New Twists on Nucleic Acids - ACS Symposium Series (ACS

Chapter 14

New Twists on Nucleic Acids

Downloaded by UNIV OF PITTSBURGH on September 13, 2013 | http://pubs.acs.org Publication Date: December 7, 1994 | doi: 10.1021/bk-1994-0580.ch014

Structural Properties of Modified Nucleosides Incorporated into Oligonucleotides Richard H. Griffey, Elena Lesnik, Susan Freier, Yogesh S. Sanghvi, Kelly Teng, Andrew Kawasaki, Charles Guinosso, Patrick Wheeler, Ventrankaman Mohan, and P. Dan Cook Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008 The structures and physical properties of oligonucleotide duplexes depend strongly on the limited conformational flexibility of (deoxy)ribose-phosphodiester linkages and the sugar. Molecular mechanics and N M R spectroscopy have been employed to understand and predict alterations in sugar pucker and conformation resulting from replacement of oxygen, hydrogen, and carbon in the sugar with other heteroatoms. Studies on model monomer, dimer, and longer single stranded nucleosides are correlated with data on duplex stability obtained from oligomers incorporating 2', 3', and 4'-modified sugars to establish a set of guidelines for the types of alterations that can be tolerated within oligonucleotide duplexes. The results suggest that increased affinity of an antisense oligomer for a complementary R N A target can be achieved by decreasing the entropic motion of the sugar while maintaining a preorganized structure with an RNA-like conformation. Utilization of antisense oligonucleotides in the treatment of disease has emerged as an exciting new therapeutic paradigm (7). Pharmaceutical "antisense" encompasses strategies where gene expression is inhibited through hybridization of an exogenous oligomer to a specific intracellular messenger R N A target (2). This provides a very high potential for specificity of action, since theoretically a small oligomer of -15-20 nucleotides has the sequence specificity required to inhibit the expression of a target gene. To be successful, antisense therapeutics have to fulfill additional criteria, including sufficient nuclease resistance, biodistribution, and ease of synthesis (J). These issues have spurred a quest for oligonucleotide replacements with favorable specificity, affinity, and stability (4). Since many types of nucleotide modifications which provide nuclease resistance reduce the stability of the resulting duplex with the R N A target, increasing affinity for the target RNA is a paramount concern (5). Conformational

Properties of Oligonucleotides

The solution conformations of nucleic acids vary dynamically on the picosecond time scale. The structure of an oligonucleotide is governed by the interaction of dipolar, torsional, electrostatic, steric, and London forces from the bases, sugar, and backbone (6). Figure 1 demonstrates the result of dynamic motion on the structure of an 8-mer

0097-6156/94/0580-0212$08.00/0 © 1994 American Chemical Society

In Carbohydrate Modifications in Antisense Research; Sanghvi, Y., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


14.

GRIFFEY E T A L .


