Applications of synthetic oligoribonucleotide analogues in studies of RNA structure and function

Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 106, No. 5, October 1994, pp. 1003-1022.

9 Printed in India.

Applications of synthetic oligoribonucleotide analogues in studies of RNA structure and function

JANE A GRASBY, CLARE E PRITCHARD* and MICHAEL J GAIT*

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

*Present address: Department of Biochemistry, University of Oxford, Oxford, OX1 3QU Abstract. The study of RNA structure and function has been considerably aided by the development of methods for the chemical synthesis of oligoribonucleotides into which have been incorporated modified nucleosides carrying site-specific alterations. Such modifications are designed to eliminate or alter individual functional groups in the R N A which potentially can take part in hydrogen-bonding or other non-covalent interactions. Comparison of the properties of the modified RNA with unmodified RNA models allows conclusions to be drawn concerning the importance or otherwise of specific functional groups within the RNA.

The methods have been applied to studies of RNA structure, RNA catalysis, and interactions of RNA with proteins.

Keywords. Oligoribonucleotides; modified nucleosides; ribozyme; protein-RNA recognition.

1. Introduction

Ribonucleic acids (RNA) are essential components of all living cells and are found also in plant and animal viruses and viroids. RNAs play vital roles as carriers of genetic information (mRNA) and form important three dimensional structures useful in cellular processes (tRNA, rRNA). More recently RNAs have been found to carry out enzymatic reactions (ribozymes). Many of these functions are mediated by specific RNA/RNA or RNA/protein interactions which in turn are determined by the chemistry of RNA and its secondary and tertiary structure (Blackburn and Gait 1990;

Gesteland and Atkins 1993).

Determination of RNA sequence is now a routine technique and this allows computer-assisted identification of possible secondary structures (Jaeger et al 1989;

Zucker 1989). Typically these consist of regions of Watson-Crick hydrogen-bonded duplex RNA interrupted by internal loops, bulges and hairpins. Although the structures of the duplex regions of RNA can be confirmed by enzymatic and chemical probing, less is known about the configurations of nucleosides within the loops and bulges, many of which are thought to contain unusual non-Watson-Crick base-pairs and other hydrogen-bonding interactions. Furthermore, very little is known about the intramolecular tertiary interactions of RNA, although some higher order structures are beginning to be defined (e.g. pseudoknot).

In the absence of structural data, an approach to the identification of functional elements in the RNA involved in non-covalent interactions is to modify the RNA

* For correspondence

1003

1004 Jane A Grasby et al

and to assess the effect of these changes on its structure, activity or recognition potential. One method involves the reaction of RNA with various chemical modifying agents and enzymes (e.g. Ehresmann et al 1987; Karaoglu and Thurlow 1991). These reagents react with limited specificity over an entire nucleic acid strand and are thus useful in probing the structural environment of particular nucleosides within the RNA.

Further insights can be obtained by mutating individual nucleosides in an RNA.

For example, site-directed mutagenesis techniques allow the generation of DNA molecules containing one or more nucleotide substitutions (Smith 1985). The resultant mutated genes are cloned within special plasmid vectors which contain a promoter recognised uniquely by a bacteriophage RNA polymerase (e.g. T7). In vitro transcription then yields the corresponding mutated RNA. Such mutant RNAs can also be generated by in vitro transcription using synthetic DNA templates (Milligan et al 1987). The mutant RNAs can then be studied in relation to their unmutated parent RNA.

However, the simple exchange in an RNA of one of the four nucleosides A,G,C or U (figure 1) for another is a relatively disruptive change, since the hydrogen-bonding and stacking capabilities of the individual bases differ substantially. More subtle changes can be brought about by modification of individual functional groups within a single nucleotide. Sometimes these can be introduced by transcription using modified nucleoside triphosphates (Jones 1979). Although a number of modified nucleoside triphosphates are accepted as substrates by RNA polymerases, it is generally not possible to direct the modification to a single position within an RNA, since the modified residue is incorporated at every position to which it complements during the transcription reaction. However, a new technique involving T4 DNA ligase- catalysed joining of two RNA transcripts in the presence of an oligodeoxynucleotide

~.N NH2

HO OH

~jN O

.o.j

NH2

HO OH

HO

Adenosine Guanosine

0 NH 2

HO OH HO OH

Uridine Cytidine

Figure 1. The four common nucleosides found in RNA.

Synthetic oligoribonucleotide analogues 1005 splint can allow certain modified nucleotides to be incorporated specifically at the site of ligadon (Moore and Sharp 1992).

An alternative and more convenient method of site-specific introduction of modified nucleotides into RNA is chemical synthesis (for a recent review see Usman and Cedergren 1992). Nucleotides which are deleted or modified at individual functional groups are synthesized and incorporated at defined locations within synthetic oligoribonucleotides. These oligoribonucleotides can be annealed and assembled into model RNAs resembling the RNA of interest. By careful comparison of the modified and unmodified RNA models, it is possible to identify those groups within an RNA which are involved in inter- or intra-molecular interactions or which are implicated as sites of interactions with proteins. Furthermore, in some cases it is possible to estimate the strengths and relative importance of such interactions (Fersht 1988).

