Proc. Natl. Acad. Sci. USAVol. 92, pp. 5798-5802, June 1995Biochemistry

Oligonucleotide N3' -* P5' phosphoramidatesSERGEI M. GRYAZNOV*t, DAVID H. LLOYD*, JER-KANG CHEN*, RONALD G. SCHULTZ*,LAWRENCE A. DEDIONISIO*, LYNDA RATMEYERt, AND W. DAVID WILSONt*Lynx Therapeutics, Inc., 3832 Bay Center Place, Hayward, CA 94545; and *Georgia State University, Department of Chemistry, Atlanta, GA 30303

Communicated by Robert L. Letsinger, Northwestern University, Evanston, IL, March 9, 1995 (received for review November 17, 1994)

ABSTRACT Synthetic oligonucleotides and their analogshave attracted considerable interest recently. These com-pounds may lead to highly specific therapeutic agents, as wellas to powerful diagnostic tools. Here, we present the synthesisof uniformly modified oligodeoxyribonucleotide N3' -> P5'phosphoramidates containing 3'-NHP(O)(0-)0-5' inter-nucleoside linkages and the study of their hybridizationproperties. Thermal dissociation experiments show that thesecompounds form very stable duplexes with single-strandedDNA, RNA, and with themselves following Watson-Crick basepairing. The duplex thermal stability was enhanced by 2.2-2.60C per modified linkage compared with phosphodiesters.The structure of complexes formed by phosphoramidatesclosely resembles that of RNA oligomers and corresponds toan A form, as judged by CD spectroscopy. N3' -> P5'phosphoramidates also form stable triplexes with double-stranded DNA under near-physiological conditions when nat-ural phosphodiesters fail to do so. Physicochemical charac-teristics of the amidates are similar to those ofRNA oligomers,even though they are composed of 2'-deoxyfuranose-basednucleosides.

Synthetic oligonucleotides have a great potential to become anew type of rationally designed therapeutic agent. Thesecompounds could interfere with the expression of selectedgenes through a specific interaction with RNA orDNA regionsof interest (1). Several modifications of the natural phosphodi-ester internucleoside bond have been introduced to improvehybridization properties of oligonucleotides as well as theircellular membrane penetration and stability and distributioninside cells (2-4). Unfortunately, the vast majority of thesemore hydrolytically stable analogs show reduced binding withRNA or DNA via duplex or triplex formation (5).

In the present report we describe the synthesis and hybrid-ization properties of the new analogs of nucleic acids-uniformly modified oligonucleotide N3' -* P5' phosphorami-dates (Fig. 1). These compounds possess some very attractivefeatures for therapeutic and diagnostic applications, such as anegatively charged and achiral phosphorus, good solubility inwater, and phosphodiesterase digestion resistance, buttressedby a high affinity for natural RNA and DNA.

MATERIALS AND METHODSOligonucleotide Synthesis. Oligodeoxyribonucleotides and

oligoribonucleotides were prepared on an ABI 380B synthe-sizer using standard DNA or RNA assembly protocols via thephosphoramidite method. Oligonucleotide N3' -> P5' phos-phoramidates were synthesized on 1 ,umol scale using an ABI394 synthesizer and 5'-4,4'-dimethoxytrityl (DMT)-N-acyl-3'-amino-2',3'-dideoxynucleosides, as described (6). Oligonucle-otides were purified by ion exchange HPLC on a PharmaciaMono Q 10/10 column at pH 12 (10 mM NaOH) with a 1%

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

03'-+P5' phosphodiester N3'-+P5' phosphoramidate

FIG. 1. Structure of the internucleoside phosphodiester and N3'PS' phosphoramidate groups; B is any nucleobase.

per min gradient of 1.5 M NaCl in 10 mM NaOH and a flowrate of 2 ml/min. Oligonucleotides were desalted immediatelyafter purification on Pharmacia NAP-5 gel filtration columnsand then lyophilized in vacuo.Thermal Dissociation Experiments. A Cary-Varian 1E

spectrophotometer equipped with a temperature programmerand data processor from Varian was used according to thereported procedure for thermal dissociation experiments (7).Buffer concentrations and compositions are listed in thelegends to Tables 1-3. Absorbance values at 260 nm wereobtained at 1-min intervals with a temperature increase of1.0°C/min. The reported tm (°C) is the temperature at themidpoint of the slope of a plot of absorbance vs. temperature.Thermodynamic Parameters Determination. Thermody-

