The genesis of microarrays.

This review provides a perspective on the initial development of microarray technologies by two independent groups in the late 1980s.

Reactive trityl derivatives: stabilised carbocation mass-tags for life sciences applications.

The rational design of novel triarylmethyl (trityl)-based mass tags (MT) for mass-spectrometric (MS) applications is described. We propose a "pK(R+) rule" to correlate the stability of trityl carbocations with their MS performance: trityls with higher pK(R+) values ionise and desorb better. Trityl blocks were synthesised that have high pK(R+) values and are stable in conditions of MS analysis; these MTs can be ionised by matrix as well as irradiation with a 337 nm nitrogen laser. (13)C-Labelled tags were prepared for MS quantitation applications. Moreover, the tags were equipped with a variety of functional groups allowing conjugation with different functionalities within (bio)molecules to enhance the MS characteristics of the latter. The MS behaviour of model polycationic trityl compounds with and without the matrix was studied to reveal that poly-trityl clusters are always singly charged under the (MA)LDI-TOF conditions. Several peptide-trityl conjugates were prepared and comparisons revealed a beneficial effect of trityl tags on the conjugate detection in MS. Trityl compounds containing para-methoxy- and dimethylamine groups, as well as a xanthene fragment, showed considerable enhancement in MS detection of model peptides; thus they are promising tools for proteomic applications. Dimethoxytrityl derivatives allow one to distinguish between Arg- and Lys-containing peptides. Maleimido trityl derivatives are suitable for the efficient derivatisation of thiol-containing peptides in pyridine.

Novel mass tags for single nucleotide polymorphism detection.

A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption/ionization of the cleavable labels. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study. Corresponding reagents compatible with automated oligonucleotide synthesis are presented. Utility of this method for SNP genotyping is demonstrated.

Multiplex target protein imaging in tissue sections by mass spectrometry--TAMSIM.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) is becoming a popular tool for imaging histological sections. Currently, this technology is used to image naturally occurring molecules. Here we report a novel development for multiplex imaging of candidate proteins. Rather than detecting whatever molecules happen to be present and above the detection threshold in the desorption pixel, we attach photocleavable mass tags to antibodies to target proteins. 'Staining' of histological sections is carried out similarly to common immunohistochemical procedures with chemiluminescent or fluorescent detection using all antibodies of a multiplex simultaneously. Mass tags with discrete masses are released from their respective antibodies by a laser pulse at 355 nm without added matrix. After scanning, mass spectrometry images are created for the mass of each tag. In contrast to fluorescent tags, mass tags do not exhibit mutual quenching. Sections of healthy human pancreatic tissue were imaged to visualize synaptophysin in neuroendocrine cells, and sections from human lymph node and liver invaded by metastatic melanoma to localize the cancer markers PS100 and HMB45 simultaneously. All these proteins are below the detection threshold of direct MALDI-MS imaging. This method is termed TAMSIM for TArgeted multiplex Mass Spectrometry IMaging.

Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication.

We demonstrate a new method for making oligonucleotide microarrays by synthesis in situ. The method uses conventional DNA synthesis chemistry with an electrochemical deblocking step. Acid is delivered to specific regions on a glass slide, thus allowing nucleotide addition only at chosen sites. The acid is produced by electrochemical oxidation controlled by an array of independent microelectrodes. Deblocking is complete in a few seconds, when competing side-product reactions are minimal. We demonstrate the successful synthesis of 17mers and discrimination of single base pair mismatched hybrids. Features generated in this study are 40 mum wide, with sharply defined edges. The synthetic technique may be applicable to fabrication of other molecular arrays.

S(O)-pixyl protecting group as efficient mass-tag.

We report herein the design, preparation and first applications of novel trityl tags with adjustable stability, efficient as protecting groups or MS analytes.

In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication.

