Search for new types of deoxyribozymes and development of human topoisomerases inhibitors on the basis of oligonucleotides

Seven naturally occurring classes of catalytic RNAs have been identified to date hammerhead, hepatitis delta virus, hairpin, Neurospora Varkud satellite, group I introns, group II intro

PART I: SEARCH FOR NEW TYPES OF

ACTIVITY UNDER HYDROLYSIS PATHWAY

CHAPTER 1 Introduction

Nature has spent more than 3 billion years to perfect the many thousands of enzymes that are utilized in living cells Yet this long and merciless process of evolution has given rise to enzymes based on only two molecular formats-protein and RNA In opting to build biocatalysts, nature has made two excellent choices Proteins exploit the different chemistries offered by their constituent 20 amino acids to form an incredible array of diverse structures and precisely configured active sites So together with their ability to adopt a seemingly endless array of tertiary structures in sequence-directed fashion, proteins are well suited to serve as enzymes The splendid catalytic potential of proteins is exemplified by enzymes, such as orotidine 5’-phosphate decarboxylase, which coverts substrate to product with a rate enhancement of 17 orders of magnitude over the corresponding uncatalyzed rate [1]

Although RNA’s role in modern biocatalysis might be an “accidental catalysts” from life’s early evolutionary history [2], catalytic RNAs (ribozymes) are certainly capable

of generating impressive rate enhancements For example, group I ribozymes catalyze RNA splicing with a rate enhancement of ~13 orders of magnitude Only eight classes

of naturally occurring RNA biocatalysts have been identified so far since the 1980s when Thomas Cech made the first surprising discovery that RNA molecules are capable of catalyzing reactions in the absence of any protein components [3] In contrast to proteins, the diversity of chemical functional groups in RNA is severely limited, and this fact is widely considered as the most significant determinant that constrains both the structural and catalytic potentials of RNA Then why is RNA

provided with the catalytic activity of enzymes from evolution? The most important reason is that RNA is capable of bringing its lesser chemical repertoire to bear on catalysis by forming surprisingly intricate three-dimensional structures [4-6] The versatilities of RNA in forming intricate and functional structures are due to the combined use of standard Watson-Crick base pairing, non-standard base pairing, and

a variety of non-covalent contacts involving phosphates, ribose oxygen and cationic metals A significant component for building complex RNA structures is the 2' hydroxyl of ribose, as the oxygen atom of this group can act both as a donor and as an acceptor for hydrogen bonding [7]

1.1 Classes of Ribozymes

Ribozymes, or catalytic RNAs, were first discovered in the laboratory of Tom Cech,

at the University of Colorado, in 1982 They found that the ribosomal RNA precursor

from Tetrahymena thermophila contained an intron, a nonencoding sequence that interrupts the gene, and was capable of excising itself, in vitro, without any protein or

external energy source [8] Shortly thereafter, Sidney Altman's group, at Yale

University, showed that the RNA component of RNase P, M1 RNA, from Escherichia

coli was likewise able to process tRNA precursors without any protein factors [9]

This seminal work ushered in a major research activity, and RNA catalysis was soon found to be widespread in nature, occurring in plants, bacteria, viruses, and lower eukaryotes

Seven naturally occurring classes of catalytic RNAs have been identified to date

(hammerhead, hepatitis delta virus, hairpin, Neurospora Varkud satellite, group I

introns, group II introns and ribonuclease P), all of which catalyze cleavage or

ligation of the RNA backbone by transesterification or hydrolysis of phosphate groups

The hammerhead, hepatitis delta virus (HDV), hairpin, and Neurospora Varkud

satellite (VS) ribozymes are small RNAs of 50–150 nucleotides that perform specific self-cleavage [10–16] The general mechanism of these self-cleavage reactions is similar to that of many protein ribonucleases in which a 2’-oxygen nucleophile attacks the adjacent phosphate in the RNA backbone, resulting in cleavage products with 2’, 3’-cyclic phosphate and 5’-hydroxyl termini Found in viral, virusoid, or satellite RNA genomes, these small catalytic RNAs process the products of rolling circle replication into genome-length strands [17]

site-Base O

O O

H H Base

H HO 5'

3'

Base O

O

-Base 5'

3'

O 1

3 O 4

Base O

O O P O O

-O

O

OH O

HO

Base 5'

3' +

A

B

Base O

OH O

3'

Base O OH O

O

OH O

O

Base 5'

3'

+ 2

Nuc O

H

1 OH

5 O H H

Base O

OH O P

3'

2 3

O 4 Nuc

OH

P O O

O

-Nuc

2

2 1

Figure 1-1 Two mechanistic classes of ribozyme phosphodiester cleavage Circles

represent divalent cations and several ways that they may participate in catalysis Although most of the ions are shown participating in the transition state of the reaction, they may also contribute to ground-state stabilization and substrate binding (represented by M2, the square labeled 2) (A) Mechanism A (mA), observed in metal catalyzed RNA hydrolysis and RNA cleavage by the hammerhead, hairpin, hepatitis

delta, Neurospora, and tRNA ribozymes The attack of the 2’-OH group proceeds by

an in-line SN2 nucleophilic displacement with inversion of the configuration at phosphorus [43] The M2 square represents a divalent cation that promotes catalysis

by the hammerhead ribozyme In addition to coordinating the nonbridging phosphate oxygen, M2 may also stabilize the oxyanion nucleophile (B) Mechanism B (mB) found in catalysis by ribonuclease P, group I and group II intron ribozymes The exogenous nucleophile (Nuc), which can be water or hydroxyl functionality, attacks the phosphorus center by means of SN2 displacement [136].The square labeled 3

represents M3, a divalent ion that promotes catalysis by the Tetrahymena ribozyme

