WO2008072933A1 - Acides nucléiques peptidiques conjugués à des lieurs multi -amine et dispositif de détection d'acides nucléiques utilisant ceux-ci - Google Patents

Acides nucléiques peptidiques conjugués à des lieurs multi -amine et dispositif de détection d'acides nucléiques utilisant ceux-ci Download PDF

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WO2008072933A1
WO2008072933A1 PCT/KR2007/006553 KR2007006553W WO2008072933A1 WO 2008072933 A1 WO2008072933 A1 WO 2008072933A1 KR 2007006553 W KR2007006553 W KR 2007006553W WO 2008072933 A1 WO2008072933 A1 WO 2008072933A1
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pna
amine
conjugated
chemical formula
group
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PCT/KR2007/006553
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English (en)
Inventor
Hyunil Lee
Hee Kyung Park
Bong Ho Um
Serka Kim
Jae Jin Choi
Jung Hyun Min
Hye Yeon Kim
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Panagene Inc.
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Priority to US12/300,235 priority Critical patent/US8133985B2/en
Priority claimed from KR1020070130798A external-priority patent/KR100938777B1/ko
Publication of WO2008072933A1 publication Critical patent/WO2008072933A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars
    • 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 peptide nucleic acid probes conjugated with multi-amine linkers at one end, a method to immobilize the same on solid surface, and a device to detect nucleic acid sequence and a kit for gene diagnosis comprising peptide nucleic acids immobilized on solid surface.
  • PNA' Peptide nucleic acids
  • a PNA/DNA double strand is more stable than a DNA/DNA double strand
  • a PNA/RNA double strand is more stable than a DNA/RNA double strand.
  • Most commonly used backbone of PNA is N- (2-aminoethyl) glycine repeatedly linked by amide bond, which is electrically neutral differently from that of natural nucleic acids with negative charge .
  • PNA nucleobases in PNA occupy almost the same space as nucleobases of DNA, having almost same distance between nucleobases as that of natural nucleic acids.
  • PNA are more stable chemically and biologically than natural nucleic acids, because they are not decomposed by a nuclease or a protease. Mhe stability of a PNA/DNA or PNA/RNA double strand is not affected by salt concentration TTecause PNA is electrically neutral. Due to these properties, PNA can better recognize the complimentary nucleotide sequence than natural nucleic acids, so that PNA can be applied for diagnosis and other biological or medical purposes .
  • the nucleotide sequence can be detected by measuring the mechanical change resulted therefrom.
  • the resonance frequency of a microcantilever or a surface acoustic wave (SAW) sensor changes after hybridization of DNA or RNA, so that it can be use for detection.
  • SAW sensors and SAW sensors using PNA have been reported [S. Manalis and T. Burg, US Patent 7,282,329 “Suspended microchannel detectors"; P. Warthoe and S. Iben, US Patent Application Publication 2004/0072208 Al "Surface acoustic wave sensors and method for detecting target analytes"] .
  • the PNA probes needs to be immobilized on solid surface.
  • Covalent bonds formed by a chemical reaction is more stable than physical attraction, and thus frequently selected as a method of immoblilization.
  • Covalent bonds generated by aldehyde-amine, carboxylic acid-amine, or epoxide-amine reaction are widely used in biochips such as PNA microarrays, DNA microarrays and protein micorarrays (M. Schena, Microarray analysis, A. John Wiley & Sons, Inc., 2003, pp95-120) .
  • a glass surface is subject to silylation by an organosilane substance having aldehyde, amine or epoxy group in order to expose the functional group on the glass surface.
  • N-terminal amine group of a PNA probe is reacted with the exposed functional group to form a covalent bond.
  • a functional group of high reactivity By using a functional group of high reactivity, the efficiency of immobilization of the probe can be enhanced.
  • a highly active functional group such as hydrazide may be used (S. Raddatz et al . , "Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation", Nucleic Acids Research, 2002, Vol. 30, pp4793-4802) .
  • a biochip with higher sensitivity can be obtained by enhancing immobilization effieiency of DNA or PNA probes to a surface.