2. Chemical Synthesis of RNA

Methodologies and practical aspects of RNA synthesis have been reviewed in detail elsewhere (Gait et al 1991; Vinayak 1993). The procedure involves the sequential introduction of nucleotide units starting from the 3'-nucleoside which is attached to

O OIBDMS O OtBDMS

DMTrO "--'~ O Base DMTrO Base

\ i / "T

DMTrO--I O Base

O

Ribonucleosidr

\ ~ / \ phosphoramidite

,.,'/~'1

P

CNCH2CH2 O /

\ O - - ~ B a s e

O OtBDMS

Figure

2. Synthesis cycle for assembly of oligofibonucleotides.

1006 Jane A Grasby et al

a solid support (figure 2). The key step is the formation of internucleoside 5'-3'-phos- phodiester bonds and the method of choice involves coupling of nucleosides as their Y-O-phosphoramidites. These and all other synthesis reagents are commercially available. As for oligodeoxynucleotide synthesis, protecting groups are required for all the other reactive moieties of the nucleotides which are not involved in the coupling reaction in order to achieve selectivity of reaction. The dimethoxytrityl (DMTr) group provides transient Y-hydroxyl protection. This is removed from the growing chain by mild non-aqueous acid treatment before further chain elongation. By contrast, T-hydroxyl protecting groups must stay in place throughout synthesis and be removed only at the very end of the oligonucleotide synthesis.

Two 2'-hydroxyl protecting groups that are currently in use are the t-butyl- dimethylsilyl group (t-BDMS) (Ogilvie et al 1974) and the 1-(2-fluorophenyl)- 4- methoxypiperidin-4-yl (Fpmp) group (Reese and Thompson 1988). The former is labile to tetrabutylammonium fluoride treatment whilst the latter is removed by aqueous acid at pH 2. The heterocyclic bases must also be protected at the exocyclic amino groups during synthesis. Acyl protecting groups developed originally for oligodeoxynucleotide synthesis, such as isobutryl and benzoyl, can be used. However, their removal requires use of quite harsh ammoniacal conditions and this can lead to partial loss of 2'-O-t-BDMS groups and subsequent chain cleavage. To circumvent this, alternatively protected nucleotide derivatives are now available that carry more labile exocyclic amino group protection for A and G, phenoxyacetyl or dimethyl- formamidine (Chaix et al 1989; Vinayak et al 1992). It should be noted that since the current methodologies of RNA synthesis are closely related to those used for DNA, the production of R N A - D N A mixed sequences is a relatively trivial exercise (Usman et al 1992).

3. The synthesis of modified oligoribonueleotides

3.1 Modified ribose moieties

RNA is distinguished from DNA by the presence of the 2'-hydroxyl groups of the ribose function. These groups possess both hydrogen-bond donor and acceptor properties and in addition have been implicated in metal binding in catalytic RNA structures. The simplest chemical probe for the importance of a particular 2'-hydroxyl group is the introduction of the corresponding deoxynucleoside into the RNA.

Although 2'-deoxynudeosides have a preference for the 2'-endo configuration over that of the 3'-endo characteristic of ribose moieties, the introduction of a single or a few deoxynucleosides into an RNA duplex to give an RNA-DNA chimera does not usually perturb the overall geometry of the duplex which remains in the A-type conformation (Egli et al 1993). Alternative probes for the T-hydroxyl function include 2'-fluoro, T-amino, T-O-methyl and 2'-O-allyl ribose compounds (figure 3) (Sproat et al 1990; Pieken et al 1991; Williams et al 1991; Benseler et al 1992; Paolella et al 1992). 2'-Fluoro and 2'-O-alkyl nucleosides adopt the Y-endo sugar pucker characteristic of ribose whilst T-amino nucleosides retain both the potential acceptor and donor properties of the T-hydroxyl function. T-O-methyl and 2'-O-allyl nucleoside phosphoramidites are commercially available.

Synthetic oligoribonucleotide analogues 1007

\ \ \

O ' ~ O B O B

O OH O F O NHz

\ e \ e \ ~ e

o ~ ' - o o,~ - ~ o ~ -

0 0 0

\ \ \

Ribose 2'-Fluoro 2'-Amino

\ \

O OMe O OCH2CH=CH2

\ o \ o

o//~ '- o o ~ ' - o

0 0

\ \

2'-O-Methyl 2'-O-Allyl

Figure 3. Some T-modified nucleosides that have been incorporated into oligoribonucleo- tides.

3.2 Modified bases

The four common ribonucleosides contain a number of exocyclic functional groups and ring nitrogen atoms which potentially may form hydrogen-bonding contacts with other nucleoside bases, proteins or metal ions. In duplex RNA these potential hydrogen-bonding sites can be divided into two groups: those oriented towards the minor groove and those orientated towards the major groove (figure 4). In order to study the roles of the various functional groups, a number of analogues of the common ribonucleosides have been incorporated into synthetic RNA (figure 5). For example, inosine can be considered an analogue of guanosine but with the 2-amino group absent (Turner et al 1987; Odai et al 1990; Green et al 1991; Fu and McLaughlin 1992b; Slim and Gait 1992; Fu et al 1993; Tuschl et al 1993) and purine riboside (nebularine) considered an analogue of adenosine but lacking the 6-amino group (SantaLucia et al 1991; Slim et al 1991; Fu and McLaughlin 1992b; Slim and Gait

1992; Fu et al 1993).