Group I introns, group II introns and ribonuclease P (RNase P) are larger, more structurally complex ribozymes with several hundred nucleotides in length [18–20] RNase P cleaves precursor RNA substrates at specific sites to generate functional 5’-

termini [21], and group I and II introns catalyze two-step self-splicing reactions [22–24] In these large ribozymes, the nucleophile and the labile phosphate are located on different molecules or are greatly separated in sequence Thus, the complex folds of these RNAs serve to orient the nucleophile and phosphate to ensure accurate cleavage

or splicing

1.1.1 Hammerhead ribozyme structure and catalysis

The hammerhead ribozyme was the first self-cleaving RNA to be discovered [25, 26], the first ribozyme to be crystallized [27, 28], and has been the focus of more studies than any other catalytic RNAs Even so, a complete understanding of its reaction mechanism still remains elusive [29-31] Hammerhead and hairpin ribozymes are found in opposite strands of the same plant virus satellite RNAs, and they catalyze identical chemical reactions [32] Nonetheless, the two ribozymes adopt different structures and have different biochemical features, including different pH and metal-cation dependencies and different proficiencies for catalyzing RNA ligation [33]

A minimal hammerhead contains three base-paired helices (helices I, II, and III) around a core of conserved nucleotides Two crystal structures of non-cleavable variants of the hammerhead ribozyme have been solved, one with a DNA substrate strand and the other with an RNA substrate containing a 2’O-methyl group at the cleavage site [34, 35] Both showed the three helices are arranged in a Y shape, as predicted by fluorescence and native gel electrophoresis data [36, 37] Stems II and III are essentially coaxial, while helix I lies at a sharp angle to helix II Backbone distortions at the junction of helices II and III force nucleotide C17 to stack on stem I rather than on stem III, placing it in the active site pocket at the three-helix junction

The scissile phosphate on the 3’-side of C17 lies above a hairpin turn of the backbone formed by the C3-A6 sequence This CUGA turn is strikingly similar to that found in the anti-codon loop of tRNA, which serves as a metal binding pocket [38]

Many experiments have been performed to determine the mechanism of hammerhead self-cleavage and the role of divalent cations in the reaction Sulfur substitution for the scissile phosphate oxygens showed that there is inversion of configuration about the phosphate during the reaction, indicating that the reaction proceeds by in-line attack of the nucleophile [32, 39-40] Further, in most of the experiments in which sulfur was substituted for the pro-Rp non-bridging oxygen of the scissile phosphate, the reduced activity was rescued by addition of a thiophilic metal ion, suggesting that

a metal ion directly coordinates this oxygen in the transition state [40-45] The reaction rate increases linearly with pH, indicating that the nucleophile is activated by

a hydroxide ion [46] Either a metal ion hydroxide deprotonates the 2’-hydroxyl directly, or a metal ion coordinated to the 2’-hydroxyl increases the acidity of the 2’-oxygen, rendering it susceptible to attack by a hydroxide ion from solution [46, 47] The ion coordinated to the pro-Rp phosphate oxygen could perform either of these functions If the hammerhead uses a two-metal ion mechanism for self-cleavage, then

an additional directly coordinated ion should stabilize the leaving group oxygen [48, 49] While sulfur substitution for the leaving group oxygen has failed to identify such

a metal ion, other observations support its existence [47, 50-51] Thus, there is debate over the number of divalent cations directly involved in hammerhead catalysis Strikingly, recent experiments demonstrate that the ribozyme can function in the complete absence of divalent cations at extremely high ionic strengths [52] This result showed that divalent cations are not essential cofactors in the reaction, though

at least one directly coordinated magnesium ion appears to be involved in catalysis

under most conditions in vitro

1.1.2 Hairpin ribozyme structure and catalysis

Another catalytic RNAs domain found in pathogenic plant virus satellite RNAs is the hairpin motif Similar to the other small ribozymes, hairpin catalysts cleave concatameric precursor molecules into mature satellite RNA during rolling-circle replication, giving rise to a 2’-3’-cyclophosphate and a free 5’-OH terminus Depending on reaction conditions, the hairpin ribozyme may also favour RNA ligation over cleavage [53] Up to now, three different hairpin ribozymes have so far been found in nature, of which the one from satellite RNA associated with tobacco ring spot virus is the best characterized [54, 55] The other two hairpin ribozymes, isolated from different satellite viruses, showed the sequence variations that preserve the overall structure of the molecules [56]

