Mapping the Conformational Landscape of the Neutral Network of RNA Sequences That Connect Two Functional Distinctly Different Ribozymes

During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000 , 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network.     

Tecto‐GIRz: Engineered Group I Ribozyme the Catalytic Ability of Which Can Be Controlled by Self‐Dimerization

RNA is a promising biomaterial for self‐assembly of nano‐sized structures with a wide range of applications in nanotechnology and synthetic biology. Several RNA‐based nanostructures have been reported, but most are unrelated to intracellular RNA, which possesses modular structures that are sufficiently large and complex to serve as catalysts to promote sophisticated chemical reactions. In this study, we designed dimeric RNA structures based on the Tetrahymena group I ribozyme. The resulting dimeric RNAs (tecto group I ribozyme; tecto‐GIRz) exhibit catalytic ability that depended on controlled dimerization, by which a pair of ribozymes can be activated to perform cleavage and splicing reactions of two distinct substrates. Modular redesign of complex RNA structures affords large ribozymes for use as modules in RNA nanotechnology and RNA synthetic biology.     

High‐Fidelity Recognition of RNA: Solution Structure of a DNA:RNA Hybrid Duplex with a Molecular Cap

Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson–Crick pairing at their termini. Here we report the three‐dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5′ terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard‐to‐suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high‐fidelity probes for RNA.     

Flexizymes: their evolutionary history and the origin of catalytic function

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.     

Catalytic strategies of self-cleaving ribozymes

[Structure: see text]. Five naturally occurring nucleolytic ribozymes have been identified: the hammerhead, hairpin, glmS, hepatitis delta virus (HDV), and Varkud satellite (VS) ribozymes. All of these RNA enzymes catalyze self-scission of the RNA backbone using a chemical mechanism equivalent to that of RNase A. RNase A uses four basic strategies to promote this reaction: geometric constraints, activation of the nucleophile, transition-state stabilization, and leaving group protonation. In this Account, we discuss the current thinking on how nucleolytic ribozymes harness RNase A's four sources of catalytic power. The geometry of the phosphodiester cleavage reaction constrains the nucleotides flanking the scissile phosphate so that they are unstacked from a canonical A-form helix and thus require alternative stabilization. Crystal structures and mutational analysis reveal that cross-strand base pairing, along with unconventional stacking and tertiary hydrogen-bonding interactions, work to stabilize the splayed conformation in nucleolytic ribozymes. Deprotonation of the 2'-OH nucleophile greatly increases its nucleophilicity in the strand scission reaction. Crystal structures of the hammerhead, hairpin, and glmS ribozymes reveal the N1 of a G residue within hydrogen-bonding distance of the 2'-OH. In each case, this residue has also been shown to be important for catalysis. In the HDV ribozyme, a hydrated magnesium has been implicated as the general base. Catalysis by the VS ribozyme requires both an A and a G, but the precise role of either has not been elucidated. Enzymes can lower the energy of a chemical reaction by binding more tightly to the transition state than to the ground states. Comparison of the hairpin ground- and transition-state mimic structures reveal greater hydrogen bonding to the transition-state mimic structure, suggesting transition-state stabilization as a possible catalytic strategy. However, the hydrogen-bonding pattern in the glmS ribozyme transition-state mimic structure and the ground-state structures are equivalent. Protonation of the 5'-O leaving group by a variety of functional groups can promote the cleavage reaction. In the HDV ribozyme, the general acid is a conserved C residue. In the hairpin ribozyme, a G residue has been implicated in protonation of the leaving group. An A in the hammerhead ribozyme probably plays a similar role. In the glmS ribozyme, an exogenous cofactor may provide the general acid. This diversity is in contrast to the relatively small number of functional groups that serve as a general base, where at least three of the nucleolytic ribozymes may use the N1 of a G.     

Enhancement of the Catalytic Activity of Hammerhead Ribozymes by Organic Cations

Catalytic turnover is important for the application of ribozymes to biotechnology. However, the turnover is often impaired because of the intrinsic high stability of base pairs with cleaved RNA products. Here, organic cations were used as additives to improve the catalytic performance of hammerhead ribozyme constructs that exhibit different kinetic behaviors. Kinetic analysis of substrate cleavage demonstrated that bulky cations, specifically tetra-substituted ammonium ions containing pentyl groups or a benzyl group, have the ability to greatly increase the turnover rate of the ribozymes. Thermal stability analysis of RNA structures revealed that the bulky cations promote the dissociation of cleaved products and refolding of incorrectly folded structures with small disruption of the catalytic structure. The use of bulky cations is a convenient method for enhancing the catalytic activity of hammerhead ribozymes, and the approach may be useful for advancing ribozyme technologies.     