Replacement of the NT-nitrogen atom of adenosine or guanosine by a C-H moiety can be achieved by the incorporation of NT-deazaadenosine (tubercidin) or N L deazaguanosine respectively into oligoribonucleotides (Fu and McLaughlin 1992a;

Seela and Mersmann 1992, 1993; Seela et al 1993). 2-Aminopurine riboside is an analogue of guanosine in which both the O6-keto function and the Nl-proton have been deleted (Doudna et al 1990; SantaLucia et al 1991; Tuschl et al 1993). The

1008 Jane A Grasby et at

Major groove X Major groove

\ o

O'" . N= N ~

o x , o

A : U G : C

Figure

introduction of this double deletion leads to the conversion of a hydrogen-bond donor into an acceptor at N 1 and this can lead to anomalous results in some circumstances (SantaLucia et al 1991). To circumvent these problems we have recently introduced the modified guanosine analogue O6-methylguanosine into oligori- bonucleotides (Grasby et al 1993a, b). Use of this analogue results in only a modest effect on the hydrogen-bonding potential at 0 6 whilst the Nl-proton is deleted. In 2-aminoadenosine (2,6-diaminopurine), an additional exocyclic amino group is added to adenosine at the 2-position (Lamm et al 1991; Sproat et al 1991). Two purine nucleoside analogues that have also been incorporated into RNA, xanthosine and isoguanosine, represent rather more drastic modifications to hydrogen-bonding and/or metal-chelating potential compared to their parent nucleosides (Tuschl et al

1993).

Compared to the purine analogues, rather fewer pyrimidine analogues have been introduced into synthetic RNA. Synthons for the incorporation of 5-bromouridine (Talbot et al 1990), a uridine analogue which introduces steric bulk at the 5 position, and N3-methyl uridine, in which the Nl-proton of uridine is replaced by a methyl group, have been reported (Slim et al 1991; Sumner-Smith et al 1991; Iwai et al 1992).

There has also been a very recent report of the incorporation of 4-thiouridine into synthetic oligoribonucleotides (Adams et al 1994).

Since introduction of a single deoxynucleotide does not greatly perturb the overall structure of RNA helices (Egli et al 1993), in some circumstances it is also possible to utilise the many commercially available deoxynucleoside analogues provided the appropriate deoxynucleoside controls are also synthesized and compared alongside.

The synthesis of modified oligodeoxynucleotides has recently been reviewed/Beaucage and Iyer 1993).

Synthetic oligoribonucleotide analogues

z o

Z - r

o

~ Z

o

I z

~ --F ~

9 I

T Z

~'C "F ~

o

o~

o 1"

- r

o

o ~. o

- r " r

e~

Z

E

0

~ Z

r

o T

0

e . 0

0

0 0

e~

0 "~

0

z ~ ~ 0

Z

.[

o - r

u~

1010 Jane A Grasby et al

4. Probing RNA structure by analogue substitution

4.1 Structure and hydrogen-bonding energies in R N A duplexes and hairpin loops The melting temperature (Tin) of nucleic acid duplexes is dependent on the number and strengths of their intermolecular interactions, which in turn depend upon their base composition (Saenger 1984). Turner and co-workers have exploited this by utilising the modified nucleosides inosine, purine riboside and 2-aminopurine to study the strengths of hydrogen bonds within RNA duplexes, internal and hairpin loops (SantaLucia et al 1991). A study involving replacement of guanosine by inosine in terminal G:C base-pairs demonstrated that the exocyclic amino group of guanosme contributes between - 1.6 and - 0 - 8 kcal mol-1 to the stability of duplex RNAs. In internal loops containing G:A mismatches, a similar energetic penalty of - 1 - 4 kcal mol-1 results upon deletion of the exocyclic amino group of adenosine via purine riboside substitution. By contrast, inosine substitution in internal G:A-containing loops has an energetically beneficial effect indicating that the 2-amino group of guanosine is not involved in hydrogen-bonding interactions within such structures and only one type of hydrogen-bonding pattern in the G:A pair was consistent with these observations. The accumulation of such thermodynamic data can lead to improvements in the ability to predict RNA structure by use of computer algorithms.

Much smaller energies of between - 0.75 and - 0-28 kcal mol- 1 are associated with the deletion of important functional groups within GNRA hairpin loops (SantaLucia et al 1992), the structure of one example of which has been previously solved using nuclear magnetic resonance (NMR) methods (Heus and Pardi 1991). Such a finding has led to the suggestion that the phylogenetic preference for GNRA tetraloops is due to a functional rather than a thermodynamic preference. The structure of the hairpin tetraloop U N C G has been investigated (Sakata et al 1990). By placing 2'-deoxy analogues in the loop region, a substantial drop in stability (measured by thermal denaturation) was observed. Substitution of the conserved guanosine by inosine (i.e.

removal of the 2-amino group) gave a further drop in Tm of 8 ~ suggesting a specific hydrogen-bonding interaction for that group within the loop structure. A subsequent structural model based on N M R data has confirmed the presence of such an interaction (Cheong et al 1990).

The stability of RNA duplexes can be increased by the incorporation of 2-aminode- nosine in place of adenosine. The extra amino group gives rise to a third hydrogen bond in U: 2-aminoA base-pairs making them stronger than normal U:A Watson- Crick base-pairs. This characteristic has been exploited to specifically identify the U5 snRNP in cellular splicing extracts in the study of messenger RNA maturation (Lamm et al 1991).