In naturally occurring hairpin ribozymes, the catalytic entity is a part of a four-helix

junction A minimal catalytic motif, containing approximately 50 nucleotides, has

been identified that can be used for metal-ion dependent cleavage reactions in trans It

consists of two domains, each of them harbouring two helical regions separated by an internal loop, connected by a hinge region One of these domains results from the

association of 14 nucleotides of a substrate RNA with the ribozyme via base-pairing

In recent work, crystal structures of hairpin ribozymes bound to a modified, cleavable substrate molecule, a transition state mimic, and a product complex have been obtained [57,58] These structures and related biochemical data showed that the

un-active site contains no tightly bound, well ordered metal ions at the site of cleavage Catalytic activity of the hairpin ribozyme thus results from distortion and precise orientation of the substrate RNA and general acid base catalysis by nucleotides in neighborhood without involvement of metal ions in catalysis [55] The requirement for metal ions may be explained by a significant role in the folding process [59, 60] It has also been shown that catalytic activity of the hairpin ribozyme can be supported

by spermine, the major polyamine in eukaryotic cells [61]

1.1.3 Hepatitis delta virus and Varkud Satellite ribozymes

The hepatitis delta virus ribozyme was found in a satellite virus of hepatitis B virus, a major human pathogen [62] Both the genomic and the antigenomic strands express

cis-cleaving ribozymes of ~85 nucleotides that differ in sequence but fold into similar

secondary structures A crystal structure of the ribozyme has been determined [63], in which five helical regions are organized by two pseudoknot structures There is strong evidence that the catalytic mechanism of the hepatitis delta virus ribozyme involves the action of a cytosine base within the catalytic centre as a general acid-base catalyst The hepatitis delta ribozyme displays high resistance to denaturing agents like urea or formamide

The Varkud Satellite (VS) ribozyme is a 154 nucleotides long catalytic entity that is transcribed from a plasmid discovered in the mitochondria of certain strains of

Neurospora [64] The VS ribozyme is the largest of the known nucleolytic ribozymes

and the only one for which there is no crystal structure available to date The global structure has been determined by solution methods, particularly Fluorescence

Resonance Energy Transfer (FRET), which revealed a formal H shape of the five helical segments [65]

1.1.4 General properties of introns

Introns, or intervening sequences (IVS), are nonencoding sequences that interrupt the coding, exon, sequences These introns must be removed, at the RNA level, in order for the gene to be expressed functionally There are five major categories of introns and splicing mechanisms These consist of nuclear tRNA introns [66], archaeal introns [67], nuclear mRNA introns [68], and the group I and group II introns [69, 70]

Of these introns, some members of the group I and group II are clearly capable of catalyzing their own excision, in vitro, in an RNA-catalyzed fashion

1.1.4.1 Group I introns

Group I introns are found in the nucleus and organelles of eukaryotes, as well as some

prokaryotes and bacteriophages These introns are composed of a strictly conserved core region essential for catalysis and moderately conserved peripheral regions that enhance their catalytic activity [71-74] The mechanism of the catalytic reaction with group I introns involves excision from precursor RNAs through a two-step splicing reaction

A prerequisite for splicing is the binding of an exogenous guanosine (exoG) cofactor

to a pocket in the catalytic core of the intron, referred to as G binding site During the first step of splicing, the cofactor attacks the 5’-splice site (SS) and attaches to the intron, resulting in the release of the upstream exon The exoG leaves the G-binding site and is replaced by the last nucleotide of the intron, which is always a guanosine

(denoted as ωG) The second step is initiated by an attack by the 3’-end of the released exon on the 3’ SS, which results in ligation of the exons and release of the intron RNA Successful catalysis is dependent on the correct folding of the intron [75-77] Group I intron splicing requires divalent metal ions for proper folding and catalysis [77] Mg2+ coordination to substrate oxygen atoms activates the nucleophile, stabilizes the scissile phosphate and stabilizes the developing charge on the leaving group The proton of the 2’-OH at the cleavage site is shared between the 2’-and 3’-oxygens in the transition state [78]

1.1.4.2 Group II introns

Group II introns are found in low frequencies in the mitochondrial genomes of fungi,

sporadically in the organellar genomes of algae They are numerous in the organellar genomes of higher plants, and are also surprisingly widespread in the bacterial world [79] One of the most interesting features of group II introns is their remarkable ability to catalyze transposition, and their own migration to specific sites in new genomes [80] These large ribozymes are organized into six domains of highly conserved secondary structure, which folds into a tertiary structure that recognizes 5’-exon (or oligonucleotide substrates) through two stretches of base pairing Furthermore, it has been evidenced that there is a functional inter-exchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result

in trans-activation of intron transposition and RNA-based exon shuffling [81] Group

II introns are true metalloenzymes that require Mg2+ specifically for folding, and substrate binding, and for direct coordination to the leaving group during chemical catalysis [82] Many studies indicated that domain 5 (D5) forms the active-site centre

of the enzyme, and that individual atoms on D5 contained specific mechanistic roles

[82-85] A variety of biochemical and spectroscopic experiments have shown that a metal ion binding platform within D5 plays an instrumental role in catalysis Available data suggested that a metal ion is directly coordinated to both the 3’-oxygen and the 2’-OH in the transition state [86]

