EMERGING TECHNOLOGIES FOR FUNCTIONAL GENOMICS

Functional genomics technologies are in rapid development, particularly those which apply genomic information by investigating large sets of molecules (DNA, RNA, proteins) in numerous samples of different origins (fluids, extracts, cells, tissues) using a widening range of tools. Those which are currently at the cutting edge include advanced array technologies, nanobiotechnology, single cell and single molecule detection, and gene knockdown by RNAi. They will be crucial to diagnose and treat cancer and other conditions, define new drug targets for the pharmaceutical industry and monitor the environment.

Advanced array technologies

Recent years have witnessed intense growth of different microarray techniques for high throughput analyses of genes, transcripts and proteins. They are already of strategic value throughout biological research as well as for industry and should also become available in clinical medicine to guide diagnosis and therapy. DNA arrays are used for genotyping and expression analysis at the transcriptional level; antibody arrays are being introduced for expression analysis at the protein level; other protein arrays are making their mark in protein interaction analysis; cell arrays enable protein function to be analysed in an in vivo setting, while tissue arrays take this one stage further and investigate the distribution of proteins within normal and diseased tissues. Nanotechnology allows arrays to be fabricated down to the level of resolution of individual molecules (see below). A major challenge in microarray technology development is in the analyses of individual genomes and proteomes. Methods for genome resequencing have the potential to become important in routine healthcare, agriculture and environmental monitoring. Applications in biomedicine will be seen in identification of genetic variation underlying common diseases, monitoring and ultimately explaining the molecular processes involved, assisting in routine checkups and diagnosis, and becoming part of the ‘predictive, preventive and personalised' revolution in medicine. The following are examples where micorarrays can make a major contribution.

Genome resequencing With the human and many other genome sequences completed, the challenge now is to rapidly and inexpensively search genomes for alterations that could explain individual phenotypic variation, particularly disease. A current ambition is to achieve a ten-fold reduction in cost and similar increase in speed and throughput, along with a substantially higher accuracy in the identification of variations. Methods in development for array-based resequencing include oligonucleotide fingerprinting, nanoliter-scale hybridizations in nanowells, iFRET technology to detect hybridisation and MALDI mass spectrometry to measure oligonucleotide composition. A high throughput “resequencing chip” may become a highly competitive tool, which would open the prospect of genome-wide sequence evaluation of patient material as a routine clinical diagnostic procedure, pathogen identification, etc.

Genotyping New genotyping technologies will enable whole-genome association studies for complex disease gene mapping. Several million common SNPs are known for the human genome, potentially allowing inheritance to be traced for factors that predispose to common conditions. The international HapMap programme will report the most frequent haplotypes in a number of human populations, further increasing the opportunity to identify important genes, given sufficiently efficient analytical methods. Further development of bioinformatics tools will be required for SNP based genotyping, haploblock analysis and haplotype tag SNP selection. Greatly increasing throughput and precision while reducing cost will also have a profound impact on applications of genotyping in DNA diagnostics and pharmacogenomics.

Transcriptome analysis Monitoring the activity of a genome by measuring mRNA expression levels provides important biological insights. Recent clinical studies of cancer and cardiovascular diseases demonstrate that expression analysis of large gene sets can identify molecular profiles correlated to disease states, which may then be developed as diagnostic tools. Very often, however, the molecular characterisation of clinical samples is complicated and limited by the available amount of samples. Strategies to overcome this problem are amplification of the starting material or of the signal to be detected, or miniaturisation of the method. Novel means are also required to measure gene expression with allele-specific and splice-variant-specific profiles. Such technologies promise to be a further big step from bench to bedside.

Protein analysis Recent years have seen a flurry of interest in protein detection on microarrays. This is clearly of central importance, since proteins are the main effector molecules, whose levels and modifications mediate cellular responses to the environment. Heterogeneity and relative instability of proteins make this a demanding undertaking. Current research concentrates both on protein microarray construction and molecular strategies for specific and sensitive detection; antibody arrays will be used for protein expression studies and as diagnostic and discovery tools in autoimmunity; whole proteome chips allow high throughput array-based characterisation of functional protein interactions. New tools for highly specific, sensitive, parallel protein analyses both in body fluids and tissue extracts will make a profound impact on clinical diagnostics.