namic parameters for the dodecamers 11-13 (see Table 1)were determined by van't Hoff analysis from the thermaldissociation experiments, performed on a Cary 4 spectropho-tometer, where tm was measured as a function of oligonucle-otide strand concentration (8). These tm measurements wereperformed in 10 mM Pipes, pH 7.0/1.0 mM EDTA. Meltingcurves were completely reversible on heating and coolingunder the conditions used. The thermodynamic values aregenerally determined in 1.0 M NaCl. However, since the tm ofthe phosphoramidate duplex is greater than 90°C in 1.0 MNaCl, tm values for the duplexes 11-13 were determined as afunction of log[Na+] (at five different NaCI concentrationsbetween 50 mM and 500 mM), and tm values in 1.0 M NaClwere determined by extrapolation. tm values generally changelinearly with log[Na+] below 0.5 M Na+, while the slopedecreases at higher Na+ concentrations (9); thus, extrapolatedtm values for 1.0 M NaCl may be slightly over estimated. Errorsin thermodynamic values at 1.0 M NaCl caused by this methodare small and close to the experimental error limit and areconsistent for compounds 11-13 (see Table 1) so that com-parisons are valid.

Abbreviation: DMT, 4,4'-dimethoxytrityl.tTo whom reprint requests should be addressed.

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RESULTS AND DISCUSSION

Synthesis of Oligonucleotide N3' -- P5' Phosphoramidates.Oligonucleotide N3' -> P5' phosphoramidates were synthe-sized on an automated synthesizer by using solid-phase meth-odology and a step-by-step elongation procedure, as generallyoutlined before (6). The synthetic cycle consists of the follow-ing chemical reactions: (i) detritylation to deprotect the 5'-hydroxyl group of a polymer-supported mono- or oligonucle-otide; (ii) phosphitylation of the 5'-hydroxyl group, followed byhydrolysis to produce a nucleoside 5'-H-phosphonate diestergroup; (iii) carbon tetrachloride-driven coupling of a 5'-DMT-N-acyl-3'-aminonucleoside to the 5'-H-phosphonate, produc-ing an internucleoside N3' -> P5' phosphoramidate diesterlinkage and extending the oligonucleotide chain by one unit.This cycle can be repeated the required number of times andafter removal of the f3-cyanoethyl phosphate-protectinggroups and cleavage from the solid support with ammonia,affords oligonucleotide N3' -> P5' phosphoramidate mo-noesters of defined base sequence (Fig. 2).

Chimeric phosphodiester-phosphoramidate oligomers weresynthesized by using a combination of the phosphoramiditemethod (10) and the one described above. Compounds werepurified by ion-exchange HPLC and analyzed by capillaryelectrophoresis and slab gel electrophoresis. The oligonucle-otide N3' -- P5' phosphoramidates which were prepared forthis study are listed in Tables 1-3.Duplex Formation and Stability. First, thermal stabilities of

hairpin-shaped duplexes and self-complementary oligonucle-otides were evaluated (Table 1). Thermal dissociation exper-iments showed that substitution of every other phosphodiestergroup by phosphoramidate in one strand increased the thermalstability of the duplex by - 1.2°C relative to the phosphodiestercounterparts (compare exps. 5 with 6 and 8 with 9; Table 1).More stabilization (4.3-7.7°C) was achieved when phosphor-amidates were placed in alternating order in both strands of the