  • the immobilizing efficiency of probes can be also enhanced by increasing the reactivity on a surface.
  • a highly active linker is chemically bonded to a simple amine or aldehyde functional group immobilized on a solid surface to expose activated ester or isothiocyanate, which would then react with DNA or PNA probes.
  • a functional group of high reactivity at a probe terminal or on solid surface is easily degraded and is difficult to keep active.
  • the object of the present invention is to provide
  • PNA probes conjugated with multi-amine linkers and a method for preparing the same .
  • Another object of the present invention is to provide a method to immobilize the said PNA probes conjugated with multi-amine linkers on a functionalized surface with high efficiency.
  • Still another object of the present invention is to provide a nucleotide sequence detecting device or a kit for gene diagnosis which is manufactured by immobilizing the said PNA probes conjugated with multi-amine linkers on a solid surface made of a functionalized plastic substrate, silica, silicon semiconductor, magnetic molecules, nylon, polymers, thin film, cellulose or nitrocellulose, as well as a functionalized glass substrate.
  • the present invention relates to PNA conjugated with multi-amine linkers, and a synthetic method for the PNA, and its utilization. More specifically, the present invention is characterized by conjugating multiple times at N-terminal of PNA a linker consisting of a monomer having multi-amine functionality to prepare PNA having multi-amine functionality, enhancing the immobilization efficiency on a solid substrate having electrophilic functional group on the surface, and improving intensity and sensitivity of detection signal of the target nucleic acid or gene that is detected by the PNA probes.
  • the present invention provides a device and a kit for gene diagnosis having enhanced sensitivity and specificity, using the PNA conjugated with multi-amine linkers.
  • the PNA conjugated with multi-amine linkers according to the present invention is represented by Chemical Formula (1) :
  • Z represents a PNA oligomer having 8-30 nucleobases; A is bonded to N-terminal of the PNA oligomer and
  • D represents alkylene having 8-200 carbon atoms, and one or more carbon atoms of the alkylene may be substituted by nitrogen, oxygen or carbonyl;
  • Li, L 2 and L 3 independently represent a chemical bond, or an alkylene having 1-10 carbon atom(s) , and the carbon atoms in alkylene may be further substituted by one or more oxygen atom(s) ;
  • Fig. 1 is a schematic of a process for conjugating a monomer having multi-amine functionality with PNA (wherein, R is resin) .
  • Fig. 2 shows synthetic process for a monomer (Chemical Formula 3-3) to be conjugated with PNA according to Example 1.
  • Figs. 3 to 5 compare the immobilization efficiency of PNA probes with different number of amine functional groups by means of PNA array.
  • Figs. 6 and 7 compare the immobilization efficiency and P/M ratio of multi-amine linkers by means of PNA array.
  • Fig. 8 compares the signal intensity of symmetric multi- amine linkers with asymmetric multi-amine linkers by means of PNA array .
  • Fig. 9 and 10 compare the degree of immobilization with respect to different slide surfaces by means of PNA array
  • Slide 1 an epoxy slide
  • Slide 2 an aldehyde slide (from Company A)
  • Slide 3 an epoxy slide (from Company A)
  • Slide 4 an amine slide coated with butanediol diglycidyl ether (from Company B)
  • Slide 5 an amine slide coated with 1, 4-phenylene diisothiocyanate (from Company C)
  • Slide 6 a slide coated with activated ester (from Company D)
  • immobilization probes PNA 7-1, PNA 8-1, PNA 9-1, PNA 10-1)
  • the PNA having the structure represented by following chemical formula, reported by Buchardt, Nielsen, Egholm, Berg et al. (Denmark) in 1991 for the first time (wherein N- (2- aminoethyl) glycine is linked via amide bond) can be employed in the present invention.
  • a PNA probe immobilized on a solid surface in order to detect nucleotide sequence or gene preferably comprises 8 ⁇ 30 nucleobases.
  • spacers may be inserted between the solid surface and the PNA probe in order to immobilize the PNA probe at a certain distance from the solid surface.
  • spacer (s) represented by one of the following chemical formulas may be linked via amide bond at the N-terminal of a PNA probe .