4.2 Guanosine tetrads

The oligoribonucleotide UG4U, like poly G and guanosine itself, is capable of forming self-aggregated structures especially in the presence of potassium ions (Kim et al 1991).

The high stability of this structure has led to the suggestion that G4 supramolecular structures may play a role in nature. This self-aggregation is thought to occur via the formation of tetrameric structures as have been observed by X-ray studies on poly G (Zimmermann et al 1974). Based on methylation experiments on DNA telomeres

Synthetic oliyoribonucleotide analogues 1011 (Sen and Gilbert 1988), a model was first proposed by Gellert and co-workers (Gellert et al 1962) for gel formation in guanosine 3'-monophosphate. The model proposes hydrogen bonds between the 0 6 and NI-H positions and N 7 and exocyclic amino groups of adjacent guanosines which are extended into polymeric structures. Seela and Mersmann (1993) have utilized N7-deazaguanosine-containing oligoribonucleo- tides to confirm these proposals. Even a single NT-deazaguanosine substitution in UG4U partially disrupts the ability to form guanine tetrads, whereas two or more NT-deazaguanosine substitutions are completely inhibitory.

5. S t r u c t u r e - f u n c t i o n relationships in c a t a l y t i c R N A

5.1 The hammerhead ribozyme

Several plant viroids and virusoids require an RNA cleavage reaction during their replication. This catalytic activity is supplied within their own RNA genomes and consists of an independently folded domain consisting of three double-helical regions, 13 conserved nucleotides and 2 semi-conserved nucleotides. The domain is known as a hammerhead (for a recent review, see Symons 1992). Although in vivo the cleavage reaction takes place in cis, the reaction may be made to occur in vitro in trans using a suitable arrangement of two or more oligoribonucleotides (figure 6) (Uhlenbeck 1987). The cleavage reaction, which requires a divalent metal ion (typically magnesium) results in the generation of oligoribonucleotide products which contain a 2',3'-cyclic

E

31 5 I

X - - X

Stem I X - - X

X - - X

ls.z C - - ~16.z

14

Stem I I 13

X i z ~

X X X Y

x I I I I

X X X X R

X A

9

8 X

15.1 z ~ - - U 16.1

/

B

S

Stem III

X X X X X

11111

X X X X X

C 3

~ U 4 N s

6

,

,

Figure 6. General secondary structure for a trans-cleaving hammerhead ribozyme. Outlined residues are those completely conserved in all hammerheads. X = any ribonucleotide, B = A, C or U,Y = U or C, R = A or G. E denotes the enzyme and S the substrate parts of the ribozyme.

1012 Jane A Grasby et al

phosphate and a 5'-hydroxyl group respectively. The reaction proceeds with inversion of configuration at phosphorus implying an in-line attack of the 2'-hydroxyl group and the development of a penta-coordinate transition state (van Tol et al 1990; Dahm and Uhlenbeck 1991; Koizumi and Ohtsuka 1991; Slim and Gait 1991). However, the structural and divalent metal ion requirements for the hammerhead cleavage reaction are still not clearly understood.

A number of modified hammerhead ribozymes have been prepared in which single or multiple deoxynucleoside substitutions have been incorporated. Multiple deoxy- nucleoside substitutions within the hammerhead conserved residues result in a drastic reduction in kea t whereas Km is largely unaffected (Perreault et al 1990 and 1991). Similar results have been obtained upon multiple substitutions with 2'-fluoro-, 2'-amino ( - ) , 2'-O-methyl- and 2'-O-allyl- nucleosides (Olsen et al 1991; Pieken et al 1991; Paolella et al 1992; Williams et al 1992). Single deoxynucleoside substitutions at G5 and Ga are particularly detrimental to catalysis (Perreault et al 1990, 1991; Fu and McLaughlin 1992b; Williams et al 1992). Incorporation of 2'-fluoro-2'-deoxy- guanosine, 2'-amino-2'-deoxyguanosine and 2'-O-allylguanosine also lead to reduc- tions in rate when substituted at G5 or at G8 although the effect is less drastic in the case of 2'-amino substitution. This has led Williams et al (1992) to suggest a possible hydrogen-bonding role for the proton of the 2'-hydroxyl groups of G5 and Ga. By contrast, replacement of G12 by 2'-deoxyguanosine or by 2'-fluoro-2'- deoxyguanosine enhances the rate of reaction. 2'-amino-2'-deoxyguanosine is less well tolerated but 2'-O-allyguanosine substitution results in a severe loss in activity. The effect of deoxynucleoside substitution at A 9 is disputed. Whereas Fu and McLaughlin (1992b) have reported a 2-fold increase in the relative rate of cleavage upon deoxy- adenosine substitution at this position, Perrault et al (1991) observed a 20-fold reduction in kea t upon similar modification.

Certain positions within the hammerhead conserved residues tolerate 2'- deoxy- nucleoside or other 2'-modifications rather well. These positions can be substituted, often in unison, with very little loss in catalytic efficiency. Since 2'-deoxy and in particular other T-modified nucleosides confer ribonuclease resistance to the RNA, these T-modified ribozymes may be useful for therapeutic applications (Heidenreich and Eckstein 1992; Paolella et al 1992).

