1. Review of Literature

Chapter 1

Chapter 1: Review of Literature

1.1 Introduction

RNA shows remarkable structural and functional versatility, despite of containing

only four different chemical subunits. RNA folds into a variety of complex tertiary

structure analogous to structured proteins and catalyzes a fairly broad range of

chemical transformation (1). Traditionally the nucleic acids were thought to be there

for storing the genetic information and their translation but as the knowledge about

the structural complexity and flexibility of the nucleic acids increased, researchers

began to think of functional capability conferred on them by these structures. Here

evolves the concept of aptamers binding with cognate ligands based on structure.

Although they are common in nature only recent advances made it feasible to select

aptamers in vitro. In vitro evolution of molecules cognate to chosen ligands can be

viewed as a model of RNA evolution in the RNA world and has been employed to

obtain many new functional RNA molecules like ribozymes, aptamers and

riboswitches (2).

Aptamers are biomolecule composed of peptides or nucleic acid (DNNRNA) that

bind tightly to a specific molecular target. Structure of the aptamers allows them to

bind tightly against the surface of their target ligand molecules. Aptamers show the

basic lock and key relationship with their binding partners. An enormous diversity of

molecular shapes exists within the universe of all possible nucleotide sequence.

Aptamers may be obtained for a wide array of molecular targets, including most

proteins, many small molecules and even entire organisms (3, 4). The surface area of

interaction between an aptamer and its molecular target is relatively large; so even

small changes in the target molecules can disrupt aptamer association. Thus an

aptamer can distinguish between closely related but non identical members of a

protein family or between different functional or conformational states of the same

protein. Selection of aptamers is performed by the technique SELEX (!ystematic

£_volution of ligands by gponential enrichment) from random sequence library of a

large population of nucleic acid sequences ( 4, 5).

1

Chapter 1

Glutathione is a tripeptide thiol made up of the amino acids gamma-glutamic acid,

cysteine and glycine and is the most abundant low molecular weight intracellular

thiol. The thiol group confers on it the function of maintaining the redox state of the

cell. Glutathione lacks the toxicity associated with cysteine ( 6), making this

compound suitable as a cellular thiol "redox buffer" to maintain cellular

thiol/disulfide redox potential. Many of the presently available anti cancer therapies

are limited in their application because of their low efficiency. There are various

mechanisms in the cell for the efflux of glutathione conjugates. The formation and

efflux of glutathione conjugates of anti-cancer drugs pertain to drug resistance of

tumour cells (7).

Cancer has become more and more prevalent over the last few years. Cancer is a

group of diseases characterized by the uncontrolled growth and spreading of abnormal

cells. This is related to dynamic changes in the genome. Cancer and normal cells are

known to respond differently to nutrients and drugs that affect glutathione status.

Tumor cells have elevated levels of glutathione, which confers resistance to

chemotherapeutic agents. One of the challenges of cancer therapy is to deplete

glutathione of tumor cells, so as to make them more vulnerable to the effects of

chemotherapeutic drugs (8, 9). Glutathione plays a critical role in cellular mechanisms

that result in cell death. It is also found that cancer cells resistant to apoptosis had

higher intracellular glutathione level. Many studies also show that lowering GSH

concentration may be convenient not only for the efficiency of chemotherapy, but also

to induce a rather fast and direct apoptosis mechanism in tumor cells (10, 11).

The association of elevated glutathione level with increased risk of relapse suggests

that glutathione depleting agents may be of therapeutic value in patients with high

WBC (12). Glutathione has been shown to affect cellular activation and proliferation,

especially in lymphocytes where modulation of glutathione levels can control T-cell

activation dependent proliferation. Apoptosis can be induced by depletion of

glutathione or conversely inhibited by elevation of glutathione levels. Evidence that

glutathione homeostasis may be more specifically involved in cell survival was

suggested by studies investigating regulation of proteins crucial to cell signaling, e.g.

the transcription factors AP-I and NF-kB (13). There is evidence that the DNA

binding capacity of these proteins is redox status dependent and can be modified by

change in intracellular glutathione concentration. More recently, attention has focused

2

Chapter 1

on the importance of the intracellular redox status in maintaining the integrity of

mitochondrial membrane. Disruption of the mitochondrial trans membrane potential

has been shown to . immediately precede the nuclear changes associated with

apoptosis. Depletion of GSH and disruption of the mitochondrial trans membrane

potential are early events (14).

Furthermore evidence of RNA's biological importance was obtained with the

completion of the human genome project that suggested a correlation between the

increased non-protein coding regions and the complexity of humans versus other

organisms. Many of these nonprotein coding regions are transcribed into RNAs that

have roles in the regulation of transcription and translation levels (15-17). From the

above research background we undertook the task of selection and characterization of

glutathione-binding RNA aptamers.

3

Chapter 1

1.2 RNA World

Our perception of the function of ribonucleic acids (RNA) has increased dramatically

since the original discovery of messenger RNA in 1961 (18). The RNA world

hypothesis assumes that modem life forms arose from RNA molecules. Today, DNA,

from which genetic information flows, is considered a modified RNA more stable

than RNA except under acidic conditions, and therefore more capable of conserving

and transferring information (19, 20). The RNA world hypothesis suggests that early

life went through a stage where RNA was responsible for both information storage

and catalytic function. The term RNA world was coined by W. Gilbert in 1986 (21,

22). The RNA world hypothesis requires that RNA is able to self replicate, catalyze

reactions and lead to the formation of peptide bonds. The discovery that the

ribosome's catalytic domain consists of RNA also confirms that RNA has the capacity

to catalyze the formation of peptide bonds (23). This discovery, along with artificially

created catalytic RNA, has provided significant support for the RNA world hypothesis

(24, 25). The existence of RNAs that are both catalysts and templates for replication

forms the basis of a consistent model for the origin of biochemical information. In the

simplest of these RNA world scenarios, RNA template- catalysts are presumed to

have been selected from pools of random sequence and length. SELEX serves as an

analogue for this presumably natural process of precellular molecular evolution and

selection (3). RNA populations of high complexity can be selected for ligand binding

or enzymatic functions in vitro, suggesting that the RNA world could emerge through

the successive steps of variation, selection, and replication seen in other evolving

systems.

1.3 RNA Aptamers

Ellington and Szostak introduced the term "aptamer" (derived from the Latin word

"aptus", meaning "to fit") to describe these nucleic acid molecules. Aptamers are

relatively short sequences (typically 15-80 nucleotides) of single stranded DNA or

RNA whose particular three-dimensional folding confers on them the ability to bind

particular target molecules (3, 26). They were discovered in the early 1990's, and

were rapidly identified as valuable tools for a wide range of applications involving

biomolecular recognition ( 4, 27). Since 1990 aptamers have been generated against

hundreds of molecular targets (Fig 1 ). There has not been any restriction on the type

4

Chapter 1

of target for aptamer generation and these have been generated against targets

including, organic dyes, metal ions, drugs, amino acids, co-factors, aminoglycosides,

antibiotics, base analogs, nucleotides and peptides to numerous proteins oftherapeutic

interest like growth factors, enzymes, immunoglobulins, gene regulatory factors and

receptors. Beside all these, aptamers are also selected using intact viral particles,

pathogenic bacteria and whole cancer cell as target. Thus, they can be used in the

same way as antibodies, their binding being equally specific and with higher affinity

(28).

Figure 1: An FMN aptamer showing binding with its target molecule (Fan P eta!. 1996)

29.

Moreover some limitations inherent to the use of antibodies can be overcome by

aptamers . First, since aptamers are generated in vitro , they can be developed to target

virtually any protein, even toxins or non-immunogenic proteins, whereas antibody

generation is limited by the need to use animals . Neither can antibodies be modified

as they arise from a natural biological process, but aptamers can be selected according

to some desired properties. For example, aptamers can be generated that are able to

work in non-physiological conditions, or within certain kinetic parameters. Reporter

molecules, such as fluorescein or biotin, can also be easily attached to aptamers at a

precise location, thus avoiding problems related to inactivated antibody binding sites.