213

D N A duplex. Four conformations of the duplex obtained from a molecular dynamics study at 25 psec intervals have been superimposed. The structures all fall within the classic Ί Γ family of helices, but illustrate the perturbations induced by the entropy of thermal motion. This random dynamic motion is balanced by the hydrogen bonds among the complementary bases, which provide the Watson-Crick sequence specificity, and by the limited range of conformations which can generate a helical structure. The small free energy of the duplex results from a cancellation of the large favorable enthalpy of the single strands, whose conformations are defined by seven backbone and glycosidic dihedral angles and their large entropy, or the energetic cost of organizing the strands into a low energy conformation. The residual thermal motion in the strands has been proposed to have an "entropie benefit", which allows duplex structures to be melted for recognition processes (7). The importance of preorganization and rigidity in the formation of stable duplexes is supported by the work of Benner, who incorporated glycerol units into a D N A backbone (8). The trisubstituted glycerol is equivalent to a ribose sugar without the C 2 ' carbon. While the glycerol moieties can adopt conformations suitable for formation of a double helix, each incorporation produced a 9-15° C decrease in the T for the duplex. Constrained cyclic nucleotides should decrease the entropy of the duplex, and two families have been synthesized (9,10). However, their incorporation into oligomers reduces the affinity of the oligo for target D N A or R N A by 1.5°-2.0° C/modification. Hence, reducing the entropy of the system is insufficient to improve the affinity unless the rigid analog adopts a conformation that is compatible with the structure of the target for duplex formation. The backbone and the sugar of an oligomer can be modified to change the entropy and enthalpy of interaction and improve the affinity. Atomic substitutions along the phosphodiester backbone generally are not well tolerated, since a limited number of dihedral solutions exist for the D N A or R N A duplexes. A variety of alternate backbones have been prepared, including substitution of sulfonates, sulfonamides, hydrazines, hydroxylamines, formacetals, alkylphosphonates, phosphorothioate, phosporodithioates, and amide linkages for the phosphate diester (77, 72,13). The affinity of these oligomers for an R N A target generally is reduced, except where substitutions with limited rotational freedom, e.g. hydrazines and hydroxylamines, are incorporated (14,15). The conformation of the sugar might be used to alter the entropy and geometry of the duplex. There are twenty possible conformations of the ribo- and deoxyribofuranose ring. These can be plotted on a "pseudorotation" cycle, which relates each "twist" or "envelope" conformation to a specific value of the five internal dihedral angles (76). Crystal structures obtained to date suggest that two low-energy states predominate, centered about the C3'-exo and C2'-exo conformations. A twostate exchange model among these forms has been proposed for R N A and D N A and is shown in Figure 2 for RNA. The two forms are separated by a relatively small barrier of 1-2 Kcal, which molecular mechanics studies suggest is traversed on the picosecond time scale (77). The same conformations also are observed for fragments of oligomers down to the nucleoside level using X-ray crystallography, NMR, circular dichroism, laser Raman spectroscopy, and other physical techniques. The furanose conformation is not "fixed", but is governed by anomeric and gauche interactions of functional groups and the ring oxygen, which stabilize certain geometries (18). In mis chapter, modified nucleosides are examined using N M R as monomers and when incorporated into oligonucleotides. The conformational properties are correlated with biophysical data including duplex stability. The conformational preferences of nucleosides are retained from monomers into single stranded oligonucleotides. The biophysical properties of oligomers and their duplexes can be predicted from analysis of monomers. The discovery of alternate structural motifs, such as peptide nucleic acids (PNAs) which have increased affinity for complimentary R N A and D N A targets, suggests that other oligonucleotide analogs have yet to be discovered (79). m


CARBOHYDRATE MODIFICATIONS IN ANTISENSE R E S E A R C H


214

Figure 2. Conformational exchange between 'Ν' and 'S' forms of R N A .


14.

GRIFFEY E T A L .

215


Results and Discussion The ribofuranose ring has been substituted at C 2 ' , C 3 ' , and C 4 ' with substituents varying in electronegativity, polarizability, and steric bulk. The pseudorotation parameters have been determined from crystal structures or in solution from an analysis of the three-bond proton-proton (HH) coupling constants observed in highresolution proton N M R spectra. The coupling constants are fit to a periodic Karplustype equation, which predicts the values of the internal dihedral angles of the sugar unit (2ft 27). The calculated pseudorotation properties can be correlated with the changes in affinity for the R N A and D N A complements that the modification produces. This correlation suggests that an increased % N conformation is associated with increased T values against R N A complements. This observation suggests that the affinity of antisense therapeutics may be improved via selective incoiporation of specific modified nucleosides into oligomers.