  • O linker eg linker, AEEA
  • the PNA conjugated with multi-amine linkers according to the invention is represented by Chemical Formula (1) : [Chemical Formula 1]
  • a spacer [wherein D represents alkylene having 8-200 carbon atoms, and one or more carbon atoms of the alkylene may be substituted by nitrogen, oxygen or carbonyl] is linked to N-terminal of the PNA oligomer to immobilize it at a certain distance from the solid surface.
  • the spacers can be exemplified as following structures, but are not limited thereto.
  • L 1 , L 2 and L 3 independently represent a chemical bond, or -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 -, X represents CH or N, m represents an integer from 2 to 7, and n is 0 or 1.
  • Chemical Formula (1) include the multi-amine linkers represented by one of Chemical Formulas (1-1) to (1-4), but are not limited thereto.
  • Each of Chemical Formulas (1-1) to (1-3) comprises 4 amine functional groups whereas Chemical Formula (1-4) comprises 8 amine functional groups.
  • Chemical Formula (1-2) and (1-3) have symmetric multi-amine groups with four amine functional groups equidistant from a branch point, whereas Chemical Formula (1-1) comprises asymmetric multi-amine groups with four amine functional groups non-equidistant from a branch point .
  • the PNA conjugated with multi-amine linkers represented by Chemical Formula (1) according to the present invention is characterized in that it is prepared by reacting a monomer having multi-amine functionality represented by Chemical Formula (3) with a PNA oligomer derivative represented by Chemical Formula (2) twice or more up to ten times, sequentially.
  • L 1 , L 2 and L 3 independently represent a chemical bond, or an alkylene having 1-10 carbon atom(s), wherein the carbon atoms in the alkylene may be further substituted by one or more oxygen atom(s);
  • X represents CH or N
  • Y represents an amine protective group,- and n is 0 or 1.
  • amine groups of the monomer having multi-amine funtionality may be protected by conventional amine protective groups.
  • protective groups include 9-fluorenylmethyl carbamate (Fmoc) , t-butoxycarbonyl (Boc) , trityl, benzyl, chloroacetyl, benzyloxycarbonyl , p-methoxybenzyloxycarbonyl, formyl, trifluoroacetyl, p-toluenesulfonyl, benzenesulfonyl, methanesulfonyl, p-nitrobenzyloxycarbonyl and 2,2,2- trichloroethoxycarbonyl .
  • Li, L 2 and L 3 independently represent a chemical bond, or -CH 2 -, -CH 2 CH 2 -, - CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 -, X represents CH or N, and n is 0 or 1.
  • Specific examples include the monomers represented by one of Chemical Formulas (3-1) to (3-3) protected by Fmoc functional groups, but are not limited thereto. [Chemical Formula 3-1]
  • the PNA oligomer which is employed for the present invention can be synthesized from PNA monomer protected by benzothiazolesulfonyl (Bts) group according to the method of Korean Patent Registration No. 10-0464261; or from the PNA monomers protected by Fmoc or t-Boc [P. E. Nielsen (Ed.)
  • the PNA conjugated with multi- amine linkers represented by Chemical Formula (1) according to the present invention is synthesized by sequentially conjugating a monomer containing one carboxyl group and two or more branched amine groups at least twice to the N-terminal of
  • the PNA oligomer or the N-terminal of the spacers conjugated to the PNA oligomer.
  • the PNA according to the present invention is characterized by at least four amine groups at its terminal.
  • Fig. 1 schematically shows a solid support synthetic process for a PNA conjugated with multi-amine linkers according to the present invention.
  • the synthetic process consists of three stages including deprotection process to remove the protective groups linked to the amine groups of the PNA oligomer; coupling or conjugation process wherein a monomer having multi-amine functionality is linked to a PNA oligomer; and capping process to remove the activity of the unreacted amine groups .
  • the first stage is to remove the amine protective groups such as Fmoc, Boc or trityl, which protect amine terminal moieties of PNA, by a conventional process.
  • the process utilizes from 10 to 20% of piperidine solution in dimethylformamide (DMF) (in case of Fmoc protective group) , or trifluoroacetic acid (TFA) solution in DMF or dichloromethane (in case of Boc or trityl protective group) .