1.1.5 RNase P

RNase P is a ubiquitous enzyme that acts as an endonuclease to generate the mature 5’-end of tRNA precursors In bacteria, RNase P exists as a ribonucleoprotein complex, consisting of a long RNA, typically 300-400 nucleotides in length, and a small protein of approximately 14 kDa The discovery that the RNA component of the

enzyme alone still possesses catalytic activity in vitro provided the first example of an RNA-based catalyst that acts in trans on multiple substrates [87] RNase P can be considered to be the only true naturally occurring trans-cleaving RNA enzyme known

to date For full enzymatic activity under in vivo conditions, however, the protein

component is essential In human cells, RNase P contains multiple proteinaceous components and in the absence of protein, the RNA moiety is thought to be catalytically inactive

But, in its quest for catalytic perfection, evolution may not have fully exploited all available molecular formats for enzyme construction Recently, enzyme engineers have attempted to construct DNA molecules that have catalytic properties, called deoxyribozymes However, the most significant difference between DNA and its catalytically powerful RNA cousin is the absence of the 2’-oxygen at each nucleotide Does the lack of this atom render DNA incapable of forming complex tertiary structures? The absence of an enchained nucleophile (the 2’-hydroxyl group) at each

phosphodiester linkage makes DNA ~100000 fold more stable than RNA under physiological conditions [88] Similarly, DNA phosphodiester bonds are 100-fold more resistant to hydrolytic degradation than the peptide bonds of proteins [89] This stability, coupled with the typical double helix structures existing in nature for DNA, makes DNA an ideal molecule for information storage and transfer Yet, it is precisely these characteristics, the ones that are most attractive for building the ideal genetic molecule, but these must be overcome if DNA is to be turned into powerful enzymes DNA can conquer the structurally restrictive effects of its typical duplex structure simply by abandoning its complementary strand, since single stranded DNA can form

a great diversity of structures by utilizing more exotic secondary and tertiary interactions including nonstandard base pairs, stabilizing hairpin loops, internal bulges, multi-stem junctions, pseudoknots, and four stranded G-quadruplex structures [90]

By using various combinatorial selection strategies, enzyme engineers have created

~100 classes of deoxyribozymes that catalyze nearly a dozen different types of reaction, including RNA cleavage and DNA modification [94, 95, 97]

1.2 Classes of Deoxyribozymes

While ribozymes consisting of RNA are distributed widely in nature, no DNA molecules with catalytic activity seem to have evolved The general impression of DNA’s potential as an enzyme is shaped by its natural role as a double-helical molecule used to store genetic information The repetitive structure of double-helical DNA is expected to restrict its catalytic potential, just as an antisense oligonucleotide would inactivate a ribozyme All deoxyribozymes are in fact single-stranded and

therefore, like single-stranded RNAs, have the potential to form higher-ordered structures

DNA, by comparison to RNA, is lacking a 2’-hydroxyl group at each nucleotide, and therefore cannot employ this group to form structure [91, 92] Without a natural deoxyribozyme to study, much effort had been spent on establishing DNA’s ability to form structures or to enhance chemistry relative to either RNA or protein What is known about laboratory-created deoxyribozymes indicates that DNA has considerable potential for folding and catalysis and exhibits true enzyme-like behavior [93] The first deoxyribozyme was discovered in 1994 [94] by Ronald R Breaker and Gerald Joyce at The Scripps Research Institute in La Jolla, CA This deoxyribozyme assists

in lead ion dependent RNA cleaving operations Catalytic amplification was found to

be 100-fold compared to the uncatalysed reaction All known DNA enzymes have

been created by using in vitro selection process (Figure 1-2) With this method, more deoxyribozymes have been created to cleave RNA, cleave DNA, cleave N-glycosidic

bonds, modify and ligate DNA, and metalate porphyrin rings [94, 97, 102-103, 106, 134] Some of these deoxyribozymes can enhance the rate constant for their

corresponding uncatalyzed reactions by 10 billion fold

DNA pool

random dna sequence

Biotin modif ication

DNA pooldouble stranded DNA

DNA cloning and sequencing

1

2

3

4

Figure 1-2 In vitro selection process of deoxyribozymes: A population of single

stranded DNA molecules (top) is chemically synthesized with regions of random DNA sequence (box) and regions of known DNA sequence (plain lines) used for example as PCR primer binding sites This population is subjected to the desired selective conditions (1) during which the portion of the population of different molecules that can perform the required function, such as self-modification in a constant-sequence region (filled circle), is allowed to react Molecules that react become substrates for PCR amplification (2) Mutations may be introduced at a low frequency under typical PCR conditions or at a higher frequency with mutagenic PCR protocols An enriched population of single-stranded functional sequences is generated that is identical in format to the starting synthetic pool by the appropriate choice of PCR primers and other molecular biology methods (3) The enriched population is subjected to the desired, perhaps more stringent, selective conditions again (1), and the cycle is repeated until catalytic activity can be detected through biochemical assay At this time, the DNA population is cloned and sequenced to

identify individual functional DNAs (4)