Not surprisingly deoxynucleoside substitutions at the site 5'-of the scissile bond in the substrate completely inactivate the hammerhead (Perreault et al 1990) as do 2'-O-methyl-, 2'-fluoro- and 2'-amino-nucleoside substitutions at this position (Koizumi et al 1988; Pieken et al 1991). Deoxynucleoside substitutions within the substrate strand of stems I and III yield reduced rates of reaction (Yang et al 1990).

These reductions are due to increases in Kin, presumably reflecting the greater stability of RNA-RNA duplexes over DNA-RNA duplexes (Walker 1988) and to decreases in k~a t. The greatest decrease in k~, t is produced upon substitution of U16.~ by dT.

This has led Yang et al (1990) to propose either a hydrogen-bonding contact with the ribozyme or a magnesium-binding function for the 2'-hydroxyl group of U~6.~.

Base modification in the hammerhead ribozyme has been concentrated on the purine residues within the conserved residues. The replacement of adenosine residues by purine riboside results in only a modest drop in catalytic efficiency at any position within the conserved region (Fu and McLaughlin 1992b; Slim and Gait 1992; Fu et al 1993). By contrast, removal of the exocyclic amino group of guanosine (replacement by inosine) causes a large drop in catalytic efficiency at positions G5

Synthetic oligoribonucleotide analogues 1013 and G12 whilst the effect at Ga is less marked (Odai et al 1990; Slim and Gait 1992;

Tuschl et al 1993). The NT-positions of guanosine residues appear to be relatively unimportant in catalysis as assessed by NT-deazaguanosine replacement (Fu et al 1993; Grasby et al 1993b). Similar modest effects of the NT-positions of adenosine have been observed except at A6 where tubercidin replacement results in a drastic reduction in the rate of reaction (Fu and McLaughlin 1992a; Seela et al 1993). The importance of the NI-H and 0 6 positions of all three conserved guanosines within the hammerhead has been demonstrated by O6-methylguanosine and 2-aminopurine substitution (Grasby et al 1993a, b; Tuschl et al 1993). In view of the behaviour of these three guanosine residues upon inosine, 2-aminopurine and O6-methyl guanosine substitution, it is perhaps not surprising that the rather less conservative xanthosine and isoguanosine replacements at these positions are detrimental to catalysis (Tuschl et al 1993).

Magnesium ion is essential for hammerhead cleavage and its possible role has been discussed by several authors. First, a solvated divalent metal hydroxide may be responsible for the initial deprotonation of the 2'-hydroxyl group of the residue at the cleavage site (Koizumi and Ohtsuka 1991; Dahm et al 1993). Secondly, experi- ments in which the scissile phosphodiester bond has been replaced by a phosphoro- thioate have suggested that divalent metal ion binds to the pro-Rp oxygen of the phosphate in the transition state of the reaction (Dahm and Uhlenbeck 1991;

Slim and Gait 1991). Furthermore, it is possible that the metal ion interacts either directly or via a water molecule to stabilize the 5'-oxyanion leaving group (Dahm and Uhlenbeck 1991, 1993; Slim and Gait 1991), a hypothesis which is supported by theoretical calculations (Taira et al 1990). Magnesium ion may also play a structural role in the hammerhead cleavage. Thus, decreases in the r-ate of reaction on deletion or modification of an important functional group may result from a decrease in the affinity for a structural or catalytically important magnesium ion or from the removal of an important hydrogen-bonding network.

These proposals have prompted investigations into the magnesium dependence of modified hammerhead ribozymes. For example, an increase in the concentration of magnesium ion used in the cleavage reactions compensates in part for the reduction in rate observed upon deoxynucleoside substitution at G s, A9 or U16.1, from which Perrault et al (1991) have suggested that the 2'-hydroxyl groups of these residues may be involved in direct or water-mediated magnesium chelation. Similar effects have been observed at positions G8 and G5 but not A9 by Fu and McLaughlin (1992b), who have also noted a substantial increase in the rate of cleavage of a ribozyme substituted at G5 by inosine as the concentration of magnesium was raised from 10mM to 50mM. Surprisingly, Tuschl et al (1993) observed only small increases in the rate of cleavage as the concentration of magnesium was raised for inosine- and 2-aminopurine-substituted ribozymes. Substitution of G5 or Ga by O6-methylgua - nosine caused a decrease in affinity for magnesium as evidenced by increases in the apparent magnesium dissociation constants (Grasby et al 1993). Fu and McLaughlin (1992a) have suggested that the N T of adenosine at A6 is a possible site of divalent metal ion binding to the hammerhead and have proposed a model for divalent metal ion binding based on this, although no magnesium dependence studies have been undertaken with this modified ribozyme. Decreases in the ability of the hammerhead to bind magnesium when a functional group is deleted may be explained in different ways. The functional group may be involved in either direct chelation or in water-

1014 Jane A Grasby et al

mediated magnesium chelation. Alternatively, an important hydrogen bond is lost which results in repositioning of a magnesium ion chelator. Magnesium titration experiments alone cannot distinguish between these two possibilities.