  • DMF dimethylformamide
  • TFA trifluoroacetic acid
  • the second stage is to react the monomer having multi-amine functionality to the N-terminal of PNA to form an amide bond.
  • the coupling process of the monomer having multi-amine functionality with PNA can be carried out by using a coupling agent conventionally used for peptide synthesis.
  • Specific examples of the coupling agent include diisopropylcarbodiimide (DIC) , dicyclohexylcarbodiimide, HATU, HOAt, HODhbt (L. A. Carpino et al . , Journal of American Chemical Society, 1993, 115, 4397-4398), HAPyU, TAPipU (A. Ehrlich et al .
  • the third stage is to carry out capping the amine groups that might not have reacted with the monomer having multi-amine functionality during the coupling process, by using acetic anhydride, in order to prevent unnecessary reactions.
  • the number of amines increases with the number of repeated processes. If the reaction of linkage is repeated m times, the number of terminal amines is increased to 2 m .
  • the PNA conjugated with multi-amine linkers prepared via those three stages is subjected to deprotection of amine protective group, and then is cleaved from solid support.
  • the cleaved PNA conjugated with multi-amine linkers is immobilized on a solid surface and used for detection of nucleic acid or gene. PNA conjugated with multi-amine linkers may be used after purification.
  • PNA conjugated with multi-amine linkers according to the present invention is immobilized on a functionalized solid surface according to conventional method to immobilize on a solid surface by using terminal amine groups.
  • the solid substance is preferably selected from silica, silicon semiconductor, magnetic molecules, nylon, macromolecular compounds such as polydimethylsiloxane (PDMS) , cellulose and nitrocellulose, but not restricted thereto.
  • the surface of the solid substance is functionalized by functional groups of aldehyde group, carboxylic group, epoxy group, isothiocyanate group, N-hydroxysuccinimidyl group or activated ester group.
  • immobilization efficiency can be improved by using PNA conjugated with multi-amine linkers. Since the immobilization efficiency increases with the number of conjugation reactions with the monomer having multi-amine functionality, the number of monomer conjugation reaction is preferably from 2 to 10, more preferably from 2 to 7.
  • a nucleotide sequence detecting device or a gene diagnosis kit can be manufactured by immobilizing the PNA conjugated with multi-amine linkers on a functionalized surface of a solid substance.
  • Those devices include, but are not restricted to, a PNA microarray wherein multiple PNA probes are arranged in two dimensions, PNA chips, PNA probes immobilized on the surface of microbeads having several ⁇ m size, a field-effect transistor using the PNA immobilized on silicon semiconductor or silicon nanowire, an impedance detector, a microcantilever, a surface acoustic wave sensor, or the like. Examples
  • PNA oligomer was synthesized via solid phase synthesis from a PNA monomer protected by Bts group and a functionalized resin, according to the process described in Korean Patent Registration No. 10-0464261.
  • a PNA monomer protected by Bts group protected by Bts group and a functionalized resin, according to the process described in Korean Patent Registration No. 10-0464261.
  • 6-dioxaoctanoic acid (hereinafter, referred to as an eg linker) was conjugated twice as spacers.
  • 8- (9H-fluoren-9- yl-methoxycarbonylamino) -3 6-dioxaoctanoic acid containing Fmoc as an amine protective group was employed as a monomer for the eg linker.
  • the resin was used for the next reaction stage with PNA attached thereto.
  • TT-V n PNA7 -1 (eg) 2 -GAC ATT ACT CAC CCG 4297.2
  • PNA7-2 (eg) 2 -GAC ATT AGT CAC CCG 4337.2
  • PNA8 -1 (eg) 2 -GAC ATT ACT CAC CCG Chemical 4688.7
  • PNA8-2 (eg) 2 -GAC ATT AGT CAC CCG 4728.7 1-1
  • PNA9-1 (eg) 2 -GAC ATT ACT CAC CCG Chemical 4811.9
  • PNA9-2 (eg) 2 -GAC ATT AGT CAC CCG 4851.9 1-2
  • PNAlO -2 (eg) 2 -GAC ATT AGT CAC CCG 4978.1
  • PNA arrays of Examples 11 to 13 were manufactured.