5

Chapter 1

It is clear that aptamers have a number of desirable characteristics for use as

therapeutics including high specificity and affinity, biological efficacy, and excellent

pharmacokinetic properties. In addition, they offer specific competitive advantages

over antibodies and other proteins. The starting point for the generation of an aptamer

is the synthesis of a nucleic acid library of large sequence complexity followed by the

selection for oligonucleotides able to bind with high affinity and specificity to a target

molecule. Randomizations of nucleotides have been used to create an enormous

diversity of possible sequences (4N different molecules while N= number of bases of

nucleotides in the oligos used) which in consequence generate a vast array of different

conformations with different binding properties and good probability to bind with the

chosen ligand. The recent development of in vitro selection and amplification

techniques and the ability of oligonucleotides sequences to assume a multitude of

shapes within a random sequence library allow harnessing this structural property of

nucleic acid to generate highly specific aptamers (4) . The secondary structure of

aptamers, consisting mainly of short helical arms and single stranded loops, is defined

by complementary base. Tertiary structure resulting from combinations of these

secondary structures, are stable and in combination with the property of nucleic acids,

form different non covalent bonds allowing aptamers to bind to targets by any or

mostly a combination of these forces like van der-Waals interactions, hydrogen

bonding, topological compatibility, stacking of aromatic rings, and electrostatic

interactions ( 30).

1.4 Designing Combinatorial Library of DNA and RNA

1.4.1 DNA Combinatorial Library

The random sequence pools consist of a region of random sequence flanked by

constant regions . The major factors which affect the pool design are:

•:• Type of randomization;

•:• Length of random sequence region;

•:• Chemistry of pool ;

6

Chapter 1

Three types of randomization can be employed: partial, segmental, and complete. Use

of partial random sequences depends on the knowledge of structural and functional

residues. It involves 'doping' mutations at constant rate into constant sequence. Thus,

an array of point mutations similar to nature are seen but at much higher frequencies.

Small nucleic acid segments of 30-50 residues can be completely randomized Table

1. The complete randomization provides a vast sequence space to be examined for

novel nucleic acid structures and functions. A short segment is completely

randomized while in segmental randomization, a compromise between partial and

complete randomization is made. This scheme has some advantages over partial and

complete randomization that it can reveal the role of "wild type" residues. It also

helps in identification of novel sequence or structural motifs which alter binding or

catalytic function. The length of random sequences depends on the function to be

explored. If a target has intrinsic non specific and weak affinity for nucleic acid then a

partial or segmental random sequence pool, of 30-60 nucleotides based on natural

ligands will yield aptamers. However, if a target is not known to bind nucleic acid

then a longer (~60 residues) and completely random pool will provide a better

opportunity for aptamer selection. The longer sequences are favored because they

allow greater structural complexity than the shorter sequences. They offer for a large

number of short structural motifs in addition to diverse and longer sequence motifs.

The ultimate complexity of population is limited by DNA synthesis chemistry to a

total of 1013-10 16 different sequences and not the shorter or longer random sequence

libraries for oligonucleotides longer than 20 residues. It is appreciated that with

increasing size of the DNA molecule, a larger mass would be needed to represent

each possible variant. This puts a practical limit on size of the pool employed for

SELEX (Table 1).

The chemistry of pool also affects the selection method. In vitro RNA selections can

also contain modified dNTPs. The T7 RNA polymerase can incorporate many

modified ribonucleotides such as 2 '-flouro and 2 '-amino dNTPs or phosphorothioate

nucleotides. The mutants can affect the chemistry which in tum can bias the course

and outcome of selection. Constant sequences are designed to ensure vigorous

amplification of all sequences in the population. The primers designed should anneal

specifically to the template without forming 'primer-dimer' or secondary structures.

20 nucleotide long primers are convenient for PCR and can be synthesized with good

yield. For generation of RNA aptamers T7 RNA polymerase promoter region

7

Chapter 1

incorporated in the 5' primer sequence are usually required. Restriction enzyme sites

are also incorporated in the non random defined sequences which help in generation

of aptamer library.

No. of MW, Maximum Minimal mass Maximum No nucleotides Daltons number of to include at of Variants

variants least one represented molecule of permg each variant (mg)

0 0 I 0 0 I 340 4 2.25801E-18 4 5 I700 1024 2.89025E-I5 1024

10 3400 1048576 5.91924E-12 1048576 15 5100 1073741824 9. 09195E-09 1073741824 20 6800 1.0995IE+ 12 1.24135E-05 1.09951E+ 12 25 8500 1.1259E+15 0.01588934 1.1259E+15 30 10200 1.15292E+ 18 19.52482043 5.9049E+16 35 11900 1.18059E+21 23325.65214 5.06134E+ 16 40 13600 1.20893E+24 27297677.48 4.42868E+16 45 15300 1.23794E+27 31446924458 3.9366E+16 50 17000 1.26765E+30 3.57796E+ 13 3.54294E+ 16

Table 1: The calculation of maximum number of sequence variants per mg of

oligoribonucleotides

1.4.2 RNA Combinatorial Library Synthesis

The original pool synthesized consists of single-stand DNA oligonucleotides. The

oligonucleotides are amplified into a dsDNA pool by PCR. The PCR amplified pool is

then transcribed into a single-stranded RNA pool. The RNA pool is then used for

selection of aptamers bound to desired ligand. This selection is usually based on the

principles of affinity chromatography or by other methods of separation of unbound

and bound fractions. To increase the specificity of selected molecule several rounds of

SELEX should be performed. The number of rounds of selection is determined by

both the type of library used as well as by specific enrichment achieved per selection

cycle. The RNA aptamers are then cloned, sequenced and characterized for any

conserved domain in all the sequences obtained.

8

Chapter 1

1.5 The SELEX Process

The main method for selecting an aptamer to a particular target is the Systematic

Evolution of Ligands by Exponential Enrichment (SELEX) process. It is based on the

screening of large combinatorial libraries of oligonucleotides by an iterative process

of in vitro selection and amplification ( 4). The general principle of the process is

shown in Figure 2 which includes the following steps: (i) incubating the library with

the target molecule under conditions favorable for binding; (ii) partitioning: the

molecules that, under the conditions employed, adopt conformations that permit

binding to a specific target are then partitioned from other sequences; (iii) dissociating

the nucleic acid-target complexes; and (iv) amplifying of the nucleic acids pool

enriched in sequences that bind to the target with high affinity and specificity. After

these steps the resulting oligonucleotides are cloned and sequenced. The sequences

corresponding to the initially variable region of the library are screened for conserved

sequences and structural elements indicative of potential binding sites and

subsequently tested for their ability to bind specifically to the target molecule. This

selection scheme works since single-stranded nucleic acids fold up into unique 3D

shapes in a similar manner to proteins, each structure being unique and dictated by the

sequence of the nucleic acid (31) .

The different methods that have been used so far for the partitioning of complexes

between RNA and protein from the pool of free RNA include nitrocellulose

membrane filtration which is simple and rapid, use of affinity surfaces such as

magnetic beads, column matrices (32), or ligands (33), gel electrophoresis (34), and

immuno precipitation (35). To improve the separation, the target aptamer complex

can be stabilized for example with UV cross-linking (36). Surface plasmon resonance

(SPR) can be used for real-time monitoring of complex formation and thus affinity

characterizations, allowing a more restricted selection (37, 38). The automation of the

process is a major advance over the time-consuming manual procedure. It allows

aptamers to be selected for several targets in parallel, and proceeds selection cycles

without any direct intervention steps (39) . A non-SELEX selection process based on

partitioning steps without amplification and using capillary electrophoresis was also

developed. The main advantage of this method is that it allows selection to be

performed within an hour instead of several days or weeks.

9

Elution o

•• ~

•

/ Random nucleic acid

.. oool

SELEX Cycle

Chapter 1

Removal of non speci IC

Figure 2: The general procedure for in vitro evolution or SELEX is shown in the picture. A

random nucleic acid library is initially incubated with the target of choice, allowing unbound

sequences to be removed. DNA or RNA molecules hound to the target are recovered and

ampl(fied by po~rmerase chain reaction. This process is repeated several times until specific

binding sequences are isolated.

