WO2001014398A1 - ACIDE NUCLEIQUE PEPTIDIQUE A HELICE ALPHA (αPNA) - Google Patents

ACIDE NUCLEIQUE PEPTIDIQUE A HELICE ALPHA (αPNA) Download PDF

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Publication number
WO2001014398A1
WO2001014398A1 PCT/US2000/021845 US0021845W WO0114398A1 WO 2001014398 A1 WO2001014398 A1 WO 2001014398A1 US 0021845 W US0021845 W US 0021845W WO 0114398 A1 WO0114398 A1 WO 0114398A1
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nucleic acid
peptide
based nucleic
surrogate
αpna
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PCT/US2000/021845
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English (en)
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Philip P. Garner
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Garner Philip P
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Priority to AU66292/00A priority Critical patent/AU6629200A/en
Application filed by Garner Philip P filed Critical Garner Philip P
Priority to US10/110,017 priority patent/US7183394B1/en
Publication of WO2001014398A1 publication Critical patent/WO2001014398A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)

Definitions

  • the present invention relates to the design, synthesis, and potential applications of ⁇ -helical peptide nucleic acids ( ⁇ PNAs) which are new molecular constructs that merge the molecular recognition elements of nucleic acids with the predictable structural features of an ⁇ -helical peptide scaffold.
  • ⁇ PNAs ⁇ -helical peptide nucleic acids
  • These new peptide-based nucleic acid surrogates bind with high affinity and specificity to complementary single stranded DNA and are capable of binding to both single stranded RNA and double stranded DNA targets.
  • the present invention is directed to the use of ⁇ PNAs for therapeutic (antisense, antigene), diagnostic (genetic), and molecular switching ( ⁇ PNA chip) applications.
  • ⁇ -helical subunits serve as the molecular scaffolding for presentation of key amino acid side chains to their specific nucleic acid binding sites.
  • Cf. Steitz, T.A. Q. Rev. Biophys. 1990, 23, 205 Sequence-specific binding of these ⁇ -helical binding domains to double- stranded DNA occurs in the major groove as a consequence of multiple interactions arrived at (combinatorially) through evolutionary selection.
  • code for molecular recognition of double-stranded DNA, it is not yet feasible to rationally design a peptide structure that will bind to any particular DNA duplex.
  • the present invention relates to the design, synthesis, and potential applications of ⁇ -helical peptide nucleic acids ( ⁇ PNAs), which are new constructs that merge the molecular recognition elements of nucleic acids with the predictable structural features of an ⁇ -helical peptide scaffold.
  • ⁇ PNAs ⁇ -helical peptide nucleic acids
  • the described ⁇ PNAs bind with high affinity and specificity to complementary single-stranded DNA and are capable of binding to both single-stranded RNA and double- stranded DNA targets.
  • peptide based nucleic acid surrogates the use of peptide secondary structure to achieve the nucleobase spacing required for base-pairing to helical nucleic acids is conceptually novel.
  • the ⁇ PNA platform has a major advantage over known art in that it allows for the ready incorporation of functionality to enhance binding (affinity, kinetics, specificity) and modify/introduce other properties (solubility, transport, stability). Also, multifunctional ⁇ PNAs may be designed for use as artificial nucleases, biosensors, and molecular switches.
  • FIGURE 1A is a Watson-Crick base pairing of the preferred ⁇ PNA module with a preferred single-stranded nucleic acid target.
  • FIGURE 1B is a preferred representation of an ⁇ PNA binding unit.
  • FIGURES 2 A-C show gel shift assay binding studies.
  • FIGURE 3 is a chart showing circular dichroism (CD) intensity versus mole percent of the preferred ⁇ PNA.
  • FIGURE 4 is a chart showing CD intensity versus wavelength.
  • FIGURES 5 A-B are charts showing T m versus calculated octanol- H 2 O partition coefficients.
  • FIGURE 6A shows a fluorescent image of a microtiter plate.
  • FIGURE 6B shows shows the quantification of hybridization as a function of fluorescence intensity.
  • the present invention relates to the design, synthesis, and potential applications of peptide-based nucleic acid surrogates having a secondary structure.
  • the secondary structure of the peptide-based nucleic acid surrogate is ⁇ -helical with the structure known as ⁇ -helical peptide nucleic acids ( ⁇ PNAs) which are new molecular constructs that merge the molecular recognition elements of nucleic acids with the predictable structural features of an ⁇ -helical peptide scaffold.