A Hexameric Ribozyme Nanostructure Formed by Double-Decker Assembly of a Pair of Triangular Ribozyme Trimers

The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units. The resulting double-decker nanostructures containing six ribozyme units were characterized biochemically and their structures were observed by atomic force microscopy. The double-decker hexamers exhibited higher catalytic activity than the parent ribozyme trimers.     

Heterodimerization of Group I Ribozymes Enabling Exon Recombination through Pairs of Cooperative trans‐Splicing Reactions

Group I (GI) self‐splicing ribozymes are attractive tools for biotechnology and synthetic biology. Several trans‐splicing and related reactions based on GI ribozymes have been developed for the purpose of recombining their target mRNA sequences. By combining trans‐splicing systems with rational modular engineering of GI ribozymes it was possible to achieve more complex editing of target RNA sequences. In this study we have developed a cooperative trans‐splicing system through rational modular engineering with use of dimeric GI ribozymes derived from the Tetrahymena group I intron ribozyme. The resulting pairs of ribozymes exhibited catalytic activity depending on their selective dimerization. Rational modular redesign as performed in this study would facilitate the development of sophisticated regulation of double or multiple trans‐splicing reactions in a cooperative manner.     

Genome Evolution from Random Ligation of RNAs of Autocatalytic Sets

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis, such as evaluating the ability of ribozyme RNA polymerases to generate random ligation products and testing the catalytic activity of linked ribozyme domains.     

Synthesis of 2'-aminoalkyl-substituted fluorinated nucleobases and their influence on the kinetic properties of hammerhead ribozymes

Hammerhead ribozymes are ribonucleic acids that catalyse the hydrolytic cleavage of RNA. They interfere with gene expression in a highly specific manner and recognize the mRNA target through Watson-Crick base pairing. To overcome the problem of point mutations (Watson-Crick "mismatches") occurring in viral genomes, we developed 2'-aminoethyl-substituted fluorinated nucleosides, which are universal nucleobases. The highly efficient synthetic pathway, which features a direct phthaloylamination of a primary alcohol under Mitsunobu conditions, leads to modified phosphoroamidites. The 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-D-ribofuranose nucleoside analogue does not differentiate between the four natural nucleosides and leads to a RNA duplex that is as stable as the unmodified parent duplex. Upon incorporation into a ribozyme, the analogue's catalytic activity is equal for all four possible substrates, and the cleavage rates for the modified ribozymes are significantly higher (up to a factor of 13) than for the natural Watson-Crick "mismatch" base pairs. In agreement with the thermodynamic data obtained by measurement of the T(m) values of the RNA 12-mers, the cleavage rates for the 2'-substituted fluorinated benzimidazole derivative 4 are slightly higher than for the corresponding fluorinated benzene derivative 3.     

RNA‐Directed Amino Acid Coupling as a Model Reaction for Primitive Coded Translation

The stereochemical theory claims that primitive coded translation initially occurred in the RNA world by RNA‐directed amino acid coupling. In this study, we show that the HIV Tat aptamer RNA is capable of recognizing two consecutive arginine residues within the Tat peptide, thus demonstrating how RNA might be able to position two amino acids for sequence‐specific coupling. We also show that this RNA can act as a template to accelerate the coupling of a single arginine residue to the N‐terminal arginine residue of a peptide primer. The results might have implications for our understanding of the origin of translation.     

Analysis of Structure–Activity Relationships Based on the Hepatitis C Virus SLIIb Internal Ribosomal Entry Sequence RNA‐Targeting GGHYRFK⋅Cu Complex

New therapeutics for targeting the hepatitis C virus (HCV) have been released in recent years. Although they are less prone to resistance, they are still administered in cocktails as a combination of drugs targeting various aspects of the viral life cycle. Herein, we aim to contribute to an arsenal of new HCV therapeutics by targeting the HCV internal ribosomal entry sequence (IRES) RNA through the development of catalytic metallodrugs that function to degrade rather than inhibit the target molecule. Based on a previously characterized HCV IRES stem‐loop IIb RNA‐targeting metallopeptide Cu‐GGHYrFK ( 1? Cu), an all‐l analogue ( 3? Cu) and a series of additional complexes with single alanine substitutions in the targeting domain were prepared and screened to determine the influence each amino acid side chain on RNA localization and recognition, and catalytic reactivity toward the RNA. Additional substitutions of the tyrosine position in complex 3? Cu were also investigated. Good agreement between calculated and measured binding affinities provided support for in silico modeling of the SLIIb RNA binding site and correlations with RNA cleavage sites. Examination of the cleavage products from reaction of the Cu complexes with SLIIb provided mechanistic insights, with the first observation of the 5′‐geminal diol and 5′‐phosphopropenal as products through the use of a Cu ? ATCUN catalytic motif. Together, the data yielded insights into structure–function relationships that will guide future optimization efforts.     

Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II

Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg(2+) concentrations were in vitro selected from a library with 18 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had the same sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mM Mg(2+) concentration in the context of several substrates with differences in the lengths of stem I and III, including the well-characterised HH16 substrate and a derivative thereof with a GUA triplet at the cleavage site, as well as a substrate used previously in a related study. For the HH16 substrate, a change of base pair 10.2-11.2 to C-G in stem II further improved activity by about 2.5-fold to 0.8 min(-1) (at 1 mM Mg(2+) concentration, 25 degrees C, pH 7.5). Interestingly, this very active variant was not identified by the selection procedure. Changing loop II from UUUU to GCAA or extension of stem II to three or four base pairs reduced the cleavage rate by 2.0-2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so far at low Mg(2+) concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degrees C and pH 7.5 in monophasic reactions, with endpoints between 75 and 90 %. Such constructs promise to be advantageous for the inhibition of gene expression in vivo.     

Site‐Specific Modification of the 6‐Amino Group of Adenosine in RNA by an Interstrand Functionality‐Transfer Reaction With an S‐Functionalized 4‐Thiothymidine

Non‐natural RNA modifications have been widely used to study the function and structure of RNA. Expanding the study of RNA further requires versatile and efficient tools for site‐specific RNA modification. We recently established a new strategy for the site‐specific modification of RNA based on a functionality‐transfer reaction between an oligodeoxynucleotide (ODN) probe and an RNA substrate. 2′‐Deoxy‐6‐thioguanosine was used to anchor the transfer group, and the 4‐amino group of cytosine or the 2‐amino group of guanine was specifically modified. In this study, 2′‐deoxy‐4‐thiothymidine was adopted as a new platform to target the 6‐amino group of adenosine. The (E)‐pyridinyl vinyl keto transfer group was attached to the 4‐thioT in the ODN probe, and it was efficiently and specifically transferred to the 6‐amino group of the opposing adenosine in RNA in the presence of CuCl₂. This method expands the available RNA target sites for specific modification.     

The 5′‐AG5CC‐3′ Fragment from the Human CPEB3 Ribozyme Forms an Ultrastable Parallel RNA G‐Quadruplex

An unusually thermostable G‐quadruplex is formed by a sequence fragment of a naturally occurring ribozyme, the human CPEB3 ribozyme. Strong evidence is provided for the formation of a uniquely stable intermolecular G‐quadruplex structure consisting of five tetrad layers, by using CD spectroscopy, UV melting curves, 2D NMR spectroscopy, and gel shift analysis. The cationic porphyrin TMPyP4 destabilizes the complex.     

Molecular basis for chiral selection in RNA aminoacylation

The chiral-selective aminoacylation of an RNA minihelix is a potential progenitor to modern tRNA-based protein synthesis using l-amino acids. This article describes the molecular basis for this chiral selection. The extended double helical form of an RNA minihelix with a CCA triplet (acceptor of an amino acid), an aminoacyl phosphate donor nucleotide (mimic of aminoacyl-AMP), and a bridging nucleotide facilitates chiral-selective aminoacylation. Energetically, the reaction is characterized by a downhill reaction wherein an amino acid migrates from a high-energy acyl phosphate linkage to a lower-energy carboxyl ester linkage. The reaction occurs under the restriction that the nucleophilic attack of O, from 3'-OH in the terminal CCA, to C, from C=O in the acyl phosphate linkage, must occur at a Bürgi-Dunitz angle, which is defined as the O-C=O angle of approximately 105°. The extended double helical form results in a steric hindrance at the side chain of the amino acid leading to chiral preference combined with cation coordinations in the amino acid and the phosphate oxygen. Such a system could have developed into the protein biosynthetic system with an exclusively chiral component (l-amino acids) via (proto) ribosomes.     

