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Structural Principles: Hierarchical Organization and Modularity

RNA and DNA modularity is hierarchically expressed at a chemical, structural and supramolecular level (Fig. 10.1).

10.2.1.1 Chemical Modularity and Stability

From a chemical point of view, RNA and DNA are modular polymers with primary (1°) sequences formed of four basic building blocks: A, U, C and G ribonucleotides for RNA and A, T, C and G deoxyribonucleotides for DNA. In cells, de-oxyribonucleotides are biologically synthesized by enzymatic modification of RNA ribonucleotides. DNA can be seen as a modified RNA that lacks the 2'-hydroxyl at the level of the sugar moiety and has an extra methyl at the level of uracil to form thymine. These minor chemical differences increase the resistance of DNA towards spontaneous hydrolysis, and the fidelity of DNA replication, making DNA a better support for the genetic information than RNA. At basic pH values (7-12),

Fig. 10.1 Supramolecular and structural modularity of nucleic acids. DNA and RNA chemical modularity is exemplified by their primary sequence (1°). The structural and conformational modularity of nucleic acids is expressed at the level of their secondary (2°) and tertiary (3°) structures.

RNA backbone hydrolyzes in the presence of divalent ions such as magnesium. Additionally, mutational events resulting from the spontaneous hydrolysis of cy-tosine into uracil cannot be repaired in RNA as uracils at mutated positions cannot be distinguished from those at non-mutated positions. At acidic pH values (4-6), however, depurination is faster for DNA than RNA [3, 4]. In living organisms, the greater chemical fragility of RNA compared with DNA essentially results from the numerous ribonucleases that biologically target RNA molecules. In a laboratory setting, this can however be easily overcome by taking basic "RNase-free" precautions.

For biotechnological and medical applications, RNA can be a good medium to build transient, biodegradable materials or molecular scaffolds for RNA drugs such as aptamers, ribozymes or siRNAs [5-7] (Section 10.2.2). Alternatively, the combination of RNA moieties with DNA and other nucleic acid analogs can offer limitless possibilities for improving and tuning the chemical and thermodynamic stability of nucleic acid foldamers that would retain the unique structural richness and thermodynamic stability of RNA (Section 10.3).

10.2.1.2 Secondary Structure Principles

In contrast to proteins, the secondary (2°) structure of RNA and DNA results from hydrogen bonding between side chains and not between backbone atoms, as for protein alpha-helices and beta-sheets. Therefore, nucleic acid 2° structures are easier to predict than those of proteins. Within the hierarchical framework that characterizes nucleic acid folding and assembly, p-stacking and Watson-Crick base pairings drive the folding and assembly of RNA and DNA 1° sequences into 2° structures through the formation of stable helical elements. Watson-Crick base pairs, with cis-glycosyl bonds, form the only set of pairs that are isosteric in anti-parallel helices. Thus, they allow formation of helices with regular sugar-phosphate backbones. RNA and DNA 2° structures are schematically represented in a planar drawing by base-paired segments that specify for various 2° structure motifs such as hairpin loops, bulges, internal loops and multi-helix junctions (Fig. 10.1).

The presence of the 2'-OH in RNA increases the structural rigidity of RNA duplexes that are locked into compact A-form helices with C3' endo sugar pucker. More polymorphous DNA helices are mostly present in the extended B-form with C2' endo sugar pucker (Fig. 10.1). As basic modular building blocks, A-form RNA duplexes are thermodynamically more stable than B-form DNA duplexes. According to the base-pair free-energy parameters determined at 1 M NaCl and 37° for RNA and DNA, RNA base pairs are, on average, —0.49 + 0.35 kcal mol—1 more stable than those of DNA [8, 9]. Note, however, that the thermodynamic stability of RNA and DNA duplexes varies as a function of the nucleic acid sequence. The higher thermodynamic stability of RNA duplexes compared with that of DNA duplexes essentially results from a higher enthalpy for duplex formation for RNA, which is consistent with much better hydration of RNA helices than DNA helices [10]. Recently, experimental measurements of the persistence length for RNA and DNA duplexes was performed by single molecule analysis using Fluorescence Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM) and magnetic tweezers techniques [11, 12]. They corroborate that RNA helices are more compact and stiffer than DNA helices, with persistence length of 55 nm and 63 nm for DNA and RNA, respectively. As the rise per helical turn is 2.9 nm for RNA versus 3.4 nm for DNA, the persistence length calculated in base pairs is 30% greater for RNA than DNA.

