Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Oligodeoxynucleotides having a loop consisting of 3'-deoxy-4'-C-(2-hydroxyethyl)thymidines form stable hairpins

Components that form stable hairpin loops are highly useful for the development of functional DNA and RNA molecules. We have designed and synthesized a sugar-modified thymidine analogue, 3'-deoxy-4'-C-(2-hydroxyethyl)thymidine (X), as a nucleosidic loop component stabilizing the hairpin structure. The ODNs I-1-4, 5'-d[CGAACG-X(n)-CGTTCG]-3' (I-1, n = 1; I-2, n = 2; I-3, n = 3; I-4, n = 4), forming the hairpin loop structures, of which the loop moiety consisted of the analogue X, and also the corresponding unmodified ODNs II-1-4, 5'-d[CGAACG-T(n)-CGTTCG]-3' (II-1, n = 1; II-2, n = 2;II-3, n = 3; II-4, n = 4), having a thymidine loop, were synthesized by the phosphoramidite method. The melting temperatures (T(m)) of the ODNs I-1-4 containing X in the loop moiety at 5 microM were 67.1, 68.1, 73.0, and 69.3 degrees C, respectively, and those of the control natural ODNs II-1-4 were 65.3, 67.0, 69.2, and 68.8 degrees C, respectively. Thus, the ODNs I-1-4 formed a more thermally stable hairpin than the corresponding unmodified ODNs II-1-4 having an equal number of loop residues. The hairpin structures of the modified ODNs I-1-4 and the unmodified ODNs II-1-4 were investigated by CD spectroscopy and molecular mechanics calculations. These results showed that the 4'-branched nucleoside X can stabilize hairpin structures when it is present in the loop moiety, probably due to the flexibility of the one-carbon-elongated 4'-branched structure.     

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5'' splice site of the Tetrahymena group I intron.

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.     

Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophosphates of clofarabine

The conjugates of anticancer nucleoside clofarabine [2-chloro-9-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)adenine] with 1,2- and 1,3-diacylglycerophosphates have been prepared by the phosphoramidite method using a combination of 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl protecting group for the sugar moiety of the nucleoside and 2-cyanoethyl protection for the phosphate fragment. Some of the synthesized conjugates exhibited cytostatic activity against HL-60, A-549, MCF-7, and HeLa tumor cell lines.     

Synthesis of very short chain lysophosphatidyloligodeoxynucleotides

Very short chain 5'-O-lysophosphatidyloligonucleotides [5'-O-(1-O-palmitoyl-sn-glycero-3-phosphoryl)oligodeoxynucleotides, (5'-LyPOdNs)] were synthesized following a two-step chemoenzymatic synthesis. 5'-O-(sn-Glycero-3-phosphoryl)oligodeoxynucleotides (5'-GPOdNs) were first prepared by simply using a phosphoramidite of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) in a further coupling step after the solid-phase elongation of each desired oligodeoxynucleotide. Next, the regioselective palmitoylation at the C-1 hydroxyl of the glycerol moiety of 5'-GPOdNs was achieved by a lipase-catalyzed transacylation with trifluoroethyl palmitate in organic solvent. Despite of the molecular bulkiness of 5'-GPOdNs, 2-, 3-, and 4-mer 5'-LyPOdNs were prepared by this procedure. Although in very low yield, 5- and 6-mer 5'-LyPOdNs were also obtained by this way.     

2'-anthraquinone-conjugated oligonucleotide as an electrochemical probe for DNA mismatch

We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be approximately 50 s (-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates ( approximately 50 s (-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G--A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.     

Di(azacrown) conjugates of 2'-O-methyl oligoribonucleotides as sequence-selective artificial ribonucleases

Functionalized 2'-O-methyl oligoribonucleotides bearing two 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane ligands attached via a phosphodiester linkage to a single non-nucleosidic building block have been prepared on a solid-support by conventional phosphoramidite chemistry. The branching units employed for the purpose include 2,2-bis(3-hydroxypropylaminocarbonyl)propane-1,3-diol, 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside, and 2-hydroxyethyl 2'-O-(2-hydroxyethyl)-beta-D-ribofuranoside. Each of these has been introduced as a phosphoramidite reagent either into the penultimate 3'-terminal site or in the middle of the oligonucleotide chain. The dinuclear Zn2+ complexes of these conjugates have been shown to exhibit enhanced catalytic activity over their monofunctionalized counterpart, the 3'-terminal conjugate derived from 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside being the most efficient cleaving agent. This conjugate cleaves an oligoribonucleotide target at a single phosphodiester bond and shows turnover and 1000-fold cleaving activity compared to the free monomeric Zn2+ chelate of 1,5,9-triazacyclododecane.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

A New Phosphoramidite Reagent for the Incorporation of Diazaphenoxazinone Nucleoside With Enhanced Base-Pairing Properties into Oligodeoxynucleotides

Abstract

New phosphoramidite reagent 7 suitable for incorporation of dC analogue was synthesized. ODNs containing diazaphenoxaziione residues in defined positions were prepared. The stability of duplexes formed was increased up to 3-5°C per modified base. Preliminary results of molecular biological testing were reported.