We and others (Grasby et al 1993; Tuschl et al 1993) have attempted to quantitate the effects of various functional group modifications on hammerhead cleavage. In cases where magnesium binding is unimpaired, it is possible to equate the energetic penalty (AAG,pptt) paid upon modification of a particular functional group with a binding energy (Fersht 1988). That these binding energies are realised in the transition state of the reaction rather than in the ground state is evidenced by the changes in kcat but not Km usually observed upon analogue substitution. Taken as a whole, the results obtained with analogue substitution of the hammerhead ribozyme point towards a complex network of hydrogen bonding and metal chelation within the catalytic core.

5.2 The hairpin ribozyme

The encapsidated linear satellite RNA of tobacco ringspot virus is considered to replicate via a rolling circle pathway. During the replication cycle, whereas the plus strand cleavage step is provided by a hammerhead structure, the minus strand contains an entirely different autocatalytic structure known as a hairpin (Symons 1990). A similar hairpin arrangement has also been shown to promote self-cleavage in satellite

S

+9

3 ' - X X X X X B

i11111

5 ' - X X X X X V

o

X -4

Y R X X - 5 '

I I I I

G Y X X X ~ X A-3' A 11 X ~ X

G A 1o X ~ X

X X

X X

X G

A

E

A 24 A X 2s C X

X I

X X

X X

X ~ X

X X

X C

A U U A H A

Figure 7. General secondary structure for a trans-cleaving hairpin ribozyme. Outlined residues are those completely conserved in all hairpins. X = any ribonucleoside, V = A, G or C, Y = U or C, R = A or G, B = U, C or G, H = A, U or C. E denotes the enzyme and S the substrate parts of the ribozyme.

Synthetic oligoribonucleotide analogues 1015 RNAs of chicory yellow mottle nepovirus and arabis mosaic virus. Unlike the hammerhead catalytic cleavage the reaction is reversible but given a suitable arrangement of two or three oligoribonucleotides, the cleavage reaction in vitro may be effected in trans (Feldstein et al 1989; Chowrira et al 1993). The residues essential for the cleavage and ligation reactions exhibited by the hairpin have recently been determined using a combination of mutagenesis and in vitro selection experiments (figure 7) (Chowrira et al 1991; Berzal-Herranz et al 1992, 1993; Joseph et al 1993).

The effect of 2'-deoxynucleoside and T-O-methyl nucleoside substitutions within the hairpin ribozyme have been studied (Chowrira et al 1993). Removal of the hydroxyl groups at Alo, GI~, A24 or C25 are detrimental to ribozyme cleavage, resulting in ribozymes with drastically lowered K,,ts. An increase in the concentration of magnesium used in the cleavage reactions partially compensates for removal of the hydroxyl groups at positions G11 and A24. Nucleoside base substitutions in the hairpin ribozyme have concentrated on the role of an essential guanosine residue at G+I, in the substrate strand of a trans-cleaving ribozyme. Substitution by 2- aminopurine riboside at this site rather modestly affects the K,, for the reaction but inosine incorporation leads to an inactive ribozyme (Chowrira et al 1991). This has led to the suggestion that the exocylic amino group of G+ ~ may be directly involved in the chemical cleavage step of the ribozyme reaction either by acting as a general base for removal of the proton from the T-hydroxyl group at position Ao or by hydrogen bonding to the phosphoryl oxygen of the scissile bond and thus stabilizing the transition state of the reaction.

5.3 The tetrahymena 9roup I intron

Splicing of the pre-rRNA of Tetrahymena thermophila is catalysed by a ribozyme consisting of.the intervening sequence (IVS) which mediates the cleavage-ligation reaction and then converts itself into a circular form. The reaction requires a guanosine substrate and a divalent cation co-factor. A shortened form of the IVS has no circularisation sites and so cannot participate in intramolecular reactions but can catalyse cleavage-ligation of exogenous substrates in trans (Zaug et al 1986). The specificities of shortened forms of the IVS are governed by complementary base pairing of the substrate with an internal guide sequence (IGS). Moreover a further truncated form of the ribozyme allows the supply of both IGS and substrate in trans.

Shortened forms of the group I intron from Tetrahymena will process both DNA or RNA substrate sequences. However, DNA sequences are bound with poorer affinity than their RNA counterparts. Although in part this is due to the decreased stability of D N A - R N A duplexes over R N A - R N A duplexes (Walker 1988), Pyle and Cech (199l) have demonstrated by deoxynucleoside substitution that the deletion of two hydroxyl groups of residues two and three nucleotides away from the cleavage site are particularly detrimental to ribozyme-substrate association. The energetic penalty of this double deoxynucleoside substitution is 2.7 kcal tool-1 and this is consistent with the formation of two hydrogen bonds between the substrate/product and enzyme.

Following the results of mutagenesis experiments (Doudna et al 1989), Green et al (1991) have investigated the requirement for a wobble base pair (G-U or C-A) at the 5'-splice site of the Tetrahymena ribozyme. They utilized a cleavage assay in which the IGS and the 5'-splice site are supplied in trans. Oligonucleotides were synthesized with the potential to form wobble (G-U or I-U) or Watson-Crick like

1016 Jane A Grasby et al

(G-C or I-C) base-pairs. Only the former wobble base-paired substrates were cleaved, further confirming the requirement for non-Watson-Crick base-pairs.