m

2'-F-Deoxynucleotides. The predominant conformation of 2'-F nucleosides shown in Figure 3a has been determined by X-ray crystallography to be C3'-endo, although 2'-F uridine crystallizes in an unusual form P=74° (22). Theoretical calculations on model systems suggest that the barrier for conversion to a C2'-endo form is increased along both the "eastern" and "western" paths (23). The P conformer is destabilized by a larger gauche effect to 0 4 ' for the more electronegative F compared to OH. This differs from R N A where the 2'-0 and 3'-0 have equal gauche effects to 04, and the conformation is governed by the anomeric effects of the C I ' and C 4 ' substituents with the ring oxygen. The conformation of oligonucleotides containing 2'-F units has been examined using H N M R analysis of coupling constants and a parameterized version of the Karplus equation for 2'-F derivatives. This represents an improvement over older studies, where a simple analysis failed to account for the alpha and beta electronic effects of the F atom (24, 25). The original N M R data on mono- and dinucleotides published by Ikehara and co-workers have been used to fit new pseudorotation parameters which are listed in Table 1.(25) These calculations show that the best amplitude of the sugar pucker is 40-43°, slightly greater than observed in crystal structures. For three 2'-F purines, the % N is calculated to be 86-92% with P =-10° to -16°. The pseudorotation of the residual P could be fit to 93° or 172°. These values are shifted toward C2'-exo by 20-30° from the previous calculations (25). Conformational analyses of fluoropiperidines also show that fluorine prefers such an axial orientation (26). The compound 3'-5'-TIPS-2'-F-uridine (Figure 3b) has been prepared as a rigid 'Ν' derivative to identify the predominant 'Ν' pseudorotation. The 3'-5'-TIPS protecting group is known to shift the pseudorotation to entirely 'Ν' (27). Calculations reveal that the derivative adopts a conformation with >99% P =-19.0° in chloroform solution at 20° C. A pentamer G*A*U*C*dT containing four 2'-F nucleotides also has been prepared and studied. Analysis of the three-bond H - H coupling constants shows that the G , C, and U 2'-F nucleotides exist in a conformation with 91-97% P = -6±1°, while the 2'-F A is 84% P„=-27±5°. The ground state P must be favored by 2-2.5 Kcal/mol over the P conformer from the ratio of P to P determined from NMR. This compares to a theoretical value of 1.5 Kcal/mol determined for model systems. The % N at 20° C would be expected to be even higher in a 2'-F-modified oligomer. The stability of oligomers containing 2'-F substitutions for R N A targets is increased by ~27modification in an R N A strand (28). This effect may result from increased preorganization of 2'-F strand for the R N A target due to a reduced rate of interconversion between 'Ν' and ' S ' forms, since the barrier to pseudorotation through Έ ' quadrant is increased (23). The 2'-F derivatives offer no increase in resistance to endo- or exonuclease degradation (29). s

l

n

s

n

n

n

n

s

s


216

CARBOHYDRATE MODIFICATIONS IN ANTISENSE R E S E A R C H


Table I. Compound fGpU* flpC fApU fGfAfUfCdT fG fA fU fC 3',5'-ΉΡ8 f U a

Pseudorotation Parameters for 2'-F Nucleosides and Oligomers Temperature (° C) 20 20 20 50

b

%N

20

87 91 93

P» -16±1 -11 -10

P 137±5 94 103

94 84 97 91 100

-6±1 -27±5 -7±1 -5 -15+1

170±5 149 168 165 161

s

D

Coupling constants taken from ref. 25. N M R experiments performed on sample

dissolved in CDCI3.

2'-0-MethyI Ribosides. This family of nucleosdes (Figure 4a) commonly occurs in tRNAs and have been proposed to stabilize R N A duplexes at specific locations in the anticodon loop (30). The conformational properties of 2'-0-Me uridine and cytidine as nucleosides and in dinucleotides have been studied in an attempt to rationalize the prevalence of this modification (31). Not all coupling constants could be determined from proton N M R spectra, and the conformations have been estimated only from the Η 1 Ή 2 ' coupling constant. The proportion of the C3'-endo conformation is increased from 55% to 60% in the 2'-0-Me derivative compared to uridine. In a UpU dinucleotide, no increase in the 63% population of the C3'-endo conformer is observed upon methylation. The stabilization of the C3'-endo form has been attributed to an unfavorable steric interaction between the 2'-0-Me and the 3'phosphate, which orients the 2'-0-Me toward the base, and a second steric interaction of the O-Me group with the C2-carbonyl of the base. Substitution of 2 ' - 0 - M e nucleotides into D N A oligomers increases the T of duplexes against R N A complement by 1.5-2° C per modification on average, but local effects can be greater (up to 3° C per point modification) (32). In a fully modified 12-mer, 2'-0-Me R N A has a greater affinity for R N A target by +1.5-1.8° C compared to a phosphorothioate D N A oligomer, and by +0.4° C compared to the same 2'-

New Twists on Nucleic Acids - ACS Symposium Series (ACS

Recommend Documents