1.6 Properties of Therapeutic RNA Aptamers

1.6.1 Efficiency and Specificity: For every therapeutic agent its specificity plays

crucial role in their efficacy. Aptamers are produced by an entirely in vitro process,

allowing for the rapid generation of initial therapeutic leads. In vitro selection allows

the specificity and affinity of the aptamer to be tightly controlled and allows the

generation of leads against both toxic and nonimmunogenic targets.

1.6.2 Pharmacokinetics and Nuclease Resistance: Natural RNAs/DNAs have poor

pharmacokinetics, primarily due to nuclease degradation and clearance via the

kidneys. But both limitations can be addressed with appropriate chemical

modifications· Nucleic acids are degraded in serum by a combination of

endonucleases and 5'-3' and 3'-5' exonucleases. To isolate modified aptamers, the

SELEX process can be carried out using random sequence pools containing

pyrimidines modified at their 2' positions. To further improve their stability, however,

10

Chapter 1

the purine nucleotides must be individually tested for the ability to accommodate

stabilizing 2'-0-methyl modifications. The lead clinical aptamer against VEGF

(Macugen; Eyetech/Pfizer) contains two natural adenosines which are required to

maintain binding affinity to the VEGF protein. Exonucleases can be blocked by

appropriate modifications at the 5'- and 3'-ends of an aptamer. Addition of a 3'-3'­

linked thymidine cap prevents 3'-5' exonuclease degradation from the 3'-terminus.

Similarly, 5'-caps (such as PEG adducts) prevent exonuclease degradation from the 5'­

terminus to increase aptamer residence times in the blood (40, 41).

1.6.3 Clearance: Even with extensive modification to block nuclease degradation,

stabilized molecules must also exhibit molecular weights greater than 40 kD to remain

in circulation for extended periods of time. A variety of studies have shown that

complexation to form high molecular weight conjugates dramatically increases the

serum half-life of aptamers. While several strategies have been used and evaluated

(including protein-aptamer complexation, tagging with lipids such as cholesterol, and

attachment to liposomes ), most efforts have been concentrated on PEGylation. High

molecular weight PEGs can be covalently attached to aptamers without substantially

altering their ability to tightly bind to targets. At the same time, these modifications

have a profound effect on aptamer half-life in animals, extending aptamer half-life

from minutes (no PEG) to several hours ( 40 kD PEG). Conjugation with 40 kD PEG

at its 5'-terminus increases the in vivo half-life of the thrombin aptamer from 24 min

to 6 h in rats, with little or no effect on thrombin binding affinity ( 42).

1.6.4 Toxicity and Immunogenicity: Another property which qualifies aptamers as

good therapeutic agent is their low toxicity and immunogenicity. Aptamers as a class

have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or

woodchucks with high levels of aptamer (lOmg/kg daily for 90 days), no toxicity is

observed by clinical, cellular, or biochemical measures. Whereas the efficacy of many

monoclonal antibodies can be severely limited by immune responses against

antibodies themselves, it is extremely difficult to elicit antibodies to aptamers (most

likely because aptamers cannot be presented by T -cells via the major

histocompatibility complex and the Immune response is generally trained not to

recognize nucleic acid fragments (43, 44).

1.6.5 Administration: Most antibody therapeutics are administered by intravenous

infusion (typically over 2-4 h), aptamers can be administered by either intravenous or

subcutaneous injection. This difference is primarily due to the comparatively low

11

Chapter 1

solubility and thus large volumes necessary for most therapeutic monoclonal

antibodies. With good solubility (> 150mg/ml) and comparatively low molecular

weight (aptamer: 10-50 kD; antibody: 150 kD), a weekly dose of aptamer may be

delivered by injection in a volume of less than 0.5 ml. Aptamer bioavailability via

subcutaneous administration is >80% in monkey studies ( 45). There is also evidence

that nucleic acid-based therapeutics can be dosed topically and via pulmonary

administration.

1.6.6 Scalability, Cost and Stability: Therapeutic aptamers are chemically

synthesized and consequently can be readily scaled as needed to meet production

demand. Therapeutic aptamers are chemically robust. They are intrinsically adapted to

regain activity following exposure to heat, denaturants, etc. and can be stored for

extended periods (more than 1 yr) at room temperature as lyophilized powders.

1.7 Applications of Aptamers

Aptamers constitute a very versatile class of molecules which can be used in a wide

range of applications. Following are some of the summarized general application of

aptamers.

1.7.1 Aptazymes: In combination with catalytic nucleic acid motifs, aptamer switches

allow the construction of allosteric ribozymes and DNAzymes, which can be

regulated by small molecule cofactors. Such nucleic acid constructs are termed

'aptazymes'. These are engineered nucleic acid whose activity can be regulated by

small molecules or co factors. For example, an AMP-activated nucleic acid ligase has

been selected from a sequence library obtained from joining an AMP-aptamer to a

random sequence. Aptazymes can serve as extremely sensitive molecular sensors in

which the aptamer domain recognizes the ligand and catalytic domain amplifies the

signal.

1.7.2 Protein Targeted Inhibitors: Like monoclonal antibodies aptamers may be

employed in a wide variety of in vitro assay formats to detect the amounts of a

particular protein or small molecule. RNA aptamers have been used as protein

targeted inhibitors which bind to a cellular protein and interfere with the function of

target. The in vivo stability of aptamers can be enhanced through modification of

sugar phosphate backbone or through use of mirror image analogs called spiege1mers.

12

Chapter 1

Spiegelmers are resistant to nuclease degradation and bind specifically to protein

target. Therefore, these are developed as potential therapeutic agent ( 46).

1.7.3 Target Identification and Validation: Many aptamers are antagonist of the

normal function of their binding partners, and in principal aptamers with agonist

activity could also be identified. The downstream effects of aptamer antagonists on

biological systems may also reveal novel molecular targets for drug design and

discovery (47).

1.7.4 Therapeutics: Due to high affinity and specificity towards their ligands they are

used as targeted molecular therapies, their chemical composition afford them unique

properties that may help to avoid many of the deficits common to competing protein

based technologies. The first commercial exploitation of aptamers was MACUGEN in

2005, for treating a form of macular degeneration. It inhibits the vascular endothelial

growth factor 165 (VEGF165) by binding it, thus preventing pathologic angiogenesis

involved in the disease ( 40).

1.7.5 Aptamers in Cancer Medicines: They are able to inhibit many of the target

molecules responsible for causing cancer in vitro and in vivo. The aptamer against

protein kinase CB11, Raf-1, RBD, VEGF165, PDGF-B, Thrombin and angiogenin

molecule selected are some of the examples ( 48, 49). Aptamers may serve as vehicle

for highly selective drug delivery when selected to bind a specific surface marker of a

cell or tissue (50).

1.7.6 Regulation of Gene Expression: Insertion of aptamers into the 5' untranslated

region of messenger RNA provides a handle to control the expression of specific genes

in living cells. Translation of such aptamer-RNA constructs can be regulated by

reversible ligand dependent conformational change ofaptamer domain (51).

1.7.7 Combating Infectious Agents: Nuclease-resistant aptamers can be selected to

block cell-adhesion events involved in some infectious diseases. For example, an

RNA-aptamer has been found to bind a glycoprotein expressed on cell-surface of the

parasite responsible for Chaga's disease. This glycoprotein is laminin and cytokeratin-

18 on host cells. The aptamer thus has the ability to prevent cell invasion. This

technique could be used with the advantage over conventional drug discovery that it

does not require a complete understanding of pathogenesis (52).