     

G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties.

Modified oligodeoxyribonucleotides (ODNs) that have unique hybridization properties were designed and synthesized for the first time. These ODNs, called selective binding complementary ODNs (SBC ODNs), are unable to form stable hybrids with each other, yet are able to form stable, sequence specific hybrids with complementary unmodified strands of nucleic acid. To make SBC ODNs, deoxyguanosine (dG) and deoxycytidine (dC) were substituted with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP), respectively. The hybridization properties of several otherwise identical complementary ODNs containing one or both of these nucleoside analogs were studied by both UV monitored thermal denaturation and non-denaturing PAGE. The data showed that while dI and dP did form base pairs with dC and dG, respectively, dI did not form a stable base pair with dP. A self-complementary ODN uniformly substituted with dI and dP acquired single-stranded character and was able to strand invade the end of a duplex DNA better than an unsubstituted ODN. This observation implies that SBC ODNs should effectively hybridize to hairpins present in single-stranded DNA or RNA.     

Synthesis of 2'-modified oligonucleotides containing aldehyde or ethylenediamine groups

Oligonucleotides carrying 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2'-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2'-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.     

Conformationally locked nucleosides. Synthesis of oligodeoxynucleotides containing 3'-amino-3'-deoxy-3'-N,5'(R)-C-ethylenethymidine

3'-Amino-3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-N,5'(R)-C-ethylenethymidine (6) was synthesized starting from 3'-azido-3'-deoxythymidine. Condensation of 6 with 5'O-(H-phosphonyl)thymidine and 5'-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Endowing RNase H-inactive antisense with catalytic activity: 2-5A-morphants

A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2'](n)()p5'A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'-->5')adenylyl(2'-->5')adenylyl(2'-->5')adenosine (p5'A2'p5'A2'](2)p5'A2', tetramer 2-5A, 9) was synthesized starting with 2',3'-O-dibenzoyl-N(6),N(6)-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N(6)-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl) adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection, generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protected tetramer 2-5 A. The title product 9 p5'A(2'p5'A)(3) (tetramer 2-5A) was obtained through phosphorylation of the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 degrees C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2'](2)p5'A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.     

Semisynthesis of 6-Chloropurine-2′-deoxyriboside 5′-Dimethoxytrityl 3′-(2-Cyanoethyl-N,N-diisopropylamino)Phosphoramidite and its Use in the Synthesis of Fluorescently Labeled Oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2′-deoxyriboside from the reaction of 6-chloropurine with 2′-deoxycytidine catalyzed by nucleoside-2′-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2′-deoxyriboside 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

Synthesis of metallointercalator-DNA conjugates on a solid support

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.     

Identification of the NADH-NAD+ transhydrogenase peptide of the mitochondrial NADH-CoQ reductase (Complex I). A photodependent labeling study utilizing arylazido-beta-alanyl NAD+

Incubation of Complex I (NADH-CoQ reductase) of ox heart mitochondria at 4 degrees C in the presence of 0.5 M NaClO4 followed by ammonium sulfate fractionation of the solubilized proteins results in the isolation of a resolved preparation still capable of catalyzing NADH-NAD+ transhydrogenation but having only low levels of NADH dehydrogenase activity. A number of NAD(H) analogues, including the photoaffinity probes, arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+ and arylazido-beta-alanyl AcPyAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]AcPyAD+ can be utilized as substrates for transhydrogenation in this preparation. A further incubation (10 min) of the resolved NADH-NAD+ transhydrogenase in the presence of 0.5 M NaClO4, but now at 30 degrees C, results in the complete loss of this transhydrogenase activity. Photoaffinity labeling experiments utilizing arylazido-[3-3H]beta-alanyl NAD+ and arylazido-[3-3H]beta-alanyl AcPyAD+ with the resolved NADH-NAD+ transhydrogenase preparation prior to and following NaClO4 (30 degrees C) treatment indicates that the 42,000 molecular weight component of Complex I is the pyridine nucleotide binding site responsible for the major NADH-NAD+ (DD) transhydrogenase activity of Complex I.     

