genuine epidemic is occurring. In addition to hereditary components, Western-style diets and overuse of antibiotics at an early age may be contributing to the problem by lowering the diversity of the gut microflora.

In terms of severe developmental ailments affecting children and young adults, autism is one of the most common, striking about one in 50 children. The disorder, often pitiless and perplexing, is characterized by an array of physical and behavioral symptoms, including anxiety, depression, extreme rigidity, poor social functioning and an overall lack of independence.

To date, studies of the gut microbiome in autistic subjects have focused primarily on pathogenic bacteria, some of which have been implicated in alterations to brain function. One example involves gram-negative bacteria containing lipopolysaccharides in their cell walls, which can induce inflammation of the brain and lead to the accumulation of high levels of mercury in the cerebrum.

A new approach

Krajmalnik-Brown and lead author Dae-Wook Kang are researchers in the Biodesign Institute’s Swette Center for Environmental Biotechnology, which is devoted to the use of microbial communities for the benefit of human and environmental health. Their new study is the first to approach autism from a different angle, by examining the possible role of so-called commensal or beneficial bacteria.  

Up to a quadrillion bacteria inhabit the human intestine, contributing to digestion, producing vitamins and promoting gastrointestinal health. Genes associated with human intestinal flora are 100 times as plentiful as the body’s human genes, forming what some have referred to as a second genome. Various environmental factors can destabilize the natural microbiome of the gut, including antibiotics and specific diets.

In the current study, a cohort of 20 healthy and 20 autistic subjects between three and 16 years of age were selected, and their gut microflora from fecal samples analyzed by means of a technique known as pyrosequencing. Pyrosequencing is a high-throughput method, allowing many DNA samples to be combined, as well as many sequences per sample to be analyzed.

Lower diversity of gut microbes was positively correlated with the presence of autistic symptoms in the study. The authors stress that bacterial richness and diversity are essential for maintaining a robust and adaptable bacterial community capable of fighting off environmental challenges. “We believe that a diverse gut is a healthy gut,” Krajmalnik-Brown says.

The new study detected decreased microbial diversity in the 20 autistic subjects whose fecal samples were analyzed. Specifically, three bacterial genera – Prevotella, Coprococcus and Veillonellaceae – were diminished in subjects with autism when compared with samples from normal children. Surprisingly, these microbial changes did not seem directly correlated with the severity of gastrointestinal symptoms.

The three genera represent important groups of carbohydrate-degrading and/or fermenting microbes. Such bacteria could be critical for healthy microbial-gut interactions or play a supportive role for  a wide network of different microorganisms in the gut. The latter would explain the decreased diversity observed in autistic samples.

Bacteria: in sickness and in health

Among the fully classified genera in the study, Prevotella was the most conspicuously reduced in autistic subjects. Prevotella is believed to play a key role in the composition of the human gut microbiome. For this reason, the group undertook a sub-genus investigation of autistic subjects. They found that a species known as Prevotella copri occurred only in very low levels in the autistic samples. The species is a common component in normal children exhibiting more diverse and robust microbial communities.

“We think of Prevotella as a healthy, good thing to have,” Krajmalnik-Brown notes. (Michael Pollan’s recent New York Times Magazine story on the microbiome points to the fact that he is proud that his gut microbiome is rich in Prevotella regarding it as a possible sign of a healthy non-Western diet. )

Jin Gyoon Park, the other lead author, who works in the Virginia G. Piper Center for Personalized Diagnostics under the direction of Joshua LaBaer, conducted a rigorous bioinformatic and statistical analysis of the intestinal microflora. He believes that the microbiome can be mined in future work to find diagnostic biomarkers for autism and many other diseases. Quantitative diagnoses of this sort have so far been lacking for autism, a disease for which subjective behavior indices are typically used to identify the disorder.  

In describing the next steps for the research group, Kang and Park point to more detailed, gene-level analyses aimed at probing bacterial function and further illuminating relationships between human health and the complexities of the microbiome. Additionally, the group will use the current results as a guide for new treatment studies for autism aimed at modifying bacterial composition in the gut.  

A new, interdisciplinary consortium (Autism Microbiome Consortium) has been formed to investigate the underpinings of autism and the gut microbiome, bringing together the combined skills of neurologists, psychiatrists, neuroimmunologists, epidemiologists, pediatricians, geneticists, biochemists, microbiologists and others.

In addition to Rosa Krajmalnik-Brown and James Adams, the group consists of: Jack Gilbert (University of Chicago); Catherine Lozupone (University of Colorado); Rob Knight (University of Colorado and HHMI); Mady Hornig (Columbia University); Sarkis Mazmanian (California Institute of Technology); Tanya Murphy (University of South Florida); Paul Patterson (California Institute of Technology); John Alverdy (University of Chicago); Janet Jansson (Lawrence Berkeley Lab); and KImberly Johnson (University of Colorado). 

Richard Harth

Science writer, Biodesign Institute at ASU

480-727-0378