a

1.5

~~~~1-

0.5

4

200 220 240 260 280 300 320Wavelength, nm

FIG. 3. CD spectra of the oligonucleotides 1-4, Table 1, recordedat 0°C in a buffer solution containing 1.0 M NaCl, 1 mM EDTA, and10 mM sodium phosphate, pH 7.0. The strand concentration was 5 ,M.

duplexes (compare exps. 1 with 2, 3 with 4, 5 with 7, and 8 with10; Table 1). CD spectra of the duplexes recorded at differenttemperatures and salt concentrations indicated structural dif-ferences between complexes containing alternating phos-phodiester-phosphoramidates and the parent phosphodiesters.CD spectra for the O-P O-P duplexes were characteristic of Bform, whereas chimeric N-P,O-P-O-P or N-P, O-P N-P, O-Pcompounds of the same nucleotide sequence gave spectrawhich more resembled A form (Fig. 3). The degree of simi-larity to A-form characteristic spectra appeared to be roughlyproportional to the level of substitution of phosphodiesterlinkages for phosphoramidates in the duplex strands.

In addition, the thermal stability of duplexes formed by theuniformly modified oligonucleotide N3' -- P5' phosphorami-

011

H-P-0

b - OCE B cb

d

FIG. 2. Synthetic scheme for N3' -* P5' phosphoramidates. R = succinyl controlled pore glass. Conditions: (a) dichloroacetic acid indichloromethane; (b) (2-cyanoethoxy)-(N,N-diisopropylamino)chlorophosphine and N,N-diisopropylethylamine in dichloromethane; (c) tetrazoleand water in acetonitrile; (d) 5'-DMT-3'-aminonucleoside, triethylamine, and carbon tetrachloride in acetonitrile; (e) repetition of steps a-d; and(f) aqueous ammonia.

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Table 1. Self-complementary and hairpin oligonucleotides and their tm values

Oligonucleotide*Exp. Structure No. tm, OCt tm, OCt

1 TATATATATA 1 o152 TnpATnpATnpATnpATnpA 2 22.03 CGCGCGCGCG 3 70.7 ND4 CnpGCnpGCnpGCnpGCnpG 4 75.0 ND5 TATATATATA 52.3 37.6

ATATATATAT 46 TnpATnpATnpATnpATnpAT 6 53.5 42.0

A TA TA TA TA T7 TnpATnpATnpATnpATnpAT 60.0 45.0

AnpTAnpTAnpTAnpTAnpT8 TACGTACGTAT 8 72.9 59.1

ATGCATGCAT9 TnpACnpGTnpACnpGTnpAT 9 74.0 62.3

A TG CA TG CA T10 TnpACnpGTnpACnpGTnpAT 10 80.0 66.5

AnpTGnpCAnpTGnpCAnpT 111 CGCGAATTCGCG 11 58.012 CnpGnpCnpGnpAnpAnpTnpTnpCnpGnpCnpG 12 84.0 78.013 r-CGCGAAUWCGCG 13 66.4 NDDashes indicate that duplex formation was not observed; ND, tm was not determined; np, a

3'-NHP(O)(O-)O-5'-phosphoramidate linkage.*All compounds are 2'-deoxyoligonucleotides, unless specified.ttm in 10 mM Tris HCI, pH 7.0/150 mM NaCI.t:tm in 10 mM Tris HCI, pH 7.0.

dates with complementary phosphodiester RNA or DNAsingle strands was evaluated. Data presented in Table 2indicate that phosphoramidates formed significantly morestable duplexes than phosphodiester compounds, regardless ofnucleotide sequence and base composition. The average en-hancement of the duplex thermal stability was 2.3-2.6°C perlinkage, resulting in an increase of melting temperature (tm) by20-30°C (compare exps. 3 with 6, 13 with 14, and 15 with 16;Table 2). One exception was observed for decaadenylic acids14 and 15, exps. 1 and 4, where the stability of the duplex withcomplementary oligo(dT) was lower for phosphoramidatethan for phosphodiester. CD spectra for some of the N-P-O-Pduplexes were also indicative of structural differences relative

to their O-P O-P counterparts. A dramatic change in thermalstability was observed for duplexes formed by two phosphor-amidate strands, where tm increased up to -2.4°C per linkagerelative to the corresponding phosphodiesters. Thus, self-complementary phosphodiester 11 and phosphoramidate 12(Table 1) have tms of 58.0°C and 84.0°C, respectively. Forcomparison, the t. of ribooligonucleotide 13 of the samesequence is 66.4°C (Table 1, exp. 13). Analogous results wereobtained for oligomers 14 and 15 (Table 2, exps. 1 and 5),which gave duplex tms of 24.4°C and 43.4°C with the phos-phodiester and phosphoramidate complements. CD analysis ofthe dodecamers phosphodiester 11 and phosphoramidate 12revealed curves that are characteristic for B form and A form,