Synthesis of multi-galactose-conjugated 2′-O-methyl oligoribonucleotides and their in vivo imaging with positron emission tomography

⁶⁸Ga labelled 2′-O-methyl oligoribonucleotides (anti-miR-15b) bearing one, three or seven d-galactopyranoside residues have been prepared and their distribution in healthy rats has been studied by positron emission tomography (PET). To obtain the heptavalent conjugate, an appropriately protected 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) precursor bearing a 4-[4-(4,4′-dimethoxytrityloxy)butoxy]phenyl side arm was first immobilized via a base labile linker to the support and the oligonucleotide was assembled on the detritylated hydroxyl function of this handle. A phosphoramidite building block bearing two phthaloyl protected aminooxy groups and one protected hydroxyl function was introduced into the 5′-terminus. One acetylated galactopyranoside was coupled as a phosphoramidite to the hydroxyl function, the phthaloyl protections were removed on-support and two trivalent galactopyranoside clusters were attached as aldehydes by on-support oximation. A two-step cleavage with aqueous alkali and ammonia released the conjugate in a fully deprotected form, allowing radiolabelling with ⁶⁸Ga in solution. The mono- and tri-galactose conjugates were obtained in a closely related manner. In vivo imaging in rats with PET showed remarkable galactose-dependent liver targeting of the conjugates.     

Synthesis of two 8-[(anthraquinone-2-yl)-linked]-2'-deoxyadenosine 3'-benzyl hydrogen phosphates for studies of photoinduced hole transport in DNA

The challenge in working with anthraquinone-2'-deoxyadenosine (AQ-dA) conjugates is that they are insoluble in water and only sparingly soluble in most organic solvents. However, water-soluble AQ-dA conjugates with short linkers are required for study of their electrochemical and intramolecular electron transfer properties in this solvent prior to their use in laser kinetics investigations of photoinduced hole (cation) transport in DNA. This article first describes the synthesis of a water-soluble, ethynyl-linked AQ-dA conjugate, 8-[(anthraquinone-2-yl)ethynyl]-2'-deoxyadenosine 3'-benzyl hydrogen phosphate, based on initial formation of a 5'-O-(4,4'-dimethoxytrityl) (5'-O-DMTr) intermediate. Because intended H2 over Pd/C reduction of the ethynyl linker in 5'-O-DMTr-protected 2'-deoxyadenosines cleaves the DMTr protecting group and precipitates multiple side products, this work also describes the synthesis of an ethylenyl-linked AQ-dA conjugate, 8-[2-(anthraquinone-2-yl)ethyl]-2'-deoxyadenosine 3'-benzyl hydrogen phosphate, starting with a 5'-O-tert-butyldiphenylsilyl protecting group.     

Semisynthesis of 6-chloropurine-2'-deoxyriboside 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and its use in the synthesis of fluorescently labeled oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2'-deoxyriboside from the reaction of 6-chloropurine with 2'-deoxycytidine catalyzed by nucleoside-2'-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2'-deoxyriboside 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

Perturbing the DNA sequence selectivity of metallointercalator-peptide conjugates by single amino acid modification.

Metallointercalator-peptide conjugates that provide small molecular mimics to explore peptide-nucleic acid recognition have been prepared. Specifically, a family of peptide conjugates of [Rh(phi)(2)(phen')](3+) [where phi = 9,10-phenanthrenequinone diimine and phen' = 5-(amidoglutaryl)-1,10-phenanthroline] has been synthesized and their DNA-binding characteristics examined. Single amino acid modifications were made from the parent metallointercalator-peptide conjugate [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2), which targets 5'-CCA-3' site-specifically. Moving the glutamate at position 10 in the sequence of the appended peptide to position 6 {[Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2)} changed the sequence preference of the metallointercalator-peptide conjugate to 5'-ACA-3'. Subsequent mutation of the glutamate at position 6 to arginine {[Rh(phi)(2)(phen')](3+)-AANVARAAWARAA-CONH(2)} caused more complex changes in DNA recognition. Thermodynamic dissociation constants were determined for these metallointercalator-peptide conjugates by photoactivated DNA cleavage assays with the rhodium intercalators. At 55 degrees C in the presence of 5 mM MnCl(2), [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2) binds to a 5'-CCA-3' site with K(d) = 5.7 x 10(-)(8) M, whereas [Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2) binds to its target 5'-ACA-3' site with K(d) = 9.9 x 10(-8) M. The dissociation constant for [Rh(phi)(2)(phen')](3+) with random-sequence DNA is 7.0 x 10(-7) M. Structural models have been developed and refined to account for the observed sequence specificities. As with much larger DNA-binding proteins, with these metal-peptide conjugate mimics, single amino acid changes can lead to single or multiple base changes in the DNA site targeted.