1.7.8 Antiidiotype Approach: Here RNA molecules can form surfaces that

functionally mimic those of proteins. This approach uses antibodies directed against

interfaces of protein-protein interactions to isolate from combinatorial libraries,

13

Chapter 1

mimics of one of the interaction domains. This strategy was used to isolate aptamers

binding to the neutralizing antibody directed against the insulin receptor. Furthermore,

an antibody raised against the HIV -rev nuclear export signal (NES) has been used to

select 'export aptamers' mimicking the NES. This export aptamers bind to the NES

receptor in vitro and inhibit Rev-dependent export in vivo. The mimic selection has

now been improved further by combining the anti-idiotype approach with a strategy

related to the blended SELEX (53).

1.7.9 Affinity Purification Medium: The major advantage of using aptamers for

purification purposes is that it can be performed in fewer steps than with conventional

methods owing to the ability to discriminate between closely related ligands.

Aptamers can thus be used as stationary phase in capillary electro chromatography or

in Western blots. For this application, they have higher affinity and specificity than

antibodies, and give a much better signal-to-noise ratio. As aptamers are generally

smaller than antibodies, they can bind epitopes sterically inaccessible to

immunoglobulins (26, 54).

1.7.10 Diagnostics and Biosensors: Aptamers are used in diagnostic tests and

probing. The property of high affinity and specificity of aptamers make them very

good diagnostic reagents. Standard immunochemical methods have been adapted for

aptamers including dot hybridization, western blotting, ELISA, affinity binding,

fluorescence polarization and flow cytometry For example; a 1231-labelled aptamer to

thrombin has been used for in vivo detection of clots. Three criteria are essential for

the development of a diagnostic sensor viz: the ability to transduce the binding

without adding an extra reagent, the ability to detect and quantify the target within

desired concentration and period of time, and the ability to tum over the sensing

capacity in order to reuse the sensor with several samples (55). A number of assay

formats have already been developed and evaluated. Aptamers are successfully used

as biosensors. Currently chip-based biosensors for multiplex analysis of proteins are

developed. The biosensor utilizes immobilized DNA and RNA aptamers, selected

against several different protein targets, to simultaneously detect and quantify levels

of individual proteins in complex biological mixtures. Aptamers were each

fluorescently labeled and immobilized on a glass substrate. Fluorescence polarization

anisotropy was used for solid- and solution-phase measurements of target protein

binding. Also, aptamers that recognize specific surface component of pathogens have

been identified e.g. anthrax spores and African trypanosomes. They can be detected as

14

Chapter 1

biosensors by fluorescent labels or radioactive labels. The aptamer is used as a probe

immobilized on a surface in order to capture the target. The first such aptasensor was

described in 1996 by Drolet (as cited in 51) and was an Enzyme-Linked Aptamer

Assay (ELAA), where the aptamer was enzyme-labeled. For this type of assay,

several factors need to be optimized. An electrochemical sensor has also been

developed, in which the aptamer was immobilized on a gold electrode surface through

a thiol group and labeled with an electroactive ferrocene group. The structure of the

protein-aptamer complex brings the ferrocene closer to the gold surface, which

increases the transfer of electrons on the electrode and thus increases the signal. This

sensor had very good sensitivity and specificity, with a nanomolecular detection limit

and a negligible response to non-specific proteins. It was also easily and rapidly

regenerated and could be used at least 25 times (56). Recently, two aptamer-based

biosensors have been developed for the detection of HIV -1 Tat protein, respectively

using the Quartz Crystal Microbalance (QCM) and Surface Plasmon Resonance

(SPR). In both cases, the biotinylated aptamer was immobilized on a streptavidin­

modified surface (57).

1.7.11 Intramers: The cytoplasmic expressions ofaptamers are called intramers. This

has been recently described for their striking potential as rapidly generated

intracellular inhibitors of biomolecules e.g. controlling small guanine nucleotide

exchange factor function through cytoplasmic RNA intramers (58). The present study

also emphasis GSH aptamer as intramers for sequestering GSH.

1.7.12 Molecular Switch Assays: Another kind of format is based on the effect of

protein binding on the conformation of the aptamer, and gives rise to label-free

assays. A fluorescing molecular switch can be obtained using ethidium bromide,

which has the property of intercalating between the base pairs of double-stranded

DNA and when this occurs, the component becomes fluorescent. In the assay, the

target molecule is put in competition with the complementary strand for the aptamer,

so that the signal is inversely proportional to the quantity of target. However this

method only works if the aptamer does not contain a secondary structure where EB

could intercalate. In addition, Faradaic Impedance Spectroscopy (FIS) has been used

to detect the binding of protein to an aptamer probe of opposite charge. The

interaction implies a reversal of charge, and thus a switch from repulsion to attraction

of the redox marker, allowing FIS detection (59).

15

•

Chapter 1

1.7.13 Biochips: The development of aptamer-based biosensors opens the way for

multiplexed sensors, also called biochips or aptamer micro-arrays. Based on the same

general principle as DNA microarrays used for the study of genomics, aptamer

biochips could help in the study of proteomics. Aptamers are immobilised on a glass

slide where they can still rotate at a rate that corresponds to their apparent volume and

mass. The binding implies a change in the mass, and consequently in the rotation rate,

which conditions the fluorescence polarisation. This technique has been proven to be

efficient even with complex biological matrices such as human serum or cell extracts

(60). However, all applications of RNA aptamers are limited by their considerable

susceptibility to endogenous ribonucleases (RNase) found in biological samples.

1.8 RNA Secondary Structure

RNA molecules share a major span of evolutionally history and have been suggested

to be important in the origin of life. There are other functions and aspects of RNA

molecules, that can be used in biotechnology and drug design. RNA molecules can be

used in drug design both as drug target and as drugs. RNA molecules have different

functions in the cell and like proteins they also need to have a certain three

dimensional structure to function properly. RNA forms diverse secondary structures;

the nomenclature for some of the common motifs is given in Figure 3. Secondary

structures are comprised of canonical (Watson-Crick) and non-canonical base pairs

that are stabilized through hydrogen bonding and base stacking interactions.In RNA,

guanine and cytosine pair (GC) by forming a triple hydrogen bond, and adenine and

uracil pair (AU) by a double hydrogen bond; additionally, guanine and uracil can

form a single hydrogen bond base pair. The stability of a particular secondary

structure is a function of several constraints e.g.

};;;> The number of GC versus AU and GU base pairs.

};;;> The number of base pairs in a stem region.

};;;> The number of base pairs in a hairpin loop region.

};;;> The number of unpaired bases, whether interior loops or bulges.

Types of single and double stranded regions in RNA secondary structures are shown

in Figure 3 Single stranded RNA molecules fold back on themselves and produce

double stranded helices where complementary sequences are present. A particular

base may either not be paired (single stranded), or paired with another base (double

16

Chapter 1

helix or stem). The double stranded regions will most likely form where a series of

bases in the sequence can pair with a complementary set elsewhere in the sequences.

The stacking energy of the base pairs provides increased energetic stability.

Combination of double stranded and single stranded regions also produce different

types of structures, with the single stranded regions destabilizing neighboring double

stranded regions (single nucleotide bulge). The loop of the stem and loop must

generally be at least four bases long to avoid steric hindrances with base-pairing in the

stem part of the structure. Interior loops, form when the base in a double stranded

region cannot form base pairs and may be asymmetric with different number of base

pairs on each side of the loop, or symmetric with the same number on each side.

Junctions may include two or more double stranded regions converging to form a

closed structure (61).

single strand

symmetric internal loop

u double helix or stem

sin&Je nucleotide bulge

asymmetric internal loop

hairpin loop

_jL -lr four stemjUJX:tion

Figure 3: Depiction of several secondary structure elements formed by RNA. In green are

Watson Crick base pairs; red represents non Watson Crick base pairing or non paired bases,

hlue represents phosphate backbone (Gesteland eta!. 1998 ) 62.

17

Chapter 1

I

Jl)~'

A. 8. c. Figure 4: Examples of known interaction of RNA secondary structural elements. (A)

Pseudokont. (B) Kissing loop. (C) Hairpin-bulge contact. (Adapted from Burkhard et a!.

1999) 63.