Table 2. Oligonucleotides and t. values of duplexes

Oligonucleotide*

Exp. Structure No. Target* tm, Oct1 AAAAAAAAAA 14 TTTTTTTTTT 24.42 Same as exp. 1 TnpTnpTnpTnpTnpTnpTnpTnpTnpT 35.83 Same as exp. 1 poly(U) 37.74 AnpAnpAnpAnpAnpAnpAnpAnpAnpA 15 TTTTTTTTTT 20.05 Same as exp. 4 TnpTnpTnpTnpTnpTnpTnpTnpTnpT 43.46 Same as exp. 4 poly(U) 50.87 TnpTnpTnpTnpTnpTnpTnpTnpTnpT 16 poly(dA) 36.08 Same as exp. 7 poly(A) 51.59 TnmpTnmpTnmpTnmpTnmpTnmpTnmpTnmpTnmpTt 17 poly(dA)

10 Same as exp. 9 poly(A) 26.511 TpnTpnTpnTpnTpnTpnTpnTpnTT§ 18 poly(dA)12 Same as exp. 11 poly(A)13 AACGTTGAGGGGCAT 19 r-AUGCCCCUCAACGUU 62.5; 48.01114 AnpAnpCnpGnpTnpTnpGnpAnpGnpGnpGnpGnpCnpAnpT 20 r-AUGCCCCUCAACGUU 90.0; 79.8w15 CCGCTTCTTCCT 21 r-GGAAGAAGCGGAGACAGCII 58.016 CnpCnpGnpCnpTnpTnpCnpTnpTnpCnpCnpT 22 Same as exp. 13 84.0Dashes indicate that duplex formation was not observed; np is a 3'-NHP(O)(O-)O-5' phosphoramidate linkage.

*All compounds are 2'-deoxyoligonucleotides, unless specified.ttm in 10 mM sodium phosphate/150 mM NaCl, pH 7.0.tnmp is a 3'-NMeP(O)(O-)0-5' methylphosphoramidate linkage.§pn is 3'-O(O-)P(O)NH-5'-phosphoramidate link.1tm in 10 mM sodium phosphate, pH 7.0.IlDuplex-forming region is underlined.

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respectively, and indicates substantial differences in theirduplex structures (Fig. 4).One of the main distinguishing features of A-form and

B-form duplexes is the difference in the topology of minor andmajor grooves (11). We used the minor groove AATT regionbinding agent distamycin to further characterize the phospho-ramidate duplex (12). While distamycin significantly increasedthe tm of phosphodiester 11 by 16.2°C due to binding to theduplex in the minor groove, no change of tm was observed forphosphoramidate 12, as well as for ribooligonucleotide 13.Again, this indicated that the phosphoramidate duplex moreclosely resembled the A-form structure ofRNA rather than theB form of DNA. Analysis of vicinal proton-proton couplingconstants (3JHH) for the model dTA dinucleoside with aphosphodiester or phosphoramidate internucleoside groupwas indicative of the N-conformational state in the penta-furanose ring of the 3'-amino-3'-deoxythymidine, rather thanthe S-conformation, which is characteristic of the 2'-deoxy-nucleosides (6). This observation is in agreement with the dataof Thibaudeau et at (13), who showed that, for 2'-deoxy-ribonucleosides, the introduction of electron-donating sub-stituents, relative to hydroxyl, at the 3' carbon leads to apredominance of the N-conformation of the furanose. Takentogether, all of these results suggest that oligonucleotide N3'

P5' phosphoramidates form A-type duplexes with comple-mentary phosphoramidates or complementary RNA strands,because of the N-conformation of their furanose rings asdictated by the 3'-amino group.Although RNARNA duplexes existing in A form are, in

general, more stable than DNADNA or DNARNA duplexes,it remains difficult to explain the remarkable thermal stabilityof complexes formed by phosphoramidates based entirely onthe adoption of an A-form, rather than a B-form duplex. Thus,the tm of self-complementary phosphoramidate 12 is 17.6°Chigher than for the RNA oligomer 13 of the same sequence(compare exps. 12 and 13; Table 1), while both are in an A-typeduplex (Fig. 4). The calculated free energy AG' of denatur-ation for dodecamers 11-13 (Table 1) was 12.8, 17.0, and 14.5kcal/mol, respectively; the corresponding AH' and AS' valuesfor duplex denaturation were 59.6, 94.8, and 67.0 kcal/mol and0.151, 0.251, and 0.169 kcal/mol.degree, respectively.

In addition, the presence of a proton in the NH groups ofN3' -> P5' phosphoramidates appears to play a very importantrole in the increased tm of duplexes. Substitution of thesehydrogen atoms by methyl groups leads to the completeabolition of binding of the 3'NMe -> P5' methylphosphorami-dates [which were prepared analogously to the N3' > P5'