1.2.1 RNA-cleaving deoxyribozymes

Most deoxyribozymes created to date promote RNA cleavage by phosphoester transfer (Figure 1-3) This group of catalytic DNAs highlights the fact that DNA is capable of forming specific contacts with a number of metal ion or amino acid cofactors that are essential for function [95] In addition, the discovery of several

“cofactor less” deoxyribozymes indicates that DNA has the chemical power to promote RNA transesterification even in the absence of external chemical groups [95,

96] The first report of an RNA-cleaving deoxyribozyme in 1994 revealed that a short DNA (38 mer) could form a complex structure that enhanced the site-specific, multiple turnover cleavage of RNA substrates by ~100,000 fold in the presence of divalent lead [94] The subsequent research showed not only that DNA could act as a true catalyst but also that such enzymes could be readily obtained Some of the more recently derived examples also showed that deoxyribozymes can be made to have an effect on biological systems and that they have promise in diagnostic and therapeutic applications One RNA-cleaving deoxyribozyme provides a striking illustration of the impact that the study of catalysis by DNA can have on biology

c

16.2-11 RNA-cleaving deoxyribozyme

C C T U G

Figure 1-3 RNA cleavage by transesterification and two deoxyribozyme motifs that

catalyze this reaction (a) RNA strand scission occurs when the oxygen of the 2’ hydroxyl group serves as a nucleophile to attack the phosphorus center of the adjacent phosphoester linkage in an SN2-like reaction, resulting in 2’, 3’-cyclic phosphate and 5’-hydroxyl products Proposed secondary structures of the 10–23 (b) and 16.2–11 (c)

RNA-cleaving deoxyribozymes

1.2.2 DNA-cleaving deoxyribozymes

The absence of 2’-hydroxyl group makes DNA approximately 105 times more resistant to hydrolysis compared with the rate constant of the related internal RNA transesterification reaction [88, 98] The extrapolated background rate of DNA hydrolysis is ~10–12 min–1 at neutral pH and at 25°C As a result, even a 100 million

fold rate enhancement during an in vitro selection reaction for DNA hydrolysis would

yield less than 1% cleaved DNA in a 12 hrs incubation It is expected that deoxyribozymes that catalyze phosphoester hydrolysis versus RNA transesterification will require a more sophisticated active site in order to permit water to make an efficient nucleophilic attack on phosphorus Therefore, deoxyribozymes that hydrolyze phosphoester must be exceedingly rare in a given random-sequence population, and as might be predicted; there have been no reports of hydrolytic self-cleaving DNAs

1.2.2.1 DNA self-cleavage reaction by oxidative mechanism

However, there are two alternative mechanisms for DNA cleavage that are exploited

by deoxyribozymes First, the spontaneous oxidative cleavage of DNA [99] by hydroxyl radicals can be induced by using redox-active metals such as Cu2+ and

reducing agents such as ascorbate [100, 101] In an earlier study, Carmi et al

identified two classes of DNA enzymes which can self-cleave by using an oxidative mechanism [102] Class I self-cleaving deoxyribozymes require Cu2+ and ascorbate to promote DNA cleavage, whereas class II DNAs require only the addition of Cu2+ as a cofactor Class II self-cleaving deoxyribozymes [103] appear to catalyze the cleavage

at one particular inter-nucleotide linkage by promoting the attack of a hydroxyl

radical at the 4’ carbon [104], thereby initiating a series of rearrangements [105] that

lead to loss of a nucleotide base and chain cleavage However, hydroxyl induced cleavage can occur at several positions along the DNA chain by variations of this mechanism, leading to a diversity of cleavage products [104] Its structure is also notable in that there is evidence to support a triplex tertiary interaction with substrate

radical-(Figure 1-4), which has no precedent in nucleic acid enzymes

Figure 1-4 DNA cleavage reaction by oxidation strategies (a) Directed attack of a

hydroxyl radical on the C4’ position induces rearrangements that lead to chain cleavage (b) Proposed structural model of the class II DNA cleaving deoxyribozyme includes two base-paired regions and a triplex interaction (dots) with substrate confirmed by compensatory mutational analysis

1.2.2.2 DNA self-cleavage reaction by depurination process

A second mechanism of DNA scission involves cleavage following a site-specific

depurination event [106] The “10–28” deoxyribozyme functions as an N-glycosylase

enzyme by catalyzing the depurination of a specific guanosine residue (Figure 1-5A) Amines or other agents that can serve as general bases will then accelerate the cleavage of the abasic DNA linkage In 2 mM Ca2+, the “10–28” enzyme catalyzes

depurination of a separate substrate oligonucleotide with a pseudo-first-order rate constant of 0.2 min–1 This corresponds to a rate enhancement for depurination of ~1 million fold over the corresponding uncatalyzed reaction The mechanism could be an

SN1 type, where protonation at the N7 position of guanine allows the base to depart

and thereby permits water to attack the 1’ carbon The resulting abasic deoxyribose

site in the DNA can undergo ring opening that would make the linkage susceptible to β-elimination, which would release the pentose while breaking the DNA strand to yield 3’- and 5’-phosphate terminated fragments The intermediate abasic site, released guanine, and phosphate-terminated fragments have all been detected experimentally for this enzyme system