An altered form of the Tetrahymena group I intron, in which the wild type G264-C311 base pair has been replaced by an A-U pair, binds 2-aminopurine with greater affinity than the usual guanosine substrate (Michael et al 1989). Guanosine binding is proposed to occur'via hydrogen bonding between the exocyclic amino group of guanosine and the N 7 of G264 and from the Nl-proton of guanosine to the 0 6 of G264. In the mutant A264-U311 enzyme, the purine possesses an exocyclic amino group which can act as a hydrogen bond donor rather than an acceptor. This can now contact the N~-acceptor of 2-aminopurine since it lacks the proton of the parent guanosine nucleoside due to a change in the tautomeric state in the analogue.

It has been shown that the mutant enzyme can catalyse a template-directed primer extension when supplied with synthetic dinucleotides of the form 2-aminopurine-pN (where N is any nucleoside) thus confirming the nature of the proposed interactions (Bartel et al 1991).

6. Protein-RNA interactions

The interactions of RNA with proteins are essential to many fundamental cellular processes (for a recent review, see Nagai 1992). In the cytoplasm, ribosomes are RNA-protein complexes essential to the translation of mRNA into proteins. Also required for translation are tRNAs which have been specifically aminoacylated by interactiofi with aminoacyl tRNA synthetases. Ribonucleoprotein complexes are involved in nuclear splicing of pre-mRNA (spliceosome) and in polyadenylation. Also in the nucleus, numerous proteins (hnRNPs), whose functions are not yet clear, are found to be bound to RNA. Many viral processes also involve protein-RNA interactions, for example the packaging of RNA viruses and in the gene regulation of retroviruses.

The principles of recognition of RNA by proteins are only now beginning to be elucidated. Very few high resolution structures have been obtained so far for RNAs or RNA-protein complexes other than for tRNA. Much useful information has been obtained in the meantime by the incorporation of synthetic RNA analogues into the RNA binding sites for proteins and by observation of the altered properties of the RNAs.

7. RNA encapsidafion of bacteriophage MS2 (R17)

Self-assembly and packaging in bacteriophage MS2 (R17) requires the interaction of a small RNA stem-loop of 19 residues within the phage genome (figure 8a) with the MS2 coat protein. The complex serves as a nucleation site for encapsidation and the interaction also acts to translationally repress synthesis of the replicase protein. A few years ago it was found that coat protein binding was inhibited by prior incubation with 5-bromouridine and the inactivation could be reversed by treatment with dithiothreitol (Romaniuk and Uhlenbeck 1985). Substitution of U_ 5 by C was found to result in 50-fold increase in binding of the coat protein and it was postulated that a transient covalent Michael adduct might be formed between a cysteine in the protein

Synthetic oligoribonucleotide analogues 1017

(.g

~ 0 " 0 (.g"

0 " . 0 A

0 qg

(.g" 0

0 (.g

(.g " 0

0 - ~ ~ -

(.g 0

0

0 (.g

m

9 , o o .

.,=.~

~.~

~'~

~ g

M ~

~.~ ~ ,, . ~

z

_ ~

~ "2, ~

9 ~ ~

1018 Jane A Grasby et al

and the 6-position of U - s in the MS2 RNA (Lowary and Uhlenbeck 1987). Further evidence for this hypothesis came from the use of a synthetic MS2 RNA fragment in which a 5-bromouridine was incorporated in place of U_ 5- The binding constant to the coat protein and dissociation kinetics were found to be similar to the C_ 5 mutant (Talbot et al 1990). More recently, a range of modifications has been chemically incorporated into the MS2 RNA at U-5. Since 5-cyanouridine incor- poration resulted in a significantly poorer binding, a transient Michael adduct now seems less likely (Stockley et al 1993).

The 5-bromouridine-substituted RNA can also be cross-linked to coat protein by use of medium wavelength UV light (Gott et al 1991). Improved results were obtained by long-wavelength irradiation of MS2 RNA substituted at U_ 5 by 5-iodouridine which was thought to cross-link to Tyr-85 in the coat protein (Willis et al 1993).

UV-induced cross-linking to proteins via 5-bromo- or 5-iodo-uridine-substituted RNA is likely to be of general use in defining the binding sites of proteins that interact with RNA.

8. Interaction of tRNA with aminoacyl tRNA synthetases

Aminoacyl tRNA synthetases recognize discrete identity elements in tRNAs which discriminate cognate from non-cognate amino acids in aminoacylation (for a review, see Lapointe and Giege' 1991). Small stem-loops that represent the acceptor end of the tRNAs (e.g. a 12 base-pair mini-helixes or a 7 base-pair microhelix) have been found to undergo aminoacylation specifically, albeit with reduced efficiency (Francklyn and Schimmel 1989). Aminoacylation can also be effected with small RNA duplexes (Musier-Forsyth et al 1991a). These shorter RNAs provide good models for the incorporation of synthetic analogues. For example, complete replacement of one strand of the duplex by deoxyribonucleotides is only tolerated on one side of the acceptor duplex (Musier-Forsyth et al 1991a). Individual substitutions by 2'-deoxy or by T-O-methyl nucleotides have pinpointed three particular 2'-hydroxyl groups in the RNA minor groove which may contact alanyl tRNA synthetase (Musier-Forsyth and Schimmel 1992). Inosine substitution in place of guanosine residues was instru- mental in the location of an unpaired 2-amino group of G3 essential for recognition in the minor groove of the RNA (Musier-Forsyth et al 1991b).