Folding and activity of the hammerhead ribozyme

The hammerhead is the smallest of the nucleolytic ribozymes, that undergo backbone cleavage by a transesterification reaction in the presence of magnesium ions. The RNA is induced to fold into its active conformation by the binding of metal ions in two stages. These generate domain 2, the scaffold on which the ribozyme is built, and domain 1, the active centre of the ribozyme. Further local structural rearrangement during the activation of the ribozyme is suggested by a number of crystal structures. The 10(5)-fold rate enhancement is probably brought about by a combination of metal-ion participation and stereochemical factors in the environment of the folded RNA structure.     

The Proto-Ribosome: An Ancient Nano-machine for Peptide Bond Formation

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A- to P-site passage of the tRNA 3′ terminus during protein synthesis is performed by a rotary motion, synchronized with the overall tRNA/mRNA sideways movement, and guided by the PTC. This rotary motion leads to suitable stereochemistry for peptide bond formation as well as for substrate-mediated catalysis. Analysis of the substrate binding modes to ribosomes led to the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, potentially in a similar manner to the modern PTC. Later in evolution, a mechanism, enabling some type of decoding genetic control triggered the emergence of the small ribosomal subunit or part of it. This seems to be the result of the appearance of reaction products that could have evolved after polypeptides capable of enzymatic function were generated sporadically, while an ancient stable RNA fold was converted into an old version of a tRNA molecule. Since in the contemporary ribosome, the symmetry relates only to the backbone fold and nucleotide orientations but not nucleotide sequences, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC may have evolved by gene fusion or gene duplication.     

Stereoselective synthesis using immobilized Diels-Alderase ribozymes

Development of artificial ribozymes by in vitro selection has so far, mostly been addressed from the viewpoint of fundamental research. However, such ribozymes also have high potential as selective catalysts in practical syntheses. Immobilization of an active and selective ribozyme is an important step towards this end. A 49-nucleotide RNA molecule that was previously found to stereoselectively catalyze Diels-Alder reactions between various anthracene dienes and maleimide dienophiles was quantitatively immobilized on an agarose matrix by periodate oxidation of the 3'-terminal ribose and coupling to a hydrazide moiety. Typical loadings were 45 pmol microL(-1) gel. The specific activity was comparable to that of soluble ribozyme, and high enantioselectivities were obtained in catalyzed cycloadditions. The catalytic matrix was found to be stable and could be regenerated about 40 times with only minimal reduction of catalytic activity. Like the soluble ribozyme, the immobilized catalyst stereoselectively converts various diene and dienophile substrates. By using either natural D-RNA or enantiomeric L-RNA, both product enantiomers were made synthetically accessible with similar selectivities.     

Cationic peptides that increase the thermal stabilities of 2'-O-MeRNA/RNA duplexes but do not affect DNA/DNA melting

Several different cationic nonapeptides have been synthesized and investigated with respect to how they can influence the thermal melting of 2′‐O‐methylRNA/RNA and DNA/DNA duplexes. Each peptide has a C‐terminal L ‐phenylalanine unit and is otherwise uniformly composed of a sequence of a specific basic D ‐amino acid that in most cases will be largely charged at neutral pH. These N‐terminal octamer stretches are composed variously of the amino acids D ‐lysine, D ‐diaminobutyric acid (D ‐Dab), D ‐diaminopropionic acid (D ‐Dap), or D ‐histidine. None of the peptides substantially affected the thermal melting of DNA/DNA duplexes, which was in sharp contrast with their effects on 2′‐O‐methylRNA/RNA duplexes. In particular, the peptides based on diaminopropionic and diaminobutyric acid units had strong positive effects on the melting temperatures of the 2′‐O‐methylRNA duplexes (up to 16 °C higher with 1 equivalent of peptide) at pH 7, whereas at pH 6 the effect was even more drastic (ΔT_m up to +25 °C). The shorter R groups of the Dap and Dab groups appear to have a better length than lysine for enhancement of the thermal melting of the 2′‐O‐methylRNA/RNA duplex, an effect that is more pronounced at lower pH but substantial even at pH 7, although the Dap derivative is not likely to be fully protonated. The dramatic difference between the influence, or lack thereof, on the 2′‐O‐methylRNA/RNA and the DNA/DNA thermal meltings suggest that, although electrostatic interactions probably play a role, there is another major and structurally dependent component influencing the properties of the duplexes. This is also seen in the observation that the oligo‐Dap and oligo‐Dab peptides give greater melting point enhancements than both the lysine peptide (with a longer side chain) and a β‐linked Dap peptide with a shorter side chain and a longer backbone.