Figure 1-5 DNA cleavage reaction by depurination process (a) The proposed

mechanism of the 10–28 deoxyribozyme involves protonation at the N7 position of guanine, which facilitates the departure of the base moiety and permits subsequent attack by a water molecule at the resulting C1’ carbocation of deoxyribose (b) Proposed secondary structure of the 10–28 N-glycosylase deoxyribozyme

Although deoxyribozymes with DNA-cleaving activity have been created by an oxidative mechanism or depurination process, a nucleotide will be eliminated from the target DNA chain during these cleavage processes, and these elimination processes will in turn result in the loss of the encoded information in sequence In contrast to oxidative DNA cleavage, which results from the facile attack of a hydroxyl radical on the base or on deoxyribose moieties, direct hydrolysis of phosphate esters is likely to be far more difficult for a deoxyribozyme to employ, because phosphate ester hydrolysis in particular is believed to be one of the most challenging objectives

considering the extraordinarily slow background rate of hydrolysis of DNA [107] Yet, this mechanism may be the most attractive for a DNA-cleaving deoxyribozyme as no loss of genetic information occurs upon DNA strand cleavage In view of the accomplishments of ribozymes, we could believe that DNA molecule could possess such catalytic capabilities too in nature

1.3 DNA Cleavage throuth Direct Hydrolysis of Phosphodiester

How can DNA, with its poor complement of chemical functionalities, be catalytic without outside help? How about cofactors? Protein based enzymes use an array of organic cofactors to provide external sources of functional groups, hydrogen atoms and electrons [109] There are examples of ribozymes that use protein cofactors for folding or stabilizing their structures [110] There are many examples of the nonspecific hydrolysis of RNA using a variety of small peptides [111-113] and organic molecules [114-116] In the past years, Roth and Breaker [95] have described

a DNAzyme that strictly requires the imidazole group of histidine for catalysis Furthermore, DNAzymes can overcome these functional limitations by utilizing divalent metal ions cofactors for catalysis [117-118] Metal ions would function in two ways First, they would partially neutralize the negative-charge density on the backbones of single-stranded DNA, and promote its folding into complex shapes Second, water molecules coordinated to divalent cations such as Mg2+, Ca2+, Zn2+ or

Pb2+ would carry out the acid/base catalysis useful for phosphodiester bond hydrolysis

Selection for deoxyribozymes that catalyze the direct hydrolysis of a phosphoester linkage is expected to be challenging However, deoxyribozymes that catalyze DNA hydrolysis might yet be amenable to isolation by taking advantage of substrate

molecules that carry analogues of the DNA phosphodiester linkage For example, replacement of the 3’-or 5’-oxygen atom of a DNA phosphodiester with an amine group yields an analogue DNA linkage that is far more susceptible to hydrolytic cleavage (due to the nature of the leaving group) The 5’-modified version of this linkage analogue has already been used as a substrate to select for deoxyribozymes that cleave phosphoramidate-containing DNAs [108] The phosphoramidate modification changes only the leaving group, whereas the geometry and chemical nature of the transition state remain largely unchanged Thus, the catalytic mechanisms that allow an enzyme to cleave this modified DNA might also be applicable to normal DNA albeit at a much slower rate Perhaps mutagenized populations of phosphoramidate-cleaving deoxyribozymes would make an opportune starting point for the isolation of variant deoxyribozymes that have the catalytic power

to efficiently cleave DNA by hydrolysis And it is expected that if deoxyribozymes that catalyze phosphoester hydrolysis versus RNA transesterification really exist in nature, a more sophisticated active site will be required to permit water molecule to make an efficient nucleophilic attack on phosphorus

1.4 G-quadruplexes

DNA can adopt several secondary structures besides the well-known canonical B-type duplex DNA sequences that contain four or more closely spaced guanine-tracts can fold to form intramolecular quadruplexes, which consist of stacked G-quartets [Figure 1-6] that are linked by three loops between the four G-strands [119] These structures are stabilized by monovalent cations, especially potassium ion [120], and can adopt a variety of different folding patterns depending on the relative orientation of the strands and the position of the loops

Figure 1-6 The guanine tetrad motif and its hydrogen bonding scheme

For intramolecular quadruplexes, the four G-tracts are separated by loops These are

of various lengths and can be as short as a single nucleotide [121] The loops can be arranged in several different ways [Figure 1-7]; external (propeller) loops link two adjacent parallel strands [122], while edgewise or diagonal loops link two antiparallel strands [123] Some structures contain both edge-wise and propeller loops [124-126]

It is known that loop length and sequence affect quadruplex stability and structure [127] Sequences with single nucleotide loops between the guanine-tracts only adopt a parallel structure, while longer loops can also adopt an antiparallel arrangement of the strands Quadruplex stability is also affected by the sequence of the loops [128], and the bases that flank the quadruplex [129]

Figure 1-7 Strand connectivity alternatives for intramolecular G-quadruplex

structures (A) All three loops run edgewise and connect adjacent-adjacentadjacent (B) One diagonal and two edgewise loops that connect adjacent-diagonal-adjacent (C)