  • ⁇ PNAs ⁇ -helical peptide nucleic acids
  • n is the number of natural or unnatural ⁇ -amino acids, wherein n > 1; and m is the number of repeating units, wherein wherein m > 1.
  • PNAs can possess different secondary structure elements (e.g., ⁇ - helix, 3 10 helix, 2.2 7 ribbon, ⁇ helix, ⁇ ribbon, etc.) and are capable of sequence- specific binding to complementary single-stranded or double-stranded nucleic acid targets.
  • a PNA can bind to complimentary double-stranded nucleic acid targets by strand invasion (i.e., partial denaturation of the double-stranded nucleic acid target) or by direct recognition of the duplex.
  • a PNA comprising an ⁇ -helical secondary structure ( ⁇ PNA) is capable of sequence specific binding to single-stranded nucleic acid targets.
  • the preferred ⁇ PNA can also act as a functional ⁇ PNA, wherein the functional ⁇ PNA can be conjugated with various molecules and molecular systems, including but not limited to, fluorescent labeling tags, other peptides, particular molecules (e.g. biotin, etc.), DNA and RNA, etc.
  • FIGURE 1A shows a W-C (Watson-Crick) base pair binding between the preferred ⁇ PNA and complimentary single-stranded nucleic acid target, such as single-stranded DNA or RNA.
  • FIGURE 1B shows a preferred structural definition for an ⁇ PNA.
  • the preferred hydroxyl-containing amino acid bound to a nucleobase B is serine, although any hydroxyl-containing amino acid is suitable.
  • Forming and identifying PNAs having a particular secondary structure include the following steps: (1) devising a practical synthesis of PNAs having particular secondary structures that incorporate the nucleoamino acid residue; and (2) purifying and characterizing of the resulting oligomeric PNAs having particular secondary structures; (3) evaluating of the ability of PNAs having particular secondary structures to bind to single-stranded nucleic acid; and (4) characterizing the structure of the resulting PNA'DNA complexes.
  • the synthesis of ⁇ PNA includes the following steps: (1) solid-phase peptide synthesis (SPPS) of an ⁇ PNA; and (2) cleaving the ⁇ PNA module from the resin with concomitant deprotection of all amino acid residues except cysteine to give a thiol-protected ⁇ PNA module.
  • SPPS solid-phase peptide synthesis
  • an optional third step of can be incorporated to link ⁇ PNA modules.
  • ⁇ PNA modules are linked by concomitant thiol deprotection and disulfide bond formation to produce a ⁇ PNA dimer, etc.
  • Protecting groups are used to mask functional groups (FGs) that would interfere with, or not survive, a particular synthetic operation.
  • a base- labile Fmoc PG is preferably chosen for the N-terminal amines.
  • Acid-labile PGs such as tert-butoxycarbonyl (Boc) and t-butyl ester, is preferably used for all sidechain functionalities except cysteine.
  • the l 2 -labile acetamidomethyl (Acm) PG is preferably chosen to facilitate a separate disulfide formation step. >/eber, D. F.; Milkowski, J. D.; Varga, S. L; Denkewalter, R.
  • Pyrimidine-containing serine derivatives are prepared by l 2 -mediated nucleosidation of Fmoc- Ser(MTM)-OBn with silylated nucleobase.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene.
  • Carpino L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398) reagent is used for all peptide couplings.
  • Either the manual or semi-automated SPPS of ⁇ PNAs can be utilized and provide ⁇ PNAs in 10-15% overall yield from commercially available Fmoc-Rink-MBHA resin after purification by gradient reverse-phase HPLC.
  • Structure assignments for the ⁇ PNAs and ⁇ PNA dimers are confirmed by any known procedure such as MALDI-TOF or ESI mass spectrometry, and circular dichroism (CD) spectroscopy.
  • ⁇ PNA modules with as few as five nucleobases can bind with high affinity to complimentary single-stranded nucleic acid targets in a sequence- specific manner.
  • the ability of an ⁇ PNA to bind to a single-stranded nucleic acid target can be evaluated via thermal denaturation and gel retardation experiments.