In addition to secondary structural interactions in RNA, there are also tertiary

interactions (Fig 4). These kinds of structures are not predictable by secondary

structure prediction programs. They can be found by detailed covariance analysis.

1.9 RNA Structure Prediction

RNA has a wonderful capacity to form complex, stable tertiary structures due to its

conformational flexibility, modularity and versatility. This capacity enables RNA

molecules to play essential roles in cellular processes of all organisms mediated by

process like ligand-binding, complementary base pairing and catalytic reactions (64-

67). Recently the regulatory roles of RNA in the control of gene expression have been

studied in details. Since ligand-binding and base pairing are fundamental aspects of

RNA interactions and activities, many such small functional RNA motifs may exist

and play roles in regulatory circuits in the cells.

Many computational methods for predicting RNA structure have been developed.

RNA structure can be predicted and chosen from many possible choices of

complementary sequences that can potentially base pair, the compatible sets that

provide the energetically most stable molecules selected for further analysis. In

another type of prediction, it accounts for conserved patterns of base-pairing that are

conserved during evolution of a given class of RNA molecules. Sequence positions

that base-pair are found to vary at the same time during evolution of RNA molecules

so that structural integrity is maintained thus it is important to analyze the structural

18

Chapter 1

alignments and to find conserved structure for predicting and analyzing the role of

RNA (including functional RNAs, non coding RNA and RNA aptamers).

Identification of base co variation that maintains secondary and tertiary structure of an

RNA molecule during evolution has been studied by many groups. Energy

minimization methods have been so well refined that a series of energetically feasible

models and the most thermodynamically probable structural models may be

computed.

Phylogenetic analysis is the most accurate method for secondary structure prediction.

It is assumed that RNAs of similar function share common structural properties and

conserved structural elements. Major limitation of this approach is that it cannot

predict noncanonical base pairs well, since the rules for these pairs are still vague.

Many RNA secondary structures like those of tRNAs (68), ribosomal RNA (69-71),

group I and group II introns (72, 73), from snRNPs (74), hammerhead catalytic RNA

(75), and RNase P RNAs have been determined by phylogenetic analyses, which were

confirmed by other methods. Sequence and structural alignment is a convenient tool

for studying the structural homology.

RNA secondary and tertiary structures are calculated using free energy minimization

techniques. Secondary structure prediction programs minimize the thermodynamic

free energies of formation (76). The thermodynamic method is best for predicting

stable double helices because of strong hydrogen bonding and stacking interactions.

These prediction programs are most useful in providing probable, low free energy

structures. They are less useful in choosing the most stable structure, as the data are

not good enough to discriminate among different structures with similar free energies

while tertiary structure prediction uses the interactions between atoms or groups to

find a structure (77). Standard bond lengths and bond angles are assumed, and then

van der Waals interactions and electrostatic interactions between non bonded groups

are calculated to obtain a structure.

Secondary-structure prediction programs use thermodynamic parameters derived from

oligonucleotides, which form simple base-paired structures. Five algorithms for

predicting secondary structures are dynamic programming (78), combinatorial (79),

Monte Carlo (80, 81 ), significance (82), and a hybrid phylogenetic-thermodynamic

approach (80, 83, 84). These programs all assume that the folded RNA molecules are

at equilibrium; there are no kinetic barriers that trap the molecules in

thermodynamically unstable states. The programs are particularly useful where only a

19

Chapter 1

single sequence is available, since several sequences are needed for phylogenetic

analysis The dynamic programming or recursive method first calculates the free

energies of all sequence fragments with five or more bases (five bases can form the

smallest secondary structure considered-a three-base hairpin closed by a single base

pair) (78). The stability of a secondary structure is quantified as the amount of free

energy released or used by forming base pairs. Positive free energy requires work to

form a configuration; negative free energies release stored work. Free energies are

additive, so one can determine the total free energy of a secondary structure by adding

all the component free energies (units are kilocalories per mole). The more negative

the free energy of a structure, the more likely is formation of that structure, because

more stored energy is released. This fact is used to predict the secondary structure of a

particular sequence. Discovering a base pair configuration with the minimum possible

free energy is the goal of most secondary structure prediction algorithms. To compute

the minimum free energy of a sequence, empirical energy parameters are used. These

parameters summarize free energy change (positive or negative) associated with all

possible pairing configurations, including base pair stacks and internal base pairs,

internal, bulge and hairpin loops, and various motifs which are known to occur with

great frequency. A number of strategies for predicting secondary structure have been

developed. The folding algorithm can be divided into two major groups and

subgroups. The major groups are Deterministic (Minimum free energy, Kinetic

folding, 5'-3' folding and Partition function) and Stochastic.

1.10 Tertiary Structure of RNA

The central biological importance of RNA has recently become more apparent

because of wide diversity of functions of RNA (85, 86); the list of known catalytic

activities has been growing rapidly (87-91). The functional diversity of RNA reflects

diversity in its three-dimensional structure also. Knowledge of the three-dimensional

structures and general rules for RNA folding are valuable for understanding the

mechanisms of all RNA functions. The field of RNA structure prediction has

experienced significant advances in the past several years, due to the availability of

new experimental data and improved computational methodologies. These methods

determine RNA secondary structures and pseudoknots from sequence alignments,

thermodynamics-based dynamic programming algorithms, genetic algorithms and

20

Chapter 1

combined approaches. Computational RNA three-dimensional modeling uses this

information in conjunction with manual manipulation, constraint satisfaction methods,

molecular mechanics and molecular dynamics. Recent developments in the

computational prediction of RNA structure have helped bridge the gap between RNA

secondary structure prediction, including pseudoknots, and three-dimensional

modeling ofRNA.

The primary structure of an RNA molecule is relatively easy to determine; however,

determination of RNA secondary structure and tertiary structure are tedious.

Therefore, improved methods for determining and predicting RNA structure are

needed. Accurate prediction of RNA structure mainly requires an understanding of

fundamental interactions such as hydrogen bonding, stacking, and hydration in

diverse structural contexts. Tertiary structure prediction of RNA is a multifarious job.

Rapid and accurate prediction of RNA tertiary structure is the core of RNA folding

problem. The flexibility of the base-sugar and sugar-phosphate linkages is large.

Interactions between bases, phosphates, sugars, and solvent add even more

complexity .

.::r-- The prediction methods use interactions between atoms expressed as distance-NJ ~ dependent or angle-dependent potential energies. The potentials allow slight

variations of standard bond lengths and bond angles. Energies between non-bonded

~ ~'- groups are characterized by van der Waals forces, electrostatic interactions and

hydrogen bonding. The basic calculation methods are molecular dynamics (92, 93),

where molecular motions are simulated as a function of time, and free energy

perturbation methods, which calculate the difference in free energies between two

states (93). Although some structures have been generated without any experimental

data (94), tertiary-structure modeling clearly relies on experimental data to ensure a

particular conformation. A model of the structure is minimized using a combined

energy function of atomic potentials and artificial forces for the restraints. A starting

conformation is generated, usually manually, that agrees with known RNA structures

and the experimental data, and this structure is energy minimized. The crucial step is

the initial structure generation that defines most of the global and local interactions.

Many methods use computer-aided structure design, where structural elements from

known structures are combined to produce a folded RNA molecule.

3D RNA structure prediction and folding presents a significant challenge in molecular

Chapter 1

limitations hinder the applications of X-ray crystallography, and nuclear magnetic

resonance for high-throughput structure elucidation and hence the prediction of RNA

tertiary structure (95) and investigating thermodynamics and mechanism of RNA

folding are important in current scenario of RNA research.

iFoldRNA (http://iFoldRNA.dokhlab.org) a web resource for rapid and accurate

predictions of 3D RNA structures and probing folding thermodynamics. iFoldRNA

performs folding simulations using the DMD engine (96, 97) and Medusa force field

(98) to simulate RNA folding dynamics by iFoldRNA, a novel web-based

methodology for RNA structure prediction with near atomic resolution accuracy and

analysis of RNA folding thermodynamics. It makes the RNA structure prediction and

folding uncomplicated as compared to previous approach. iFoldRNA rapidly explores

RNA conformations using discrete molecular dynamics simulations of input RNA

sequences. RNA folding parameters including specific heat, contact maps, simulation

trajectories, gyration radii, RMSDs from native state, fraction of native-like contacts

are accessible from iFoldRNA. iFoldRNA really serve as a useful resource for RNA

structure prediction, folding and thermodynamic analyses (95, 99).