  • Example 11 the probes of Examples 2 to 4 (see Table 1) were diluted to a immobilization concentration of 50, 75 and 100 ⁇ M, and immobilized.
  • Example 12 the probes of Examples 2, 3, 5 and 6 (see Table 1) were employed to manufacture the PNA arrays.
  • Example 13 PNA array was manufactured by using the probes of Examples 7 to 10 (see Table 1) .
  • Employed were a slide whereon epoxy functional groups are exposed (manufactured by the inventors themselves) (Slide 1) ; a slide from Company A whereon aldehyde groups are exposed (Slide 2) ; a commercial slide whereon epoxy groups are exposed (Slide 3) ; a commercial slide from Company B, prepared by treating an amine slide with butandiol diglycidylether (Slide 4) ; a commercial slide from Company C, prepared by treating an amine slide with 1, 4-phenylene diisothiocyanate (Slide 5); and a commercial slide from Company D whereon activated ester groups are exposed (Slide 6) .
  • Target DNA oligomer 1 was designed to specifically hybridize with PNA oligomers 2-1, 2-2, 3-1, 3-2, 4-1, 4-2, 5-1, 5-2, 6-1 and 6-2 of Examples 2 to 6.
  • Target DNA oligomer 2 was designed to specifically hybridize with PNA oligomers 2-3, 2-4, 3-3, 3-4, 4-3, 4-4, 5-3, 5-4, 6-3 and 6-4.
  • Hybridization process of synthetic DNA oligomer a) A silicon rubber sealing mat manufactured to contain 100 ⁇ l of hybridization solution was adhered to a PNA array. b) The target DNA oligomer was diluted with 100 mM Tris- buffer (pH 7.6) containing sodium chloride (150 mM) . c) To the well formed in the silicon rubber sealing mat to which the PNA array has been adhered, buffer solution (100 ⁇ l) , in which the target DNA oligomer has been diluted, is added. d) Hybridization reaction is carried out at 4O 0 C for 2 hours . e) After removing the silicon rubber sealing mat on it, the PNA array is washed twice with 10 mM phosphate buffer solution for 5 minutes. f) The PNA array is dried. g) Intensity of fluorescence is measured by using a fluorescence microarray scanner.
  • Example 14 the PNA array manufactured according to Example 11 was hybridized with target DNA oligomer 1 and target DNA oligomer 2, according to the hybridization process described above, and the intensity of fluorescence was shown in Figs . 3 to 5.
  • Fig. 3 shows the results of hybridization of labeled target DNA oligomer 1 with the PNA array from Example 11.
  • the fluorescence emitted from spots of probes PNA 4-1 and 4-2 was more intense than that from spots of probes PNA 4-3 and 4-4 (noncomplementary base sequences) .
  • the fluorescence emitted from spots of probes PNA 3-1 and 3-2 was more intense than that from spots of probes PNA 3-3 and 3-4
  • the intensity of fluorescence emitted from spots of probes PNA 3-3 and 3-4 was higher than that from spots of probes PNA 3-1 and 3-2 (noncomplementary base sequences) .
  • the fluorescence signal increases in the order of probes PNA 2-3, 3-3 and 4-3.
  • the intensity of fluorescence signal increases in the order. Since this order corresponds to the number of amine groups at the terminal of PNA probes (1, 4, 8 in the order) used for immobilization, it is concluded that the intensity of detection signal increases with the increase with the increase of the number of terminal amine groups .
  • the fluorescence signal increases in the order of probes PNA 2-1, 3-1 and 4-1; and in the order of PNA 2-3, 3-3 and 4-3.
  • Example 15 the target DNA oligomer 2 was hybridized with the PNA array from Example 12 by using the hybridization process described above. The results are shown in Figs. 6 and 7.
  • Fig. 6 shows the results of intensity of fluorescence measured after hybridization of labeled target DNA oligomer 2 and the PNA array from Example 12.