Functional cell microarrays Another developing area is the high throughput technology for assessing gene function in living cells, leading to cell biological studies on a genomic scale. Typically, transcriptome and proteome profiling methods generate results that are correlative and descriptive. The present challenge is to identify direct causalities, such as the effects of gene expression and networks in living cells. Cell arrays are produced by spotting cDNAs onto coated microscope slides, which are covered with suspensions of adherent growing cells to be transfected. Of particular interest are their use with high throughput RNAi-based methods using siRNA transfection and single-cell imaging techniques to analyse knockdown phenotypes in mammalian cells. The ability to explore, in high throughput, the effects on cellular phenotypes and signalling is of fundamental importance in biology, and at the centre of attention by the biopharmaceutical industry.

Tissue micorarrays To extend functional genomics analysis to the next level of organisation, tissue microarrays (TMAs) are becoming increasingly used high throughput tools. They are prepared by drilling out cores from selected regions of paraffin embedded tissue and assembly into an array of several hundred individual sections. TMAs were originally introduced for high throughput molecular profiling of tumour specimens, their dominant use to date, but are equally useful for the analysis of gene expression across tissues and in cells within the individual tissue. They can be used to survey hundreds of clinical specimens in a single experiment using DNA, RNA, peptide, protein or antibodies as probes. As an example, in a project supported by 30M€ from the Wallenberg foundation, TMAs are being used in a systematic immunohistochemical analysis of human protein expression using antibodies against each individual ORF, which aims to produce an atlas of 5000 proteins or 20% of the human proteome within 5 years (The Swedish Human Proteome Resource Programme, www.hpr.se). TMAs will similarly be an indispensable element in the high throughput analysis of the tissue transcriptome.

Nanobiotechnology: single cell and single molecule analysis

Nanotechnology in general involves the manipulation of material at the atomic or molecular scale and the creation of useful materials at this scale, while nanobiotechnology is an interdisciplinary collaboration between life scientists, physical scientists and engineers to construct technological platforms for biochips, proteomics, and cell analysis. While current analyses typically yield statistical measures across time, involving large numbers of cells and millions of molecules, elucidation of biological function will require targeting of individual cells and even individual DNA, RNA or protein molecules. The goal of nanobio-technology is to develop high speed analytical approaches exploiting nanofabrication to engineer structures approaching the single molecule level for spatial addressing, manipulation, imaging and detection of biomolecules and cells. Microfluidics and nanotechnology are set to transform many analytical techniques in biology and medicine. In regard to the latter, developing any technology intended for clinical sample analysis will require the miniaturisation, integration and automation, which in turn will lead to more sensitive and cost-effective analyses. The fabrication of micro- and nanoscale devices for assessing biological processes - an area of development that has advanced rapidly over the past five years - will be the key to achieving these goals.

Coupling of microfluidics, using nano- and picolitre volumes, with molecular separation and analytical methods in a form that can be miniaturized in ‘chip-like' formats (‘lab-on-a-chip' devices) allows high throughput quantitative analysis of dynamic bioprocesses. The combination of micro- and nanoscale fabrications, unique surface chemistries and biological components provides novel structural frameworks for experimental work, allowing previously unattainable control over spatial and temporal distributions of soluble or fixed biological components, as well as the capability to measure dynamic events within very brief time frames. Microfluidic circuits can be designed to accommodate a number of analytical biochemical applications and to support parallelized, high-throughput screening. Given the dimensions of these devices, they will be suitable for single-cell analysis. Representative functional genomics applications that will be carried out at the single cell level to obtain multiple measurements using a single device include protein and/or gene expression analyses, protein-protein or protein-DNA interaction studies and electrophysiological recordings. The results will provide knowledge and devices that can advance drug development and increase opportunities for commercial transfer. As technologies mature, they will accommodate smaller sample volumes and will be more economical, in turn supporting personalized medicine. Emerging nanostructured materials will provide new and powerful tools for biology and healthcare. For instance, semiconductor nanocrystals, or quantum dots, in the size range of 2-8 nm, that are highly light absorbing over a broad spectral range, promise to transform in vitro imaging. They can be linked to biological molecules such as peptides, proteins, or nucleic acids and are emerging as a preferable class of biological label with properties superior to traditional dyes and fluorescent proteins.