An example of a loop that runs on the outside of the guanine tetrad core

There is considerable variation in quadruplex structures, depending on the DNA sequence and the ionic conditions [121] The biological function of quadruplexes may well depend on the folded conformation that is adopted, especially if this involves interaction with specific proteins Such an effect has been suggested for the NHE element of the c-myc promoter, which can in principle adopt multiple conformations Since the loops can have a considerable effect on quadruplex folding and stability, many experiments have been designed to examine how changes in loop length affect quadruplex properties One very stable intramolecular quadruplex contains four guanine-tracts that are linked by single T residue [130-132] and this is known to be an inhibitor of HIV integrase

1.5 Guanine Quartets as Structural Components for Some DNA Aptamers and DNA Enzymes

Natural DNA serves almost without exception as an informational macromolecule that exists primarily as a base-paired duplex Single- tranded DNAs are rarely encountered in nature, and even less frequently do these single-stranded DNAs form shapes (other than the ususal duplex) that serve a functional role Yet a number of compelling arguments can be made to support the notion that certain DNAs are capable of forming structures that mimic those of folded RNAs, including ribozymes The G-quartet motif has been identified or implicated in a number of DNA aptamers and deoxyribozymes, although it is not unique to DNA, and is at the top of the putative list of recurrent DNA structural motifs The primary role for the guanine-quartet in folded DNAs might be to serve as a stable center around which other structural domains can be assembled The first example of a protein-specific DNA

aptamer was reported by Bock et al [133], who identified a 15-nucleotide DNA that

binds to and inhibits the function of human thrombin This DNA consists of stacked guanine-quartet structure Guanine quartets structure in particular appear in many single-stranded DNAzymes, possibly including the porphyrin-metalating deoxyribozyme, the HD RNA-cleaving DNA enzyme and the ATP-utilizing kinase deoxyribozyme [95, 97, 134] Any attempts to exploit a systematic approach to the rational design of DNA aptamers and enzymes would be further progressed if we have grasped the ability to examine examples of structured DNAs and thereby have gained a better understanding of the principles that regulate DNA folding

1.6 Objectives

Since the 1980s when Thomas Cech made the first surprising discovery that RNA molecules are capable of catalyzing reactions in the absence of any protein component, seven naturally occurring classes of catalytic RNA have been identified to date, all of which catalyze cleavage or ligation of the RNA backbone by transesterification or hydrolysis of phosphate groups While ribozymes consisting of RNA are distributed widely in nature, no DNA molecules with catalytic activity seem to have evolved DNA, by comparison to RNA, is lacking a 2’-hydroxyl group at each nucleotide, and always exists in a double-helical molecule to restrict its catalytic potential

By using various combinatorial selection strategies, enzyme engineers have created

~100 classes of deoxyribozymes that catalyze nearly a dozen different types of reaction including RNA cleavage and DNA modification [95, 96] For example, deoxyribozymes with DNA-cleaving activity have been created by an oxidative mechanism or depurination process By oxidative mechanism or depurination process,

a nucleotide will be eliminated from the target DNA chain during the cleavage process, and the elimination process will in turn result in the loss of sequence-encoded information

Yet, in contrast to oxidative DNA cleavage, which results from the facile attack of a hydroxyl radical on the base or on deoxyribose moieties, direct hydrolysis of phosphate esters may be the most attractive for a DNA-cleaving deoxyribozyme as no loss of genetic information occurs upon DNA strand scission But, as we know, phosphate ester hydrolysis in particular is believed to be one of the most challenging objectives, so direct hydrolysis of phosphate esters is likely to be far more difficult for

a deoxyribozyme to employ to catalyze DNA, considering the extraordinarily slow background rate of hydrolytic cleavage of DNA

DNA can overcome the structurally restricting effects of its typical duplex structure simply by abandoning its complementary strand Single stranded DNA can form a great diversity of structures by exploiting more exotic secondary and tertiary interactions including nonstandard base pairs, stabilizing hairpin loops, internal bulges, multi-stem junctions, pseudoknots, and four stranded G-quadruplex structures [90] For example, guanine quartets, which are distinguished by their exceptional rigidity and stability, are likely to be a recurrent structural element of folded DNAs Guanine quartets in particular appear in many single-stranded DNA structures, possibly including the porphyrin-metalating deoxyribozyme, the HD RNA-cleaving DNA enzyme and the ATP-utilizing kinase deoxyribozyme [134-135] Each tier of a stacked guanine quartet requires only four nucleotides to build, thereby serving as an economic way of creating a stable structural core from which to constitute an active

center Ribozyme makes an extensive use of contacts with 2’-hydroxyl groups to bring distal regions into proximity to help constrain independently folding subdomains DNA, of couse, must invent alternative ways to link otherwise disparate structural components, and guanine quartets may be one element that serves this purpose

In addition, a number of metal and non-metal ion cofactors such as Mg2+ and histidine will also be the excellent candidates to trigger the cleavage reaction, because they both are found to play significant roles in the reactions involving RNA and DNA phosphoester transfer and hydrolysis by some kind of enzymes, where such metals as

