一、研究新知

Ten years of Methods

Nature Methods期刊選出近十年影響最大的十項生命科學技術
Our choice, among many candidates, of the ten areas of methods development with the most impact on biological research over the last decade. Visit Methagora to browse Nature Methods papers in some of these areas.

Next-generation sequencing
The advent of next-generation, or massively parallel, sequencing has affected nearly every aspect of biology, enabling scientists to sequence genomes, assess genetic variation, quantify gene expression, study epigenetic regulation, survey microscopic life and scale up countless assays and screens with relative ease. In addition to technological innovations that drive the relentless rise in sequence data quantity and quality, sequencing library construction has evolved to probe limited material or degraded samples, to flexibly target portions of a sequence space, to tag diverse molecules in the cell and to capture molecular interactions and genomic structure. Computational tools have been an indispensable aid for interpreting the tomes of data, revealing fundamental aspects of sequence variation, regulation and evolution.

Single-molecule methods
The study of the behavior of individual molecules such as protein or DNA provides powerful insights into biological mechanisms that cannot be gleaned by studying averaged molecular properties. Several single-molecule methods have matured over the past decade. These include force spectroscopy to probe the binding, folding or mechanical behavior of molecules as well as fluorescence microscopy to detect and track individual molecules in vitro or in the cell. Newer technologies such as nanopores promise the ability to sequence single molecules; optical and plasmonic devices can detect single molecules without labeling. These diverse methods allow a myriad of deep mechanistic investigations of molecular function.

Genome engineering
Tools for genome engineering allow customized alterations in cultured cells and in model and nonmodel organisms, vastly increasing the ease with which researchers can knock out genes, introduce mutations or make fusions to an endogenous gene product. In all such tools, a designed nuclease cleaves a genomic sequence of choice, initiating a repair process that results in the intended sequence changes. Meganucleases, zinc fingers and transcription activator–like effectors engage the target sequence via their respective DNA-binding domains. Most recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, which uses RNA instead of proteins to target the nuclease, has gained popularity for its ease of design and ability to alter almost any genomic sequence.

Light-sheet imaging
The old technique of light-sheet imaging is having a renaissance, largely because of substantial improvements in instrumentation, including microscopes and cameras, as well as better fluorescent probes and developments in image analysis. In this approach, a sample is illuminated with a thin sheet of light rather than with a point source or full field illumination. This means that the entire volume of three-dimensional biological objects can be rapidly imaged at high resolution and with relatively low phototoxicity. Scientists in neuroscience and developmental biology are applying light-sheet imaging to many organisms to study fundamental biological processes such as embryo development and brain function.

Mass spectrometry–based proteomics
Ten years ago, mass spectrometry–based proteomics was a relatively specialized field, eschewed by many traditional cell biologists. But rapid advances in the speed and robustness of mass spectrometry instrumentation, as well as tremendous development in sample preparation, experimental design and data analysis, have addressed many questions about data reproducibility and comprehensiveness and have allowed the field to blossom. Deep, quantitative proteome profiling of cellular state, which used to take days of instrumental time, is now possible in just a few hours. This enables researchers to investigate protein function on a systems scale, such as by profiling protein post-translational modifications and protein interactions.

Optogenetics
Cellular behavior can be altered non-invasively by illuminating light-sensitive proteins that are ectopically introduced into the cells. Optogenetic tools are particularly popular in neuroscience, where they are used to activate or inhibit neural activity with precise temporal and spatial control. These tools have been applied both in vivo and in vitro:for example, to study the function of individual cells in a neural ensemble or cellular properties such as neuronal excitability and synaptic transmission. In addition, light-sensitive tools are available to dimerize proteins or to activate transcription. Discovery of new light-sensitive proteins as well as ongoing improvements to existing
ones are expanding the optogenetic toolbox together with improved illumination procedures, including two-photon excitation and stimulation using patterned light.

Structural biology
The ability to solve the atomic structures of small, soluble proteins by X-ray crystallography is now nearly routine, thanks in part to structural genomics efforts aimed at optimizing all parts of the structure determination pipeline, from protein expression to crystallization. Building on these advances, researchers have recently been tackling the structures of challenging membrane proteins and large protein complexes, which are difficult to produce in quantity and resistant to crystallization. The last ten years have seen improved methods for sample preparation, crystallization and data analysis. Together with developments in alternative
technologies, including nuclear magnetic resonance spectroscopy and single-particle cryo-electron microscopy, and the rise of new technologies such as X-ray free-electron lasers, these improvements are enabling researchers to solve challenging biomolecular structures.

Synthetic biology
The ambitious goals of this field—from engineering microorganismal metabolic pathways for drug and biofuel production, to creating synthetic organisms, to equipping mammalian cells with new capabilities—have been met in part owing to improvements in experimental and computational methods. Advances in gene synthesis and assembly have yielded synthetic bacterial genomes and yeast chromosomes. Better circuit design is enabled by the characterization of regulatory elements that control transcription and translation and by software to aid circuit assembly. Models to predict how building blocks can be combined to reach a desired output are being continually developed and will be the foundation for synthetic biology’s success in the next decade.

Cellular reprogramming
The discovery that cells from the body can be turned back in time and rendered
pluripotent has stretched imaginations across many research disciplines. The resulting
induced pluripotent stem cells (iPSCs) can be expanded to large numbers and in principle used to make cells of any type, which can then be used to study normal and disease biology and to screen for drugs. Because iPSCs are created with standard techniques, the ability to generate human cell types with specific genetic backgrounds is now within reach for many laboratories, though good differentiation methods are still being developed for most cell types. This discovery
has also resulted in renewed attention to so-called direct reprogramming, in which exogenous transcription factors are used to change somatic cell fates.

Super-resolution microscopy
For centuries, the ‘diffraction limit’ of light microscopy was considered inviolable: it was
thought to be impossible to resolve structures that are closer together than roughly half the wavelength of the illuminating light. Several ways to ‘break’ this diffraction limit, collectively called super-resolution microscopy or nanoscopy, have been developed and applied to biology within the last decade. This means that tiny objects within cells—organelles or even macromolecular complexes—can now be discriminated, whereas previously they appeared as an unresolved blur. Methods development, particularly in the analysis of super-resolution data, continues fast and furious, but these techniques have already opened up entirely new vistas for scientists studying molecules and cells.

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