Nanoarrays To detect single molecules and their interactions on arrays, micro- and nanofabricated tools are being applied in conjunction with high resolution optical and electrochemical imaging techniques. Ultraminiaturised versions of the traditional microarray that can measure interactions between individual molecules to a resolution of one nanometre are being devised, e.g. the technique of dip-pen nanolithography, which ‘writes' molecules on a surface by using the tip of an atomic force microscope coated with ‘molecular ink', is being used to make arrays of proteins with features more than 1,000 times smaller than those used in conventional arrays. Antibodies and other proteins have been immobilised on nanoarrays, using conventional optical fluorescence, mass spectrometry or atomic force microscopy as readout options. This allows very small quantities of individual proteins to be effectively screened against a large set of drug or diagnostic targets. A novel concept is the use of microcantilevers to study molecular interactions; when biomolecular interactions take place on one surface of a microcantilever beam, the nanomechanical forces of the interaction cause the cantilever to bend by 10-20 nanometers, which can be detected by lasers. While at a very early stage, there is impetus for creating microcantilever arrays. Chemical sensors have been described based on single-walled carbon nanotubes or semiconductor nanowires, in which an antibody or single strand of DNA is attached such that binding of its cognate molecule leads to measurable changes in the conductivity of the nanowire. Measurements are taken in real time and do not require any modifications or reporter groups so that rapid physiological processes (approximately 0.1 s) can be captured. Such a detection device has the potential to be ultrasensitive, down to single molecules, and could be constructed in parallel arrays. One concept is of ‘nanolaboratories', each capable of analyzing single cells and their contents, which can be integrated together with a microfluidics device that will import cells, thus integrating biology and nanotechnology into the systems biology approach.

RNA interference: gene knockdown for functional genomics
Gene functions can be revealed by loss of function assays, of which gene targeted deletion is one method and knockdown by modulation of mRNA turnover is another. The recently introduced RNA interference (RNAi) is a particularly effective mechanism for selective inhibition of gene expression which, in a very short time, has become the preferred method for inhibiting expression of targeted genes. As well as functonal genomics applications, it also shows tremendous potential for diagnostics and therapeutics .

The RNAi phenomenon is activated when dsRNA molecules enter the cell, causing the degradation of ssRNA of identical sequence, including endogenous mRNA. This occurs after cleavage of the dsRNA into small interfering (si) RNAs, 21-23 nt in length. In mammalian cells, where the use of large dsRNA is prevented by the protein kinase interferon response, the introduction of small 21-23 nt sequence-specific RNA duplexes can initiate post-transcriptional gene knockdown. RNAi libraries covering the entire genome are being developed to provide a functional validation of gene targets and potential RNA-based therapeutics. The ease and speed of RNAi as an experimental technique have made it feasible to rapidly determine the loss of functional phenotypes for large numbers of genes. For example, libraries of 12,000 different dsRNAs have been used to screen C. elegans for genes involved in obesity and ageing, by inhibiting the function of approximately 86% of the ~19,000 predicted nematode genes. Many of the newly identified fat-regulatory genes in the worm have mammalian homologues, some of which are known to function similarly. Synthetic RNAi molecules are expensive and only inhibit gene expression temporarily. Therefore vector systems have been designed, such as pSUPER from the Netherlands Cancer Institute, that direct expression of siRNAs in mammalian cells, making it possible to study loss of function phenotypes over longer periods of time. There are also issues regarding target selection and the design of both RNAi fragments and experimental conditions. When fully elucidated, the RNAi pathway will provide further scope for manipulation.

Recently, RNAi has been combined with cell microarray systems for screening. Up to 10,000 spots of siRNA oligos are arrayed on a slide and overlaid with cells; phenotypic effects are quantified by sensitive, digital image analysis at a single cell level with special instrumentation, and the readouts include fluorescence, cell survival and apoptosis, immunohistology and calcium release, allowing analysis of the impact of thousands of genes on many cell biological functions in a single experiment. This high throughput array-based cell biology is an excellent tool for systems biology, enabling a systematic gene function analysis and development of mathematical models of how the cells actually work.