  • the fluorescence emitted from spots of probes PNA 2-3 and 2-4 was more intense than that from spots of probes PNA 2-1 and 2-2 (noncomplementary base sequences) ; and the fluorescence emitted from spots of probes PNA 3-3 and 3-4 (complimentary base sequences) was more intense than that from spots of probes PNA 3-1 and 3-2 (noncomplementary base sequences) .
  • the fluorescence emitted from spots of probes PNA 5-3 and 5-4 was more intense than that from spots of probes PNA 5-1 and 5-2 (noncomplementary base sequences) ; and the fluorescence emitted from spots of probes PNA 6-3 and 6-4 (complimentary base sequences) was more intsnse than that from spots of probes PNA 6-1 and 6-2 (noncomplementary base sequences) .
  • Fig. 7 shows the perfect match/mismatch signal ratio (P/M ratio) of the fluorescence signal measured after hybridization of the PNA array from Example 12 with labeled target DNA oligomer 2.
  • P/M ratio shows the value in the range of 7-8 (PNA 2-3/PNA 2-1, PNA 2-4/PNA 2-2) .
  • PNA 5- 3/PNA 5-1 the number of amine group at the terminal of PNA probe is four (4)
  • all P/M ratios are more than 10, with a maximum of 35 (PNA 5- 3/PNA 5-1) . Accordingly, it is concluded that use of PNA conjugated with multi-amine linkers enhances specificity as well as intensity of detection signal.
  • Probe PNA 3-1 and probe PNA 5-1 have identical PNA base sequence but different multi-amine linkers. Though there are the same number of amine groups, the four amine groups are equidistant from a branch point in probe PNA 5-1, whereas they are not in probe PNA 3-1. Likewise, though the base sequence and the number of amine groups are the same for probes PNA 3-3 and 5-3, the four amine groups are equidistant from a branch point in probe PNA 5-3, whereas they are not in probe PNA 3-3.
  • Fig. 8 shows the results of hybridization of probes PNA 3-1 and 5-1 with DNA oligomer 1 (a complimentary base sequence) in different concentrations, and the results of hybridization of probes PNA 3-3 and 5-3 with DNA oligomer 2 (a complimentary base sequence) in different concentrations. It is found that probes PNA 5-1, 5-3 having equidistant amine groups from a branch point exhibited more intense signal than probes PNA 3-1 and 3-3 having non-equidistant amine groups from a branch point (Fig. 8) .
  • Example 16 PNA-DNA hybridization by using E. coli polymerase chain reaction product DNA was amplified by using 16S rDNA gene of E. coli as a template. DNA of E. coli was extracted by using a DNA extraction kit (Qiagen) , and a synthesized DNA oligomer having the base sequence shown in Table 3 was employed as a primer for polymerase chain reaction (PCR) . A PCR solution having the composition as shown in Table 4 was prepared, and subjected to PCR according to the process listed in Table 5.
  • PCR polymerase chain reaction
  • the PNA arrays obtained from PNA-DNA hybridization as described above were compared in intensity of fluorescence emitted from the spots of the probes immobilized on each slide, by means of a fluorescence microarray scanner. The results are shown in Figs. 9 and 10.
  • probes PNA 7-1, 8-1, 9-1, 10-1 the sequences of 15 nucleobase are identical, but the number of terminal amine groups are different.
  • PNA 7-1 has one terminal amine group, whereas probes PNA 8-1, 9-1 and 10-1 have four amine groups at the terminal.
  • probes PNA 7-2, 8-2, 9-2 and 10-2 the sequence of 15 bases are identical, but only one base at the center is different from the base sequences of probes PNA 7-1,
  • the signal of probes PNA 8-1, 9-1 and 10-1 having four terminal amine groups is far more intense than that of probe PNA 7-1 having only one terminal amine group.
  • Probes PNA 8-2, 9-2 and 10-2 having single mismatch base at the center among the 15 bases show little fluorescence signal.
  • the signal of PNA array with probes PNA 8-1, 9-1 and 10-1 having four amine groups are very specific (Fig. 9) .
  • the PNA conjugated with the multi-amine linkers is also effective on a slide with other functional groups than epoxy groups.
