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432 PARE AND HOBMAN

robust and specific silencing of the unc-22 gene after they introduced a 742

base pair segment of homologous RNA into Caenorhabditis elegans (Fire et al.,

1998). Because of their lengths, it was not economically feasible to chemically

synthesize these reagents for routine laboratory use. However, the dsRNAs can

be easily produced using T7 RNA polymerase in in vitro transcription reactions.

Long dsRNAs can be introduced into cells or organisms by microinjection or

transfection with cationic lipids. In addition, RNAi can be induced in nematodes

through ingestion of bacteria that express long dsRNAs (Timmons and Fire, 1998).

These reagents work very well for gene-silencing in well-characterized invertebrate

systems such as C. elegans and D. melanogaster. However, there are two major

limitations with the use of long dsRNAs. First, the RNA-dependent silencing is

inherently transient. Unless the target cell has a means to amplify the siRNAs

(e.g. RNA dependent RNA polymerase), they will be degraded or their effects lost

as a result of dilution after cell division. Second, this method is not suitable for

use in most mammalian cells because they activate the innate immune system.

Perhaps because the replication intermediates of many RNA viruses are double

stranded RNAs, the appearance of long dsRNAs (over 30 bp) in mammalian cells

can trigger a double-stranded RNA-dependent protein kinase (PKR)-dependent

anti-viral response. In this case, total cellular translation is inhibited following

phosphorylation of eIF2 (Manche et al., 1992; Minks et al., 1979). The exceptions

are cell lines that lack the ability to mount PKR or interferon responses (e.g.

embryonic cell lines).

4.2. Synthetic siRNAs

A major advance in the use of dsRNA for gene-silencing followed the realization

that Dicer cleavage products (siRNAs) are important effectors of RNAi (Bernstein

et al., 2001; Hammond et al., 2000). This led to the wide-spread use of chemically

synthesized siRNAs as silencing triggers. The rationale for this approach was that

synthetic siRNAs resemble Dicer cleavage products and that their incorporation into

RISC particles would direct the accurate targeting and destruction of homologous

mRNAs. The procedure involves transfection of 21–22 nt synthetic siRNAs into

target cells. Typically, the siRNAs share 100% sequence identity to a region of

the targeted mRNA, often within the coding region. These dsRNAs are synthesized

as two separate 21-mers that share 19 bp of sequence complementarity and form

19 bp of dsRNA with 2 nt 3’ overhangs. The separate 21-mers are then annealed

before transfection. Because of their length and the characteristic 3’ overhangs, the

annealed 21-mers mimic Dicer cleavage products and provide robust and specific

gene-silencing (Elbashir et al., 2001a). Since they are under 30 base pairs, trans-

fected siRNAs, in most cases, do not induce a PKR-dependent global shutdown

of cellular translation. The siRNAs can be purchased from commercial suppliers

at a reasonable cost and are very easy to use. As a result, many laboratories with

no prior knowledge of RNAi biology have been able to exploit RNAi as a gene-

silencing tool.

DICER: STRUCTURE, FUNCTION AND ROLE 433

There are, however, two significant drawbacks to this method. First, unlike

nematodes and plants, mammalian cells do not have a mechanism to amplify

the siRNA triggers and, therefore, sustained gene-silencing can only be achieved

through repeated transfection with siRNA. Second, different siRNAs directed

against the same mRNA often exhibit different silencing efficiencies. This problem

has been alleviated in large part by the use of algorithms for design and selection

of siRNAs. A number of these programs are freely available online. One need only

enter the nucleotide sequence or genebank accession number of the mRNA into the

algorithm and a number of candidate siRNA sequences are provided.

Based on the canonical RNAi pathway, 21–22 nt siRNAs are not expected to

require the action of Dicer to effect gene-silencing. However, in addition to its well

recognized role in initiation of RNAi, evidence points to a downstream role for Dicer

in RISC function (Pham et al., 2004). Indeed, down-regulation of Dicer appears to

decrease the efficiency of siRNA-mediated gene-silencing in mammalian cells (Doi

et al., 2003). It is also interesting to note that slightly longer siRNAs (25–30 nt) are

reportedly up to 100-fold more potent than 21-mers of similar sequence (Kim et al.,

2005). Given that Dicer and Argonaute proteins interact in the context of a RISC,

it was hypothesized that the longer siRNAs, which are substrates for Dicer, are

better silencing reagents because the production of 21 nt siRNAs by Dicer is now

directly coupled to their loading onto the Argonaute subunit of RISC. Thus, for a

slight increase in the cost of the siRNAs, researchers can benefit from a significant

increase in gene-silencing efficiency.

Finally, a third siRNA-based method involves the in vitro production of

transfectable pools of siRNAs. Long dsRNAs, synthesized in a T7 polymerase-

dependent reaction, are subjected to Dicer cleavage in vitro and the resulting pools

of siRNAs are transfected into target cells (Kawasaki et al., 2003).Recombinant

Dicer is available from a number of commercial suppliers. The major advantage

of “in vitro dicing” is that unlike using pre-designed chemically synthesized

siRNAs, this method does not require optimizing target sites within mRNAs.