  • binding affinities of ⁇ PNA to complimentary nucleic acid targets see Garner, P., Dey, S., and Huang, Y., J. Am. Chem. Soc. 2000, 122, 2405-2406, herein incorporated by reference.
  • 3 ) possesses different a secondary structure from the antiparallel counterpart Ac-CTCCT'd AjAGGAGAj), which is evident from their CD profiles, particularly in the 230-290 nm region where the interaction of asymmetrically-disposed nucleobases is expected dominate the CD spectrum.
  • Ac-CCTCC symmetrical base sequence
  • ⁇ PNAs may be N- capped to obtain enhanced affinity and orientational specificity with complementary nucleic acid targets.
  • N-capped ⁇ PNAs see Garner, P., Huang, Y, Dey, S. "Enhancement of ⁇ PNA Binding Affinity and Specificity via Hydrophobic Interactions" (unpublished).
  • Binding studies show that changing the peptide backbone can affect both the affinity and kinetics of ⁇ PNA binding to a nucleic acid target such as DNA.
  • Combinatorial techniques are useful to explore the relationship between ⁇ PNA amino acid diversity and sequence-specific hybridization. The success of combinatorial techniques will depend largely on the development of an adequately sensitive and specific screen for hybridization. While there are numerous examples of binding assays for nucleic acid and peptide libraries, (Pirrung, M. C. Chem. Rev. 1997,97, 473-488; Schultz, J. S. Biotechnol. Prog. 1996, 12, 729-743; Gold, L; Polisky, b.; Uhlenbeck, o.; Ya s, M. Annu. Rev.
  • Immobilized nucleic acid hybridization has become a common technique in molecular biology. This assay is based on Watson-Crick base- pairing and can be customized to screen ⁇ PNA libraries so that one can quickly identify compounds exhibiting high affinity towards immobilzed complementary nucleic acid targets. Since only the structure of the peptide backbone is to be varied, the nucleobase sequence of the ⁇ PNA will remain fixed. Thus, the effect of ⁇ PNA structure variation on hybridization using a single immobilized oligonucleotide target can be evaluated. The ⁇ PNAs can be "tagged" with a suitable fluorophore and, after hybridization, the high-affinity ⁇ PNAs bound to the nucleic acid would give an easily-detected fluorescent signal.
  • flourophores may be utilized in the fluorescence-based in situ hybridization assay for ⁇ PNA libraries.
  • the BODIPY (Aleshkov, S. B.; Fa, M.; Karolin, J.; Strandberg, L.; Johansson, L. B.-A.; Wilczynsha, M.; Ny, T. J. Biol. Chem. 1996, 271, 21231-21238) fluorophore is used since it has no residual charge that may influence the hybridization of the ⁇ PNA and nucleic acid.
  • Either thiol- or amine-reactive BODIPY dyes can be used depending on which stage the fluorophore is to be incorporated into the ⁇ PNA.
  • T 5 (b2) module hybridized with d(A 10 ) - albeit weakly as expected for a complex held together by only A « T base pairs (entry 5).
  • b2 ⁇ PNAs that contained cytosine resulted in remarkably stable ⁇ PNA»DNA complexes.
  • C 5 (b2) » d(rA 3 G 5 A 3 7) exhibited a T m of 54 °C in TE-buffer and no hysteresis was observed in the cooling curve. This implies that equilibration is being achieved during the cooling cycle.
  • This T m is 20 °C higher than that of C 5 (b1) « d(7A 3 G 5 3 T) in H 2 O (compare entries 6 and 7) and 35 °C higher than the corresponding DNA'DNA duplex (in TE-buffer + 150 mM NaCI).
  • Added salt was expected to disrupt favorable charge-charge interactions between cationic ⁇ PNAs and anionic DNA.
  • FIGURE 2 shows gel shift binding studies on (A) CCTCC(b2) + d(A 3 GGAGGA 3 ), (B) CTCCT(b2) + d(A 3 GAGGAA 3 ) (lanes 1-4) and CTCCT(b2) + d(A 3 AGGAGA 3 ) (lanes 5-8), and (C) T 5 (b2)-dimer + d(C 3 rcrC 2 2 A 10 CCrC 2 ) 3 ). Solutions were made up by combining DNA (40 ⁇ M) with varying amounts of ⁇ PNA in 7.5 ⁇ L of TE-buffer.