  • the slides prepared by treating an epoxy slide (slide 1, 3) and an amine slide treated with butanediol diglycidylether (Slide 4) the PNA probes conjugated with multi-amine linkers exhibited far more intense signal than the PNA probes having single amine group.
  • the PNA conjugated with multi-amine linkers exhibited more intense detection signal than the PNA containing single amine group.
  • the PNA conjugated with multi-amine linkers according to the present invention comprises linkers prepared by conjugating monomers having multi-amine functionality at the N-terminal of PNA, so that the PNA can be effectively immobilized on a solid surface.
  • linkers prepared by conjugating monomers having multi-amine functionality at the N-terminal of PNA so that the PNA can be effectively immobilized on a solid surface.
  • the PNA conjugated with multi-amine linkers can be immobilized on a functionalized plastic substrate, microbeads, membrane, a semiconductor substrate, or the like, as well as a functionalized glass substrate, and used for extensive applications .

Abstract

La présente invention concerne un acide nucléique peptidique (ANP) conjugué à des lieurs multi-amine, et un procédé de préparation de celui-ci et son utilisation. Le procédé se caractérise, plus spécifiquement, par la conjugaison de monomères présentant une fonctionnalité multi-amine séquentielle au niveau d'un terminal ANP, et l'immobilisation efficace de l'ANP conjugué à des lieurs multi-amine sur une surface solide. Un réseau ANP préparé à l'aide de l'ANP conjugué à des lieurs multi-amine présente une sensibilité et une spécificité améliorées des signaux de détection des acides nucléiques cibles par rapport à un réseau ANP utilisant des sondes ANP ne présentant qu'un groupe amine. L'ANP conjugué à des lieurs multi-amine peut être utilisé dans des dispositifs ou des kits de détection d'acides nucléiques pour le diagnostic génétique, tels que des jeux de microéchantillons ANP, des puces ANP, des transistors à effet de champ ANP et des détecteurs d'impédance.
PCT/KR2007/006553 2006-12-15 2007-12-14 Acides nucléiques peptidiques conjugués à des lieurs multi -amine et dispositif de détection d'acides nucléiques utilisant ceux-ci WO2008072933A1 (fr)

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KR1020070130798A KR100938777B1 (ko) 2006-12-15 2007-12-14 다중 아민기를 갖는 펩티드 핵산과 이를 이용하는 핵산검출 장치
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015112438A1 (fr) * 2014-01-21 2015-07-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Affichage de ligand acide nucléique peptidique polyvalent cgap-pna

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002558A1 (fr) * 1994-07-15 1996-02-01 Isis Pharmaceuticals, Inc. Acides nucleiques peptidiques lies
US20020188101A1 (en) * 1993-04-26 2002-12-12 Isis Pharmaceuticals, Inc. Conjugated peptide nucleic acids having enhanced cellular uptake
US6887664B2 (en) * 2000-06-06 2005-05-03 Applera Corporation Asynchronous primed PCR

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020188101A1 (en) * 1993-04-26 2002-12-12 Isis Pharmaceuticals, Inc. Conjugated peptide nucleic acids having enhanced cellular uptake
WO1996002558A1 (fr) * 1994-07-15 1996-02-01 Isis Pharmaceuticals, Inc. Acides nucleiques peptidiques lies
US6887664B2 (en) * 2000-06-06 2005-05-03 Applera Corporation Asynchronous primed PCR

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG J.: "DNA biosensors based on Peptide Nucleic Acid (PNA) recognition layers", BIOSENSORS & BIOELECTRONICS, vol. 13, 1998, pages 757 - 762, XP055140811, DOI: doi:10.1016/S0956-5663(98)00039-6 *
WANG W.H. ET AL.: "Solid-phase Synthesis of PNA Monomer by Ugi Four-component Condensation Reaction", CHINESE CHEMICAL LETTERS, vol. 16, no. 5, 2005, pages 585 - 588 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015112438A1 (fr) * 2014-01-21 2015-07-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Affichage de ligand acide nucléique peptidique polyvalent cgap-pna

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