Besides the classic Watson-Crick pairs, eleven distinctive ''non-canonical'' base pairs, which involve at least two hydrogen bonds, can potentially occur between complementary nucleotides [13]. In RNA, they can contribute significantly to the rigidity and thermodynamic stability of RNA structural elements [14]. Design and prediction of RNA and DNA 2° structures can presently be achieved by energy minimization with a reasonable degree of accuracy [14, 15] using software like mfold, RNAfold or RNAsoft [16-19]. Because the formation of mismatches is allowed between strands that are not perfectly complementary, RNA helices have a lower selective informational content than their DNA counterparts. Consequently, for RNA, positive and negative design is particularly critical to maximize the stability of the desired 2° structure while minimizing folding into stable alternatives.

10.2.1.3 Tertiary Structure Principles

Entropic gain through water and ion release is the main driving force for the tertiary (3°) folding of a protein. It leads to a hydrophobic core mostly devoid of water molecules. By contrast, for nucleic acids, both entropy gain through ion and water release and enthalpy gain via the formation of intricate solvent networks lead to the 3° folding mostly devoid of hydrophobic pockets. At the 3° structure level, the 2° structural elements can associate through numerous van der Waals contacts, p-stacking, metal coordination and specific hydrogen bonds via the formation of a small number of additional Watson-Crick and/or non-canonical base pairs that involve single-stranded regions, loops or bulges. For 3° nucleic acid structures, such as large stable RNA molecules, the assembly occurs by metal-ion induced collapse of the 2° structure into compact conformations (reviewed in Refs. [20-22]) (Fig. 10.2). Metal ions essentially screen the negative charges on the phosphate groups. The loosely folded intermediates then undergo further conformational rearrangements before adopting the final 3° structure [22] (Fig. 10.2). This conformational search is essentially dependent on the local folding of recurrent and specific set of nucleotides that specify for modular 3° structure motifs that adopt unique local 3-D shapes and mediate stereochemically precise quaternary (4°) interactions (Fig. 10.3). These structure motifs can be seen as the minimal information for folding a nucleic acid sequence into a specific 2° or 3° structure. As such, they can be seen as basic foldamer modules that can be combined and encoded within a nucleic acid sequence to specify for more complex 3-D shapes. The proper folding of 3° structure motifs requires the formation of 3° interactions between specific nucleotide positions, which are highly dependent on temperature, salts and divalent ion concentration. For instance, without

Metal ion Collapse Conformational condensation (helix assembly) search

Fig. 10.2 Metal ion-induced folding of RNA. The association of the positive ions (grey spheres) with the unfolded RNA rapidly neutralizes more than 90% of the phosphate charges and induces the collapse of the RNA into more compact conformations. The conformational search which is the time-limiting step, is dependent on the formation of 3C structure motifs. Cylinders symbolize helical elements. Double arrows indicate helical motions. The diagram is based on Fig. 2 from Ref. [20].

>90% charqe Compact Stable tertiary

Secondary structure neutralized intermediates structure

Fig. 10.2 Metal ion-induced folding of RNA. The association of the positive ions (grey spheres) with the unfolded RNA rapidly neutralizes more than 90% of the phosphate charges and induces the collapse of the RNA into more compact conformations. The conformational search which is the time-limiting step, is dependent on the formation of 3C structure motifs. Cylinders symbolize helical elements. Double arrows indicate helical motions. The diagram is based on Fig. 2 from Ref. [20].

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