  • the ratios in (B) for lanes 1-4 as well as 5-8 are 0/1, 1/1 , 2/3, and 4/7.
  • the ratios in (C) from lanes 1-8 are 0/1, 1/2, 1/1 , 3/2, 2/1, 5/2, 3/1 , and 4/1.
  • Gels were developed using the PlusOneTM DNA silver staining kit (Pharmacia Biotech). To investigate the stability of the ⁇ PNA*DNA hybrids as well as their binding stoichiometry and structure, both gel-shift mobility and circular dichroism (CD) titration studies were performed.
  • FIGURE 3 shows job plots for CDs of C 5 (b2) + ⁇ TA 3 GgA 3 T) (at 258 nm, squares), CCTCC(b2) + d(A 3 GGAGGA 3 ) (at 258 nm, circles), and T 5 (b2)- dimer + d(A 10 ) (at 261 nm, triangles).
  • Spectra were recorded at 5 °C using a JASCO J-600 CD spectro-polarimeter.
  • Samples having a total concentration ([ ⁇ PNA] + [DNA]) of 12 ⁇ M were made up in doubly deionized water and placed in a stoppered optical quartz cell (1 cm pathlength). Dry air was purged through the sample compartment.
  • FIGURE 3 shows further support for the binding stoichiometries of the ⁇ PNA « DNA complexes comes from CD titration studies on C 5 (b2) + d(7 ⁇ 3 G 5 A 3 T) and CCTCC(b2) + d(A 3 GGAGG 3 ), which both showed intensity minima at 50 mol% ⁇ PNA as expected for a 1 :1 binding stoichiometry.
  • the inset of FIGURE 3 shows that in addition to a duplex, T 5 (b2)-dimer + d(A 10 ) also showed evidence of a 2:1 complex.
  • FIGURE 4 shows the comparative CD spectra of (a) DNA d(A 3 GGAGGA 3 ) alone, (b) ⁇ PNA CCTCC(b2) alone, and (c) a 1:1 mixture of d(A 3 GGAGGA 3 ) + CCTCC(b2) (6 ⁇ M each) in distilled H 2 O. Spectra were recorded as described in FIGURE3.
  • the CD spectrum of a solution containing equimolar amounts of CCTCC(b2) and d(A 3 GGAGGA 3 ) shows the characteristic CD signatures of both a peptide ⁇ -helix (minima at 220 and 206 nm, maximum at 196 nm) (Woody, R. M. Methods Enzymol. 1995, 246, 34-71) and a DNA B-helix (maximum at 280 and minimum at 255 nm). (Gray, D. M.; Ratliff, R. L; Vaughan, M. R. Methods Enzymol 1992, 211, 389-406).
  • binding properties of a series of N-capped ⁇ PNAs that incorporated additional ⁇ -stacking interactions were examined. This experiment was based on the assumption that tighter binding would result if "end-fraying" of ⁇ PNAs were minimized by incorporating such interactions at one or both ends of the ⁇ PNA chain. Such an effect could be important since the described ⁇ PNA modules rely on only 5 base-pair interactions and end-fraying could adversely affect up to 50% (in CTTTC) of the interstrand Watson-Crick hydrogen-bonds.
  • N-capped ⁇ PNAs lead to both enhanced affinity as well as orientational specificity with complementary ssDNA targets. Unexpectedly, the observed N-cap effects in the preferred parallel series were found to be better correlated to hydrophobicity rather than ⁇ -stacking.
  • N-capped ⁇ PNAs were synthesized via the SPPS protocol but substituting an appropriate carboxylic acid in the N-capping step. All of the ⁇ PNAs possess the backbone shown in FIGURE 1B and are described by the N- cap followed by the nucleobase sequence (N- to C-terminus from left to right).
  • Electrospray ionization mass spectral (ESI + -MS) characterization data for N- capped ⁇ PNAs 4-phenylbutyryl-CTCCT calcd 2811.41 , found 2812.61 ⁇ 0.41; 4- (p-methoxyphenyl)butyryl-CTCCT calcd 2841.42, found 2842.36 ⁇ 0.13; 4-(p- nitrophenyhbutyryl-CTCCT calcd 2856.40, found 2857.55 ⁇ 0.24; 4-(2- naphthyl)butyryl-CTCCT calcd 2861.43, found 2862.68 ⁇ 0.40; 4-(1- pyrenyl)butyryl-CTCCT calcd 2935.44, found 2937.02 ⁇ 0.39; n-butyryl-CTCCT calcd 2735.38, found 2736.52 ⁇ 0.54; n-hexanoyl-CTCCT calcd 2763.41, found 2766.01 ⁇ 1.19; 3-
  • N-caps investigated consisted of a series of arenyl and aliphatic carboxylic acids. Both sets of N-caps included a flexible methylene linker to allow for the anticipated cap-base interaction without significantly disrupting the complex structure.