4.3. Small Hairpin RNA (shRNA)

The third generation of RNAi-based gene-silencing reagents is based on the

use of plasmid or viral vectors for the production of Dicer substrates. Because

selectable markers can be included in the vectors, these reagents allow for prolonged

gene-silencing through the production of stable cell lines that express the Dicer

substrates. The first plasmid-based silencing systems typically employed RNA pol

III promoters for production of short hairpin RNAs (shRNAs). The shRNAs are

designed to encode inverted repeats of 19–29 bp separated by a 6–9nt loop. The

resulting transcripts fold into hairpin structures that resemble pre-miRNAs, which

are Drosha cleavage products (Fig. 3). Accordingly, these hairpins are recognized

as substrates for Dicer and concomitant cleavage by this enzyme yields 21–25 nt

products that are incorporated into RISCs. Alternatives to this scheme include

co-transfection of two plasmids that each encode one strand of a RNA duplex.

434 PARE AND HOBMAN

Recently, a new generation of shRNA-based vectors has been developed and

these reagents appear to be significantly more potent than first generation shRNA

reagents (Dickins et al., 2005; Silva et al., 2005). These new vectors encode

shRNAs that resemble pri-miRNAs, as opposed to the first generation vectors that

encode pre-miRNA-like transcripts. Therefore rather than serving directly as Dicer

substrates, the pri-miRNA-like silencing triggers are first substrates for Drosha,

followed by a Dicer-dependent processing step. Through coupling of the Drosha and

Dicer processing steps, the pri-miRNA-like shRNAs are reportedly up to 12 times

more potent than comparable first generation shRNAs (Silva et al., 2005). Making

life even easier for scientists is the fact that a number of libraries containing pre-

designed shRNA constructs, which cover most of the human and mouse genomes,

are now available (Silva et al., 2005). Finally, because the pri-miRNA-like shRNAs

can be expressed from RNA pol II promoters, RNAi-dependent gene-silencing can

now be tightly regulated. For example, tetracycline-responsive promoters have been

used to drive the expression of pri-miRNA-like shRNAs in both cultured cells and

mice (Dickins et al., 2005). The creation of inducible RNAi tools adds a whole new

dimension to this technology. Specifically, by introducing tissue-specific promoters,

one can limit knock down of genes to tissues of interest. In addition, by fine tuning

the level of knock down, it should be easier to study genes that are critical for

viability.

ACKNOWLEDGEMENTS

This work was funded by grants from the Canadian Institutes of Health Research,

the Natural Sciences and Engineering Research Council and the Cancer Research

Society. T.C.H. is the recipient of a Medical Scientist award from the Alberta

Heritage Foundation for Medical Research.

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SECTION E

OXIDOREDUCTASES AND OTHER ENZYMES

OF DIVERSE FUNCTION

SECTION E

OXIDOREDUCTASES AND OTHER ENZYMES

OF DIVERSE FUNCTION

CHAPTER 25

HYDROGEN PEROXIDE PRODUCING

AND DECOMPOSING ENZYMES: THEIR USE

IN BIOSENSORS AND OTHER APPLICATIONS

NÓRA ADÁNYI

, TERÉZ BARNA

, TAMÁS EMRI

, MÁRTON MISKEI

AND ISTVÁN PÓCSI

3∗

Central Food Research Institute, Budapest,

Department of Biochemistry and

Department of Microbial Biotechnology and Cell Biology, Faculty of Science, University of Debrecen,

Debrecen, Hungary

∗

istvanpocsi@yahoo.com

1. INTRODUCTION

In this chapter H

-producing enzymes including glucose, galactose and choles-

terol oxidases will be presented together with the H

decomposer catalase.

Industrial production of these redox enzymes is increasing rapidly owing to their

established use in the construction of biosensors and novel applications, e.g. in

the food and textile industries. We will cover three research areas to give a broad

overview of these very important and remarkable enzymes, namely (i) studies

on their structure and mechanism of action, (ii) the construction of oxidase- and

catalase-based biosensors and (iii) novel applications.

2. ENZYME STRUCTURE AND MECHANISM OF ACTION

2.1. Glucose Oxidases

Glucose oxidase (-D-glucose:oxygen 1-oxidoreductase, GOx, EC 1.1.3.4.) is

a flavoprotein which catalyses the oxidation of -D-glucose by O

to -

gluconolactone and H

(Fig. 1). The -gluconolactone subsequently hydrolyses to

gluconic acid. Industrial production of GOx is mainly carried out using Aspergillus

niger or Penicillium amagasakiense (Crueger and Crueger, 1990), however other

Penicillium and Aspergillus spp. including P. notatum, P. funiculosum, P. piceum,

441

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 441–459.