2

4-~ ~ ~ ~ -1

U- -12

l

-4 &--13-5 1. I

200 220 240 260 280 300 320

Wavelength, nm

FIG. 4. CD spectra of the oligonucleotides 11-13, Table 1, re-corded at 15°C. Buffer conditions were as described in the legend toFig. 3. The strand concentration was 2.5 ,uM.

phosphoramidates by using 5 '-DMT-3 '-methylamino-3'-deoxythymidine (14) as a building monomer] to single-stranded DNA and a dramatic reduction of thermal stability(by 25°C) of the duplex formed by the 10-mer with single-stranded RNA (compare exps. 7 with 9 and 8 with 10; Table2). Interestingly, the structural isomers of the N3' -> P5'phosphoramidates, P3' -> N5' phosphoramidates, synthesizedaccording to the published method (7, 15), do not formduplexes with single-stranded DNA or RNA or triplexes withdouble-stranded DNA (Table 2, exps. 11 and 12). We suspectthat a possible interaction between the 4'-O of 2'-deoxyfuranose and the hydrogen of the 5'-NH group mayseverely alter the structure of the sugar-phosphate backboneand lead to the inability of nucleobases to engage in Watson-Crick hydrogen bonding.

It is also important to mention an observed increase inapparent rigidity of the N3' -> P5' phosphoramidate linkages(relative to phosphodiesters) that is probably derived from aN(n) -- P(3d) electronic conjugation. Thus, N3' -- P5' phos-phoramidates exhibit slower mobility in denaturing and non-denaturing PAGE (Fig. 5) than phosphodiesters of the samesequence. Retention times on ion-exchange and reversed-phase HPLC resins are shorter for N3' -> P5' phosphorami-dates than those for the corresponding phosphodiesters, sug-gesting a reduced net negative charge of the molecules and anincreased hydrophilicity, respectively (data not shown).

Triplex Formation Properties. It was reported that RNAoligopyrimidines oriented parallel to a polypurine strand of aduplex form more stable triple-stranded complexes with dou-ble-stranded DNA, double-stranded RNA, and mixedRNADNA duplexes than do DNA oligopyrimidines (16, 17).This difference in behavior of RNA and DNA molecules canbe attributed to the N-sugar puckering of RNA nucleotideswhich favors Hoogsteen-type hydrogen bond formation withpolypurine tracts in double-stranded nucleic acids. Since N3'

> P5' phosphoramidates appeared to adopt RNA-like N-sugar conformation, it was desirable to evaluate the triplexformation properties of these compounds. We have usedthermal-dissociation experiments in solution and mobilityanalysis of nucleic acid complexes in nondenaturing polyacryl-amide gels (gel-shift assay) to study triplex formation. Themelting temperatures of the complexes studied are summa-rized in Table 3.The melting curves obtained show that the thymidine- and

cytidine-containing 11-mer phosphoramidate 24 (Table 3) canform a stable triplex with the duplex region of the monomo-lecular hairpin DNA target (Table 3, exp. 2) where the tm of

1 2 3 4 5

FIG. 5. Gel electrophoretic analysis of the oligonucleotide triplexformation under nondenaturing conditions: 0.75 mm thick, 20% acryl-amide/5% bisacrylamide gel in 80 mM Tris/160 mM boric acid/10MM MgC.2 at pH 7.2 and 10eC. A total of 0.1 A260of OligonucleotidesinS ,ul of buffer were loaded per lane. Lane 1, phosphodiester 23; lane2, phosphoramidate 24; lane 3, phosphodiester monomolecular hairpintarget duplex (slow-moving minor band is likely to correspond to thebimolecular duplex); lane 4, hairpin target and 23; lane 5, hairpintarget and 24. Mobility of the triplex is denoted by arrow. Gel wasstained with Stains-all (Kodak) and imaged on a Molecular Dynamicsdensitometer.

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Table 3. Oligonucleotides and tm values of triplexesOligonucleotide* tm, tm,

Exp. Structure No. Target* OCt Oct1 CTTCTTCCTTA 23 TTCCTTCTTCT-