  • Thermal UV denaturation TJ data for the equilibria between N-cap-CTCCT and d(A 3 GAGGAA 3 ) (N/5' or parallel orientation) and N-cap- CTCCT and d(A 3 AGGAGA 3 ) (N/3' or antiparallel orientation) are collected in Table 2.
  • cap CH 3 CO 37.0, 32.0 32.0
  • Table 3 shows analogous 7 ⁇ m enhancements with the symmetrical sequences N-4-(cyclohexyl)butyryl-CCTCC*d(A 3 GGAGGA 3 ) (60.8, 51 °C, entry 1) and N-4-(cyclohexyl)butyryl-CCCCC'd(7A 3 GGGGGA 3 7) (64.9, 66 °C, entry 2), thus excluding the possibility that these trends were related solely to the base sequence.
  • FIGURE 5 shows a plot of T m versus calculated octanol-H 2 O partition coefficients (log P values) for each N-cap.
  • FIGURE 5 shows that the melting temperatures of ⁇ PNA'DNA complexes is proportional to N-cap hydrophobicity.
  • FIGURE 6 shows a preferred hybridization of fluorescence-labeled ⁇ PNAs with gel-immobilized DNA d(A 4 GGAGGA 4 ) on microtiter plate.
  • the ratio of the average intensity of C1-C5 to B1-B5 is 2 to 1 after subtracting the background A1-A5 signal.
  • the thiol-reactive dye, BODIPY ® FLIA is introduced to Ac- Cys Acm -Lys-(Ser c -Ala 2 -Lys) 2 -Ser T -(Ala 2 -Lys-Ser c ) 2 -Gly-Lys-NH 2 (CCTCC) after SPPS.
  • the course of the labeling reaction can be easily followed by analytical HPLC and only a single HPLC purification is needed for the whole process.

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Abstract

La présente invention concerne des substituts d'acide nucléique peptidique (PNA) comprenant une structure à répétition de (AAB-aan)m et une structure secondaire particulière pouvant se lier à des cibles d'acide nucléique monocaténaire particulières. De préférence, un substitut d'acide nucléique peptidique comporte une structure secondaire à hélice alpha (αPNA). Par ailleurs, la présente invention concerne un procédé de production de substituts d'acide nucléique peptidique comprenant une structure secondaire particulière. Ces substituts d'acide nucléique peuvent être utilisés à des fins thérapeutiques (anti-sens, antigène) et diagnostiques (génétique), et dans des applications de commutation moléculaire (puces à αPNA).
PCT/US2000/021845 1999-08-25 2000-08-11 ACIDE NUCLEIQUE PEPTIDIQUE A HELICE ALPHA (αPNA) WO2001014398A1 (fr)

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WO2010118243A2 (fr) 2009-04-08 2010-10-14 Genentech, Inc. Utilisation d'antagonistes de il-27 pour traiter le lupus
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US8962247B2 (en) 2008-09-16 2015-02-24 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
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US8097422B2 (en) 2007-06-20 2012-01-17 Salk Institute For Biological Studies Kir channel modulators
EP3699291A1 (fr) 2008-01-17 2020-08-26 Sequenom, Inc. Procédés d'analyse de séquence d'acide nucléique à molécule unique et compositions
US11708607B2 (en) 2008-01-17 2023-07-25 Sequenom, Inc. Compositions containing identifier sequences on solid supports for nucleic acid sequence analysis
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US10144966B2 (en) 2008-01-17 2018-12-04 Sequenom, Inc. Methods of nucleic acid sequences analysis using solid supports containing identifier sequences
US9034580B2 (en) 2008-01-17 2015-05-19 Sequenom, Inc. Single molecule nucleic acid sequence analysis processes and compositions
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US10738358B2 (en) 2008-09-16 2020-08-11 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
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