1.11 Advancement of Aptamer Research and Technology

In slico selection of RNA aptamers that bind to a specific ligand usually begins with a

random pool of RNA sequences. Computational approach is used for designing a

starting pool of RNA sequences for the selection of RNA aptamers for specific

analyte binding. The in silico selection approach consists of three steps: (i) selection

of RNA sequences based on their secondary structure, (ii) generating a library of

three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual

screening of this library to select aptamers with binding affinity to a desired small

molecule. This allows one to select a sequence with potential binding affinity from a

pool of random sequences. As verification, the test performance of in silico selection

on a set of six known aptamer-ligand complexes was also established (100).

Computational approach reduces the RNA sequences search space by four to five

orders of magnitude significantly accelerating the experimental screening and

selection of high-affinity aptamers. In the in silico approach, RNA sequences are

screened at two levels. At the first level, selection of RNA sequences is based on

analysis of secondary structure ofthe generated sequences. At this screening level, the

22

Chapter 1

selected sequences are not target specific. While the second screening level, needs

computational docking· to identify RNA molecules that bind to a specific target

ligand. At this point, the selected RNA molecules are specific to the desired target

molecule and they are placed into a pool of sequences for experimental verification

and selection of high-affinity aptamers. This could be used to create the initial pool of

RNA sequences for experimental selection of high-affinity aptamers, thus greatly

accelerating the process of finding the desired aptamer sequence (101).

The aptamer-based therapeutic, Macugen, is derived from a modified 2-fluoro

pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now

being used to treat the wet form of age-related macular degeneration. This VEGF165

aptamer binds specifically to the VEGF165 isoform, a dimeric protein with a

receptor-binding domain and a heparin-binding domain (HBD). The VEGF aptamer is

a potent inhibitor of VEGF mediated angiogenesis and effectively blocks the

interactions with the cell surface tyrosine kinase receptors VEGFR-1 and VEGFR-2

((102, 103).

Surface plasmon resonance (Biacore) has been used for selecting aptamers that bind

to hemagglutinin (HA) of human influenza virus. This procedure allowed to monitor

and select the target-bound aptamers specifcally and simultaneously. These studies

not only yielded an aptamer that binds to the HA of influenza virus with high affinity

but also revealed the consensus sequence, 5 _-GUCGNCNU(N)2-3GUA-3, for HA

recognition (104).

A method for the rapid collection and detection of leukemia cells using a novel two­

nanoparticle assay with aptamers as the molecular recognition element was developed

in 2006. An aptamer sequence was selected using a cell-based SELEX strategy for

CCRF-CEM acute leukemia .cells. Aptamer-modified magnetic nanoparticles were

used for target cell enrichment, while aptamer-modified fluorescent nanoparticles

were simultaneously added for sensitive cell detection. Combining two types of

nanoparticles allows for rapid, selective, and sensitive detection not possible by using

either particle alone. Fluorescent nanoparticles amplify the signal intensity

corresponding to a single aptamer binding event, resulting in improved sensitivity

over methods using individual dye-labeled probes. In addition, aptamer modified

magnetic nanoparticles allow for rapid extraction of target cells not possible with

other separation methods (105).

23

Chapter 1

Methods similar to SELEX have been used to develop nucleic acid sequences with

catalytic ability, artificial ribozymes . Artificial ribozymes have been selected for the

catalysis of a variety of reactions including transesterification, alkylation, and peptide

bond formation (106-109). This modified SELEX uses the presence of catalyst to

select sequences to be amplified. This method usually requires that the detection of

catalysis must also allow for isolation of catalytic sequences. For example, random

sequences were mixed with iodoacetyl derivative of biotin, self alkylation biotinylates

the sequences allowing for separation from non catalytic sequences (107). Another

approach to obtain catalytic sequences has been to develop aptamers that bind to

transition state analogs. This method has been successfully used to develop catalytic

antibodies. A 35 nucleotide aptamer was developed to target N-methylmesoporphyrin

(NMMP), which is an analog of a possible transition state of the metalation reaction

of mesoporphyrin (11 0, 111 ).

1.12 Glutathione

Glutathione (GSH) is a tripeptide. It contains an unusual peptide linkage between the

amine group of cysteine and the carboxyl group of the glutamate side chain.

Glutathione, an antioxidant, protects cells from toxins such as free radicals. The thiol­

containing tripeptide, glutathione (GSH), is one of the most abundant and ubiquitous

antioxidant in nature. This endogenous antioxidant is found in high concentrations

(0.5-8.0 mM) in nearly all living cells and has many important functional roles that

have been extensively reviewed. Glutathione is found almost exclusively in its

reduced form, since the enzyme that reverts this from its oxidized form (GSSG),

glutathione reductase, is constitutively active and inducible upon oxidative stress.

H,"J· I

HSG

HB22 ;=... r 11

Hfl N~ I

Figure 5: Structure of Glutathione. (Source: RCSB PDB).

24

Chapter I

Glutathione plays important roles in antioxidant defense, nutrient metabolism, and

regulation of cellular events (including gene expression, DNA and protein synthesis,

cell proliferation apoptosis, signal transduction, cytokine production, immune

response, and protein glutathionylation). New knowledge of the nutritional regulation

of GSH metabolism is critical for the development of effective strategies to improve

health and to treat several diseases. It has several vital roles within a cell including

antioxidation, maintenance of the redox state, modulation ofthe immune response and

detoxification of xenobiotics (112-114). Impaired GSH status has been implicated in

the pathogenesis of a number of cancers (115,116).

1.12.1 Glutathione and Cancer

Cancer is a term used for diseases in which transformed cells by passing cell cycle

control and are able to invade other tissues. Cancer cells can spread to other parts of

the body through the blood and lymph systems (Fig 6) . The GSH content of cancer

cells is particularly relevant in regulating mutagenic mechanisms, DNA synthesis,

growth, and multidrug resistance. Drug resistance against cancer in most cases

associates with higher GSH levels. Thus, approaches to cancer treatment based on

modulation of GSH should control possible growth-associated requirement in cancer

cells. With respect to cancer, glutathione metabolism is able to play both protective

and pathogenic roles. It is crucial in the removal and detoxification of carcinogens,

and alterations in this pathway, can have a profound effect on cell survival. However,

by conferring resistance to a number of chemotherapeutic drugs, elevated levels of

glutathione in tumour cells are able to protect such cells in bone marrow, breast,

colon, larynx and lung cancers (117).

The specific involvement of GSH in the carcinogenic process is supported by the

major role of this compound in detoxification of carcinogens by conjugation (108).

The reactivity of GSH with electrophilic compounds formed from the metabolism of

carcinogens by the mixed function of oxidase system (115, 116). Since these

electrophiles are often genotoxic and have the capacity to react with and damage

DNA, their efficient detoxification is a critical step in the prevention of

carcinogenesis. The reaction of GSH with electrophilic compounds may either be

spontaneous or catalyzed by one of the GSH S-transferases, and usually results in the

formation of a nontoxic GSH conjugate. GSH conjugation has been implicated as an

25

Chapter 1

important detoxification pathway for a variety of chemical carcinogens and mutagens

(118)

Elevation of cellular GSH levels has been frequently observed in cell lines rendered

resistant to alkylating agents, cisplatin and anthracyclines by in vitro exposure. Recent

evidence shows that the intracellular and/or intra mitochondrial redox state may

influence the apoptosis. The fact that redox potential may be influenced by GSH

depleting compounds offers the exciting prospect that it may be possible to alter the

sensitivity of cancer cells to cytotoxic drugs and thereby reduce the problem of drug

resistance in future (119).