Gasparutto et al (1992) have reported the chemical synthesis of the entire alanyl tRNA containing three minor bases, ribothymidine, pseudouridine and dihydrouridine.

They also demonstrated that it was aminoacylated by alanyl-tRNA synthetase as well as naturally produced alanyl tRNA. The ability to introduce individual minor bases into chemically synthesized tRNA should now make it possible to study the role of minor bases in synthetase recognition and in translation.

9. The HIV-1 tat-TAR interaction

The trans-activator protein tat from Human Immunodeficiency Virus -1 (HIV-1) binds specifically to an RNA stem-loop located at the 5'-end of the viral mRNAs (Dingwall et al 1989). The RNA element is known as the trans-activation response element (TAR). Tat-TAR binding is an important step in trans-activation by tat and

Synthetic oligoribonucleotide analo#ues 1019 leads to a boost in viral expression (reviewed in Gait and Karn 1993 and Karn 1991).

Tat recognises TAR in the vicinity of a U-rich bulge located near the apex of the stem-loop and it has been found that near wild-type tat binding can be obtained using a synthetic duplex RNA containing this bulge and the surrounding base-pairs (figure 8b) (Sumner-Smith et al 1991; Hamy et al 1993).

Nucleotide substitution analysis has established that mutations to U23 in the bulge or to the two base-pairs above the bulge (G26:C39 or A 27:U38) result in considerably reduced binding but that alterations at positions 24 or 25 have little effect (Churcher et al 1993). U24 and U2s may also be replaced by propyl linkers without a drastic reduction in tat binding (Delling et al 1992). Replacement of U23 by dU or dT has no effect on tat binding. However, binding was abolished when N3-methyl ( - ) dT was placed at the same position (Sumner-Smith et al 1991). Similarly, replacement of U23 by O4-methyl ( - ) dT resulted in substantially reduced tat binding (Hamy et al 1993). These results indicate that the Nl-proton and/or 0 4 of U23 is important for tat recognition.

Substitution of TAR at G26 by NT-deaza-2'-deoxyguanine or of A27 by NT-deaza - 2'-deoxyadenosine drastically reduced binding of tat (Hamy et al 1993). These data suggest that tat recognises each of these nitrogen atoms which are expected to be located within the major groove of the RNA duplex. Although in regular A-form RNA duplexes the major groove is thought to be inaccessible to proteins, it has been proposed that the bulged residues serve to widen the major groove to allow protein binding (Weeks and Crothers 1991) and the chemical substitution results support this hypothesis.

10. The HIV-1 rev-RRE interaction

The HIV-1 rev protein is responsible for controlling the cytoplasmic expression of partially and unspliced viral mRNAs encoding the viral structural proteins and enzymes. Rev activity is mediated through interaction with an RNA structure located within the env gene known as the rev-response element (RRE) (Gait and Karn 1993).

The rev protein binds initially with high affinity to a duplex region containing a purine-rich "bubble" structure at the base of one of the RRE stems. Further co-operative binding of rev to neighbouring duplex regions of the RRE takes place and this is correlated with biological activity.

Rev binds stoichiometrically and with high affinity to a short model duplex RNA containing the purine rich bubble (figure 8c) (Iwai et al 1992). The roles of individual functional groups within the RNA in rev recognition have recently been established by chemical substitution experiments (Iwai et al 1992; Pritchard et al, manuscript in preparation). Substitution of individual residues by deoxynucleosides had no effect on rev binding except for G12 9 and A131. However, other data suggest that this is likely to be due to structural perturbation rather than direct contact of rev with the T-hydroxyl groups at these positions.

Major groove recognition by rev at base-pairs flanking the bulge is suggested by the finding that substitution of either Glo5 or G12s by NT-deaza-2'-deoxyguanosine in each case resulted in substantially reduced rev binding. However, no evidence has been obtained so far of direct rev recognition of any of the 5 bases in the bulged residues of the bubble. Instead, substitution experiments with a range of nucleoside

1020 J a n e A G r a s b y e t al

a n a l o g u e s ( 2 ' - d e o x y i n o s i n e , N 6 - m e t h y l - 2 ' - d e o x y a d e n o s i n e , N 7 - d e a z a - 2 ' - d e o x y a d e n o - sine, N V - d e a z a - 2 ' - d e o x y g u a n o s i n e , O 6 - m e t h y l g u a n o s i n e ) h a v e p r o v i d e d s t r o n g e v i d e n c e s u p p o r t i n g t h e e x i s t e n c e o f c r o s s - s t r a n d n o n - W a t s o n - C r i c k p a i r s b e t w e e n G l o s : A 1 3 1 a n d G l o 6 : G 1 2 9 (Iwai e t al 1992; P r i t c h a r d et ale m a n u s c r i p t in p r e p a r a t i o n ) . By c o n t r a s t , U13o s e e m s o n l y to be r e q u i r e d as a s p a c e r s i n c e it c a n be r e p l a c e d b y C, N 3 - m e t h y l U o r b y a n o n - n u c l e o t i d i c p r o p y l s p a c e r w i t h o u t l o s s o f r e v b i n d i n g .

Acknowledgement

W e t h a n k K a r i n M e r s m a n n f o r a d v i c e o n s e c t i o n s o f t h e m a n u s c r i p t .

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