AAGGAAGAAGT42 CnpTnpTnpCnpTnpTnpCnpCnpTnpTnpA§ 24 Same as exp. 1 57.0 62.03 TTTTCTTTTCCCCCCT 25 AAAAGAAAAGGGGGGA -19

TTTTCTTTTCCCCCCT4 TnpTnpTnpTnpCnpTnpTnpTnpTnpCnpCnpCnpCnpCnpCnpT 26 Same as exp. 3 24.0 40.5S TTTTCTTTTGGGGGG 27 Same as exp. 3 32.06 TnpTnpTnpTnpCnpTnpTnpTnpTnpGnpGnpGnpGnpGnpG 28 Same as exp. 3 36.4 55.47 TnpTnpTnpTnpTnpTnpTnpTnpTnpTCAc 29 poly(dA) 38.2 58.6

T T T T T T T T T TCA8 Same as exp. 5 poly(A) 41.7 58.6Dashes indicate that triplex formation was not observed; np is a 3'-NHP(O)(O-)O-5' phosphoramidate linkage.

*All compounds are 2'-deoxyoligonucleotides, unless specified.ttm in 10 mM sodium phosphate buffer (except exps. 1 and 2, where 10 mM Tris HCl was used)/150 mM NaCl, pH 7.0.*tm in 10 mM sodium phosphate buffer (except exps. 1 and 2, where 10 mM Tris HCl was used)/150 mM NaCl/10 mM MgCl2, pH 7.0.§Ten nucleosides except 3'-A are available for triplex formation with the duplex target.

the triplex was 57.0°C or 62.0°C in buffers containing 150 mMsodium chloride in the absence and presence of 10 mMmagnesium chloride, respectively. Gel-shift experiments alsoshowed formation of the triplex in this system (Fig. 5). Incontrast, the corresponding phosphodiester 11-mer 23 did notform a triplex under identical conditions (Table 3, exp. 1, andFig. 5). Analogous results were obtained for another polypyri-midine 16-mer phosphoramidate, 26, and for purine-pyrim-idine oligomer 28, both of which formed stable triplexes witha bimolecular duplex (compare exps. 3 with 4 and 5 with 6,Table 3).

In addition, we found that the chimeric phosphoramidate-phosphodiester oligomer 29 formed stable clamp-shaped, tri-ple-stranded complexes with single-stranded DNA and single-stranded RNA (Table 3, exps. 7 and 8). It has been shown thatpyrimidine DNA oligomers cannot form stable triplexes withheteroduplexes where the polypurine strand is RNA (16, 17),but oligopyrimidine RNAs can. Since N3' -> P5' phosphor-amidates appear to adopt an N-sugar conformation, it is likelythat they can successfully mimic RNA oligomers and formtriplexes with RNADNA or RNA-phosphoramidate hetero-duplexes.

CONCLUSIONSOligonucleotide N3' -* P5' phosphoramidates were synthe-sized by using solid-phase methodology and 5'-DMT-base-protected-3'-amino-2',3'-deoxyribonucleotides as key mono-mer units. We have shown that N3' -- P5' phosphoramidatesare capable of forming extremely stable duplexes with com-plementary amidates, phosphodiester single-stranded DNAand single-stranded RNA, and triplexes with double-strandedDNA, as well as RNA-DNA heteroduplexes. In spite of being2'-deoxyribooligomers, N3' -> P5' phosphoramidates are sim-ilar to RNA in sugar puckering and hybridization properties,exhibiting even significantly enhanced binding affinities. CDspectra and molecular modeling indicate that duplexes formedby N3' -> P5' phosphoramidates are in A rather than B form.

It is possible to propose that at an early stage of terrestriallife some negatively charged phosphorus derivatives other thanphosphodiesters may have linked nucleosides together. Thegreater nucleophilicity of a 3'-amino group (relative to a

3'-hydroxyl group) would have allowed such monomers tomore effectively compete for an activated nucleoside-5'-phosphate in the presence of a vast excess of water during theassembly of nucleotide polymers (18). Furthermore, the en-hanced duplex and triplex formation properties observed forphosphoramidate oligonucleotides would have been valuableattributes in any self-replication scheme. Consequently, onecan suggest, on the basis of the disclosed physicochemicalproperties, that nucleic acids containing N3' -> P5' phospho-ramidate linkages may have existed in prebiotic systems onEarth and later evolved into 03' -- P5' phosphodiesters.

The communicating member is a consultant for Lynx Therapeutics,Inc.

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