Figure 6: Acquired capabilities of cancer (Hanahan D, Weinberg RA. 2000) 120.

26

Chapter I

1.12.2 Glutathione and Apoptosis

The apoptotic machinery can be broadly divided into two classes of components:

Sensors and effectors. The sensors are responsible for monitoring the extracellular and

intracellular environment for conditions of normality or abnormality that influences

whether a cell should live or die. These signals regulate the second class of

components which function as effectors of apoptotic death (Fig 7). The ability of

tumor cell population to expand in number is determined not only by the rate of

population but also by the rate of cell attrition and apoptosis represent a major source

of this attrition. The acquired resistance towards apoptosis is a hallmark of most and

perhaps all types of cancer. There are two basic pathways or mechanisms of cell

death: necrosis and apoptosis. Necrosis is characterized by swelling of the cytoplasm

and mitochondria, rapid loss of cellular membrane integrity, followed by swelling and

rupture of entire cell. In contrast, apoptosis, programmed cell death, is characterized

morphologically and biochemically by cellular shrinkage, membrane blebbing,

chromatin condensation and cellular DNA fragmentation. During apoptosis, genomic

DNA is degraded by means of specific endonucleases in a series of reactions that

could be triggered by at least two interconnected pathways: the extrinsic and the

intrinsic apoptotic induction systems. The role of free radicals has been shown in both

cases. There is a direct relationship between glutathione oxidation and mtDNA

damage in apoptosis (121). It has been shown that over-expression of the p53 gene

results in induction of multiple redox-related gene products and initiation of apoptosis

(122). High glutathione levels increase cellular resistance to apoptosis. There is a

direct relationship between glutathione oxidation and mtDNA damage in apoptosis

(123). The mechanism of oxidative stress-induced apoptosis appears to involve loss

of mitochondrial transmembrane potential (124) and increased intracellular calcium

concentration (125).

27

Death Receptor Pathway Mitochondrial Pathway

oxidant ceramlde other

DNA . . . ·.. .

damage

DISC

/

Cellular targets - apoptosome

Chapter 1

• • Cytochrome

c

Figure 7: Major apoptotic pathways (Extrinsic and Intrinsic pathway) (Hengartner, MO.

2000) 126.

Release of cytochrome c to the cytoplasm (127) and degradation of DNA have also

been reported (128). However, one of the earliest and most conspicuous events during

apoptosis is depletion of GSH concentration (123, 129). Cellular redox status is an

important regulator of apoptotic potential. The effect of modulating the redox status

on apoptotic potential has been studied using GSH depleting agents. It is known that

GSH is an important regulator of the cellular redox state. However, the relative

importance of cytosolic and mitochondrial GSH levels as mediators of apoptotic

signaling has not been clearly defined. Mitochondria do not possess the enzymes

required for de novo GSH synthesis, (130) but utilize cytosolic GSH derived from a

multi component, A TP dependent, mitochondrial transporter that translocates GSH

from the cytosol into the mitochondrial matrix (131, 132). The transporter has a high

affinity component which functions at low cytosolic GSH levels, to maintain

mitochondrial GSH levels during periods of cytosolic GSH depletion (131). The

regulation of cell death by apoptosis studies suggest that changes in mitochondrial

permeability frequently precede the development of morphological features such as

chromatin condensation, phosphatidylserine inversion of the outer cell membrane and

28

Chapter 1

the activation of endonucleases. There is evidence of intermediate role of intracellular

redox status of the cell. Role of glutathione in the regulation of apoptosis associated

redox changes and the control of mitochondrial membrane permeability is also

reviewed by A. G. Hall (1999) (133). The loss of mitochondrial GSH appears to be a

key regulator of apoptotic potential in PW cells, since the subsequent increase in ROS

production precedes the induction of apoptosis. GSH depletion in PW cells is

sequentially associated with a decline in total cellular GSH, along with NF kB

activation, the release of cytochrome c from mitochondria, and the expression of a

truncated form ofp53. ROS production is then increased, at which time pro-caspase 3

is cleaved and apoptosis is irreversibly induced. Since ionizing radiation and

chemotherapy alter redox status, elucidation of the role of redox modulation on

apoptotic signaling pathways may have broad clinical relevance and ultimately allow

for the development of novel therapeutic strategies to improve the efficacy of

cytotoxic therapies. As GSH is the major determinant of the intracellular redox

potential, the role of this system in the commitment to cell death is of clear

significance, although it may not be the only factor involved. For example,

transfection of cells with the low-molecular weight redox protein thioredoxin is

capable of protecting cells against apoptosis induced by a wide range of agents (134).

1.12.3 Glutathione Depletion and Cancer Therapy

ROS such as free radicals are capable of causing massive cell injury if left unchecked,

which can lead to a number of pathologies including cancer. Restoration of

intracellular GSH levels may help in protecting cells from this damage. GSH within

cells may enhance detoxification of cells, and help reduce cancer incidence, many

GSH-based therapeutic strategies have focused on lowering GSH levels in order to

increase sensitivity of cells to ionizing radiation, and to decrease the resistance to

many chemotherapeutic drugs (135). Buthionine sulfoximine (BSO), a selective

inhibitor of GSH synthesis, and oxothiazolidine-4-carboxylate (OTZ), which

stimulates GSH synthesis in normal cells, have been used in many of these studies in

the modulation of cellular GSH. Modulation of GSH level within a human lung

adenocarcinoma cell line compared to a normal human lung fibroblast line, using

BSO and OTZ, and subsequent exposure to the chemotherapy drugs Melphalan,

Cisplatin and Bleomycin were investigated by Russo et al. (136). These studies

29

Chapter 1

showed that modulation of GSH levels produced different chemotherapy responses in

normal versus tumour cells, which if true in vivo, may provide a GSH-based strategy

for the protection of normal cells, and sensitization of tumour cells to

chemotherapeutic drugs. On the same line GSH-binding RNA aptamer could play

important role in this regard and could be used as RNA therapeutics, while it will be

noteworthy to search natural motifs having homology with these aptamers in the

genome and further investigate its role in regulation.

GSH depletion has been seen to enhance the anti tumour cytotoxicity of various drugs

without increasing toxicity to normal tissue. OTZ was able to decrease the GSH levels

in MCF 7 and in rat mammary tumors, and sensitize the tumours to the alkylating

agent melphalan thereby increasing its cytotoxicity beyond that of melphalan alone

(137). Buthionine sulfoximine (BSO) is a specific inhibitor of GSH biosynthesis and

it was developed as an anticancer drug candidate since it is a potential sensitizer of

alkylating agents in different neoplasic cell lines. Treatment with BSO induces a

severe GSH depletion causing overproduction of reactive oxygen species (ROS) and

DNA damage, thus leading to apoptosis. It is known that there is a decrease in cellular

and mitochondrial glutathione during apoptosis and that this decrease opens the

permeability transition pore inducing apoptosis. In another study BSO was used to

modulate the levels of GSH within two squamous oesophageal carcinoma cell lines

that were resistant to alkylating agents ( cisplatin), but not Vinca alkaloids and

doxorubicin (138). It has been used to sensitize previously resistant human ovarian

cancer cells to cyanomorpholino doxorubicin (MRA-CN) by altering GSH levels

(139) and in a neuroblastoma cell line, enhanced cytotoxicity was observed when

treated with a combination of both melphalan and BSO compared to melphalan alone

(140). Clinical trials of these inhibitors are currently underway with phase I trials of

BSO in advanced cancers reporting a relatively non-toxic depletion of tumour

glutathione with continuous BSO infusion (141). GSH depletion in human leukaemic

cell lines by BSO converts cell death induced by a variety of agents from apoptosis to

necrosis (142), suggesting that the uncontrolled production of ROis will overwhelm

the apoptotic machinery. In addition, under certain circumstances, including F AS­

induced death in human T cells (143) and etoposide exposure in monocytes (144),

GSH levels may fall because of extrusion across the cell membrane. It has been

demonstrated that inhibition of glutathione efflux is able to rescue U937 and HepG2

30

Chapter 1

cells from puromycin induced apoptosis (145), suggesting that in diverse cell types,

the control of GSH homeostasis has a central role in the regulation of cell death.