Mg2+ can serve as Lewis acids or as general acid/base catalysts in the form of metal hydroxides and it can also partially neutralize the negative charge density on the backbones of single strand DNA to promote its folding into complex shapes Histidine

is chosen as a candidate cofactor because of the potiental for the imidazole side chain

to function in both general acid and general base catalyses near neutral pH Histidine

is one of the residues most frequently used to form the active sites of protein enzymes [136]

So in view of the exceptional structural versatility, rigidity and stability inherent with G-quadruplex, we are planning to utilize guanine quartets as the template to construct new types of deoxyribozymes with self-splicing activity Firstly, certain guanine-rich oligonucleotide squence which could form G-quadruplex with proper folding structure and strand connectivity through self-assembly is designed and its self-splicing activity is tested accordingly Further information obtained from polyacrylamide gel electrophoresis and Mass Spectrometry analysis shows that

instead of a self-splicing reaction, a self-cleavage reaction occurs in the middle of the designed sequence with a highly site-specific feature Secondly, with the purpose to confirm that the occurrence of self-cleavage reaction is fully dependent on the proper conformation of G-quadruplex assembly, and to explore more catalytic potentials with these G-quadruplex assemblies, additional oligonucleotide sequences with different catalytic loop geometry are designed and tested for their catalytic activities Finally, a number of metal and non-metal ion cofactors such as Mg2+ and histidine which are found to make excellent cofactors for reactions involving RNA and DNA phosphoester transfer and hydrolysis, will also be searched and examined in my study

So, it is our expectation that the results shown in the current studies could serve as a starting point for the further development of a DNA enzyme that more closely mimic the function of ribozyme and protein-enzyme

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CHAPTER 2 Search for New Types of Deoxyribozymes with Self-Cleaving

Activity under Hydrolysis Pathway

2.1 Background Information

A large number of self-cleaving ribozymes have been identified in biological systems and generated in the laboratory Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able

to function as an enzyme as well with self-cleaving activities

DNA, by comparison to RNA, is lacking a 2’-hydroxyl group at each nucleotide, and always exists in a double-helical molecule to restrict its catalytic potential DNA can overcome the structurally stifling effects of its typical duplex structure simply by abandoning its complementary strand Single stranded DNA can form a great diversity of structures by exploiting more exotic secondary and tertiary interactions including nonstandard base pairs, stabilizing hairpin loops, internal bulges, multi-stem junctions, pseudoknots, and four stranded G-quadruplex structures [1]

The guanine-rich sequences that exhibit potential for the formation of G-quadruplex are found in a number of applications, such as targets for anticancer drug development,

In addition, some aptamers, such as an inhibitor of HIV integrase [2] and the thrombin-binding aptamer [3], are formed through the utilization G-quadruplex as their structural cores as well The importance of the quadruplex as a DNA secondary structure motif is established beyond contest Taken altogether, the available data

point to the important roles for DNA quadruplexes in biological systems It consequently becomes apparent that the unimolecular structure of G-quadruplex plays certain significant roles in various types of biological processes [23] So we set out to utilize G-quadruplex as the template to construct deoxyribozymes with self-cleaving activity The self-cleavage reaction may result from the nucleophilic attack by actived water molecule on the phosphodiester bond since the formation of a sophisticated active center

2.2 Results

2.2.1 Emergence of self-cleavage activity

RNA splicing is a fundamental part of RNA processing in many organisms The

pre-rRNA of Tetrahymena thermophila was found to undergo “self splicing” in vitro

without the need for protein catalysts This and other “group I intron” self-splicing ribozymes promote two RNA phosphoester transfer reactions (Figure 2-1) that result

in the removal of an intervening sequence and the splicing of adjacent RNA domains The initial transesterification reaction that is promoted by this ribozyme uses the 3’-hydroxyl of guanosine or one of its 5’-phosphorylated derivatives as the nucleophile for an SN2 attack on the phosphate of the target internucleotide linkage The second transesterification uses the newly formed 3’-hydroxyl of the 5’-exon as the nucleophile in the subsequent attack at the second splice-site junction [4]

Figure 2-1 The first and second steps of RNA splicing reaction by the group I

ribozymes

Since such activity only was observed in RNA world, no such DNA enzyme has been found in nature Our initial goal of this study was to design a DNA sequence that is able to mimic the self-splicing activities of ribozyme upon the formation certain assemblies of G-quadruplex

Protein and RNA enzymes must fold into stable structures to create active sites by using precise spatial positioning of functional groups It is likely that DNA will have its own set of frequently used motifs that are functionally analogous to those recurrent RNA motifs The G-quartet motif has been identified or implicated in a number of DNA aptamers and deoxyribozymes The primary role for the G-quartet in folded DNAs might be to serve as a stable center around which other structural domains can

be assembled

A linear sequence (5’-TGGGGTTAGGGGAAAAGGTTAGGGGTTAGG-3’) which contains 5 stretches of two and four consecutive guanines was designed to form G-quadruplex with proper folding structure and strand connectivity through molecular self-assembly It was anticipated that under certain conditions, four-stranded structures with a guanine tetrad core could be formed by stacking four consecutive