In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 microm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling. High, reproducible yields were achieved for both 6mer and 21mer probes as assessed by hybridization to fluorescent oligonucleotides. Oligonucleotide surface density was comparable with that obtained on glass substrates. These results suggest PDMS as a stable and flexible alternative to glass as a suitable substrate in the fabrication and synthesis of DNA microarrays.

Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy.

We show that binding of an antisense oligonucleotide can lead to considerable changes in the target mRNA structure. The approaches described here are not only useful in the study of intra-molecular interactions in RNAs but can also be used to design oligonucleotides that facilitate binding of other antisense reagents. Such "cooperatively acting" antisense reagents have the potential to overcome several problems faced in their use, for example, low efficacy and non-specificity. To provide proof-of-principle, radiolabelled cyclin B5 transcript, a model mRNA, was hybridised with an antisense oligonucleotide array. An oligonucleotide sequence was selected from the array hybridisation data and was used in an RNase H/oligonucleotide library (dN12) assay to assess its ability to enhance cleavage of target RNA. This oligonucleotide ("facilitator") greatly enhanced cleavage of B5 RNA at a neighbouring site. The precise position and sequence of this "new" site was determined by further hybridisation of RNA-facilitator mixture to the B5 antisense array. Antisense oligonucleotides designed from the new region were used in combination with the facilitator in a cell-free system. The presence of the facilitator considerably enhanced cleavage of B5 RNA with these oligonucleotides. These approaches may be useful in designing antisense reagents against sequences of specific interest, such as, gene fusion sites, splice variants, mutant alleles and tightly structured RNA sites.

A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides.

The optimal design of hybridisation-competent antisense oligonucleotides (ODNs) coupled with an efficient delivery system appear to be important prerequisites for the successful use of antisense reagents for gene silencing. We selected an antisense ODN complementary to an accessible region of the epidermal growth factor receptor (EGFR) mRNA with the aid of an antisense oligonucleotide scanning array. The scanning array comprised 2684 antisense ODN sequences targeting the first 120 nts in the coding region of EGFR mRNA. The array-designed antisense ODN was covalently conjugated to a novel anionic dendrimer using a pentaerythritol-based phosphoroamidite synthon via automated DNA synthesis and the ability of this conjugate to effectively deliver and down-regulate EGFR expression in cancer cells was evaluated. Each dendrimeric structure had nine ODN molecules covalently linked to a common centre at their 3' termini. This dendrimer conjugate was markedly more stable to serum nucleases compared to the free ODNs and the cellular uptake of ODN-dendrimer conjugates was up to 100-fold greater as compared to mannitol, a marker for fluid phase endocytosis, and up to 4-fold greater than naked ODN in cancer cells. ODN-dendrimer uptake was energy-dependent and mediated, at least in part, via binding to cell surface proteins; a process that was inhibited by self-competition and by competition with free ODN, salmon sperm DNA, heparin and dextran sulphate. Fluorescent microscopy studies showed a combination of punctate and more diffuse cytosolic distribution pattern for fluorescently labelled ODN-dendrimer conjugate in A431 cells implying internalization by endocytosis followed by release and sequestration of the conjugate into the cytosol. Little or no conjugate appeared to be present in the nuclei of A431 cells. In vitro RNase H-mediated cleavage assays confirmed that covalently conjugated antisense ODNs in the dendrimer conjugate were able to hybridize and cleave the array-defined hybridisation target site within the EGFR mRNA without the need for ODN dissociation from the conjugate. In cell culture, ODN-dendrimer conjugates were effective in inhibiting cancer cell growth that correlated with a marked knockdown in EGFR protein expression. These data highlight a novel anionic dendrimer delivery system for gene silencing oligonucleotides that improved their biological stability, cellular delivery and antisense activity in cultured cancer cells.

Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.

A simple and cost-effective method for producing small interfering RNAs with high efficacy.

Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity. This approach is expensive due to the cost of chemical synthesis of RNAs. We report a technically simple, quick and cost-effective method for the production of siRNAs that makes use of in vitro transcription and deoxyribozyme digestion of the transcripts to produce the desired sequence and length. The method allows for several siRNAs to be produced in parallel at much reduced costs. The siRNAs produced with this method were tested in MDA-MB-231 human breast cancer cells for efficacy against the type 1 insulin-like growth factor receptor (IGF1R) mRNA and they caused dose-dependent inhibition of IGF1R expression comparable to that induced by chemically synthesised siRNAs of the same sequence. This method is also useful for producing long RNA fragments of defined length and sequence that may be difficult to synthesise chemically, and also for producing large quantities of RNAs for applications including structural studies and the study of interactions between RNA and other molecules, such as proteins, other nucleic acids and drugs.

The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.

The type 1 insulin-like growth factor receptor (IGF1R) is often overexpressed by tumors and mediates growth and apoptosis protection. We previously showed that antisense reagents complementary to the IGF1R translation start site enhance radio- and chemosensitivity and impair Atm function. However these agents induce relatively modest IGF1R down-regulation and affect insulin receptor levels. To identify alternative sites for molecular targeting, we utilized scanning oligonucleotide arrays to probe the secondary structure of IGF1R mRNA. This strategy enabled selection of antisense oligonucleotides that generated high heteroduplex yield with IGF1R but not insulin receptor transcripts. Antisense oligonucleotides that hybridized strongly to IGF1R mRNA caused IGF1R down-regulation within intact tumor cells, whereas weakly hybridizing oligonucleotides were inactive. Furthermore, the ability of small interfering RNAs (siRNAs) to block IGF1R expression correlated with the accessibility of the target sequence within the transcript. Thus, siRNAs corresponding to weakly hybridizing oligonucleotides caused minor IGF1R down-regulation, whereas siRNAs homologous to accessible targets induced profound sequence-specific IGF1R gene silencing, blocked IGF signaling, and enhanced tumor cell radiosensitivity. This indicates that secondary structure in the target transcript has a major effect on siRNA efficacy. These findings have implications for siRNA design and suggest that IGF1R-targeting agents incorporating this mode of action have potential as anticancer therapy.

An electrochemical redox couple activitated by microelectrodes for confined chemical patterning of surfaces.

Microelectrodes, printed as an array on the surface of a silicon chip, generate chemically active species in a solution of electrolyte held between the electrode array and a glass plate. The active species induce chemical change in molecules coupled to the surface of the glass plate, which is separated from the electrode array by a gap of several micrometers. This paper explores the nature and pattern of the induced chemical change. The patterning is discussed with respect to the electrolyte composition and the magnitude and duration of current applied to the microelectrodes. We show that under suitable conditions the active species is confined to micrometer-sized features and diffusion does not obscure the surface pattern produced.

Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than thermus thermophilus DNA ligase.

To mimic large numbers of nicked DNA duplexes we used a technique that produces nicked duplex DNA substrates by hybridization of complementary oligonucleotides, adjacent to an initiating primer, which are ligated together by a thermostable DNA ligase. Sequential ligation of nonanucleotides to this primary duplex results in the formation of polymers that can be analyzed by gel electrophoresis. The extent of polymerization is a measure of the efficiency of ligation. We determined the efficiency of ligation of nonanucleotides, using various length initiating primers, with three thermostable DNA ligases: Thermus thermophilus (Tth), Thermus scotoductus (Ts), and Rhodothermus marinus (Rm). Analysis of the effect of temperature for each ligase, and for each directing primer length, revealed that at 37 and 41 degrees C there was variation between ligase efficiency in the order Rm > or = Ts > or = Tth. The higher temperature of 46 degrees C was optimal for polymerization with each of the ligases and Rm ligase was the most efficient. Analysis of directionality of the ligations reactions suggests that for each of the Thermus ligases we tested, there was a bias to polymerization of nonanucleotides in a 5'-3' direction.