1.12.4 Glutathione Homeostasis

ROis are constantly produced as a by-product of the process that produces ATP to

facilitate energy-dependent biochemical pathways. As an outcome, such cells are

under constant attack from reactive species which are capable of causing serious

damage to macromolecules such as lipid and DNA. To manage this, the cells have

evolved mechanisms, such as antioxidants system. Glutathione (GSH) is particularly

important in this respect. As the most abundant low-molecular-weight thiol within the

cell, it represents an easily mobilized system for the removal of peroxides by a

reaction that results in the generation of oxidized glutathione (GSSG). The product of

GSH oxidation, GSSG, is known to be toxic and is rapidly converted back to GSH by

the enzyme glutathione reductase. As a consequence, the ratio of GSH/GSSG is held

at about 100:1 (146) and the balance of this ratio inside the cell plays crucial role in

cell survival and cell death. GSH levels within a cell are controlled by the balance

between the rate of production or salvage and the rate of consumption or loss. De

novo synthesis of GSH is an energy-dependent process involving two enzymes:

gamma-glutamyl cyteine synthetase (GGCS) and glutathione synthase. GGCS activity

is subject to feedback inhibition by GSH.

1.12 5 Glutathione and Glutathione-S-transferases (GSTs)

Glutathione-S-transferases (GSTs) are a family of Phase II detoxification enzymes

that catalyze the conjugation of glutathione (GSH) to a wide variety of endogenous

and exogenous electrophilic compounds. GSTs have been implicated in the

development of resistance toward chemotherapy agents. It is plausible that GSTs

serve two distinct roles in the development of drug resistance via direct detoxification

as well as acting as an inhibitor of the MAP kinase pathway. Recently GSTs have

emerged as a promising therapeutic target because specific isozymes are

overexpressed in a wide variety of tumors and may play a role in the etiology of other

diseases. Some of the therapeutic strategies so far employed are described in the

review (147).

31

Chapter 1

Glutathione conjugation is the first step in the mercapturic acid pathway that leads to

the elimination of toxic compounds. GSTs have evolved with GSH, and are abundant

throughout most life forms. Development of drug resistance is a key element in the

failure of chemotherapy treatment and GSH/GSTS are responsible for drug resistance.

GSTs have been implicated in the development of resistance toward chemotherapy

agents, insecticides, herbicides, and microbial antibiotics. Overexpression of GSTs

and increased levels of GSH are often associated with an increased resistance to

cancer chemotherapeutic drugs via GSH conjugation and detoxification, involving the

combined mechanisms ofGSH, GST and glutathione S-conjugate export pump (GS-X

pump) (148, 149). GSH and y-GCS levels have been shown to be elevated in a

number of different human cancer tissues. Since the original observation of an

increase in GSH levels in melphalan- resistant murine Ll210 cells (150) numerous

studies have been carried out on various drug-resistant cell lines with many

identifying a relationship between GSH/GSH-associated enzyme levels and the extent

of drug resistance. Contribution of GSH, GST activity and the GS-X pump to the

resistance of HepG2 cells to cisplatin cytotoxicity was investigated. It was shown that

while inhibition of GST and the GS-X pump (using ethacrynic acid, quercetin, tannic

acid and indomethacin) had no effect on cisplatin cytotoxicity, modulation of GSH

levels (using Buthionine sulfoximine (BSO) and monoethyl GSH ester) were able to

regulate the cytotoxic effects of cisplatin. This suggests that intracellular GSH is the

rate-limiting factor and highlights a possible role for regulation of GSH in tumour cell

sensitization to cisplatin (151). The development of further inhibitors for specific

molecules in the GSH synthesis/GST detoxification pathway may help to further

elucidate the complex role of GSH in cancer, provide better strategies in cancer

prevention and enhance the effectiveness of current chemotherapeutic approaches.

1.12.6 Glutathione Depletion and Bcl-2

Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies

that include conditions characterized by periductal inflammation and cholangiocyte

apoptosis. Because inflammation is associated with oxidative stress, the hypothesis

was developed which states that cholangiocytes exposed to oxidative stress will be

depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis.

The hypothesis was tested by exploring the relationships among glutathione depletion,

32

Chapter 1

expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a

nonmalignant human cholangiocyte cell line. Monolayers of human bile duct

epithelial cells, derived from normal liver and immortalized by SV 40 transformation,

were depleted of GSH using BSO. Bcl-2 expression was assessed. Maintenance of

GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the

BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also

prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not

change steady-state levels ofbcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2

protein half-life decreased 57% in BSO-treated vs. untreated cells. The data

demonstrate that reduction in the cellular levels of an antioxidant such as GSH results

in increased degradation of Bcl-2 protein and an increase in apoptosis. This also

provides a mechanistic link between the consequences of oxidative stress and

cholangiocyte apoptosis, an observation that may be important in the pathogenesis of

the inflammatory cholangiopathies (152).

1.12. 7 Glutathione and Telomerase Activity

Telomerase plays a key role in cellular homeostasis, because it maintains the length of

the telomeres. The eukaryotic chromosomes are capped by telomeres. These

structures play an important role in the stability and the complete replication of the

chromosomes. Conventional DNA polymerases cannot fully replicate the 3 '-end of

the lagging strand of linear molecules, and therefore telomeric sequences are lost in

every cell division (153). Telomerase is an important enzyme that ensures the

maintenance of normal telomere length. This activity is high in human cancers (154)

but virtually absent in normal human tissues, except germinal cells (155). Telomerase

regulation is not completely understood, but its changes are related to both cancer and

aging (156). This is especially important in germinal cells in which it is necessary to

keep a normal telomeric length after many cellular divisions. Mammalian telomeres

and subtelomeric regions are enriched in epigenetic markers that are characteristic of

heterochromatin. Glutathione may play a major role in epigenetic control (157).

Little is known about the short term regulation of the activity of telomerase (158)

using vascular smooth muscle cells, reported that hypoxia up-regulates its activity.

Hypoxia is known to lower oxidative stress and thus to increase levels of GSH. On the

other hand, 2-[3-(trifluoromethyl) phenyl] isothiazolin-3-one, a specific inhibitor of

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Chapter 1

telomerase, reacts with a key cysteine residue, which is essential for telomerase

activity and must be kept reduced. Consequently, it has been reported that dithiotreitol

reverses this inhibition. In conclusion, a critical cysteine residue must be kept reduced

in order to maintain full telomerase activity. It is likely that the glutathione redox

potential may be important in this process. It has also been found that cellular

glutathione level correlates with telomerase activity in 3T3 fibroblasts. The peaks of

telomerase activity coincide with the peak of GSH. Depletion of GSH with BSO

reduces the telomerase activity and that of regulators of cell cycles such as Id2 and

E2F4 after 24h and 48h of treatment. Changes in the GSH/GSSG redox potential

modulate telomerase activity. GSH levels in cells subsequently oxidative stress

regulate, at least in part, the activity of tel om erase as well as that of other relevant cell

cycle-related proteins (159).

In conclusion, literature shows that glutathione is important regulator of redox status

and playing important role in apoptosis in cancer cells. On the other hand aptamers

can specifically bind to cognate ligands with high affinity and specificity. GSH

depleting agents are important in inducing apoptosis and playing significant role in

cancer therapy. Recently the importance of non coding RNA in regulation explores

the area of RNA studies. With the above background of knowledge we decided to

select and characterize the glutathione-binding RNA molecules from the random pool

and investigate its effects in cancer cells with respect to GSH sequestering, ROS

generation and its involvement in apoptosis. The summary of the work plan is shown

in Figure 8. The selected glutathione-binding molecules can be of therapeutics

importance.

34

Chapter 1

Figure 8: Summary of Work Plan. We proposed to select and characterize glutathione­

binding RNA aptamers and investigate its effect with respect to GSH sequestering and its

involvement in apoptosis.

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