DNA-NH_2酰化反应:分子量还是结构占主导地位?
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摘要
DNA的化学修饰可以使DNA分子具有其他功能,如构建DNA编码化合物库(DNA-encoded chemical libraries,DECLs)、制备用于生物传感的DNA探针、提高DNA适配体的结合活性等.对DNA化学修饰反应效率已有的研究集中在对反应条件(溶剂、温度等)的优化,为了确定影响修饰效率的重要因素,我们以DNA-NH_2的酰化反应为模型进行了系统研究.结果表明,影响DNA-NH_2酰化反应的主要因素是DNA和酰化试剂的分子量.相反,DNA的结构在反应中起着次要的作用.
DNA has the unique chemical and physical characteristics as a carrier of genetic information. With the development of structural DNA nanotechnology, it has been recognized that DNA can perform versatile functions with promising applications in nanomachine, computing, sensing and nanoarchitectures. In addition, the chemical incorporation with various functional groups brings in a diverse range of additional properties to DNA molecules for broadened applications. Therefore, post-synthetic modification of DNA has been widely used to introduce additional functionalities to DNA molecules, for example, design and synthesis of DNA-encoded library, preparation of DNA-based probes for biosensing, improving binding activities of DNA aptamers, etc. The modification efficiency is determined by both the intrinsic factors of the molecules involved(DNA and reagents) and the external factors(experimental conditions). However, previous studies on the modification focused on the optimization of reaction conditions. To determine the important intrinsic factors that affect modification efficiency, we have conducted a systematic study on the reaction with a model system— acylation of amino-DNA. Nine amino-modified DNAs with two different DNA conformations(a–i) were reacted with two carboxylic acids with different molecule weights(12-oxo-2,5,8,11-tetraoxapentadecan-15-oic acid, M_W 264.1209, and 30-oxo-2,5,8,11,14,17,20,23,26,29-decaoxatritriacontan-33-oic acid, M_W 528.2782), respectively, and characterized by polyacrylamide gel electrophoresis(PAGE). The bands were analyzed and calculated by Image J. In the present study, three factors are considered for the design. First, acylation is one of the most widely methods used for chemical modifications of DNA. The acylation reactions afford amides and these amides binding to DNA form more structurally complex and diverse DNA building blocks. Therefore, we explored the acylation reaction between amino-modified DNA with carboxylic acids. Second, two DNA conformations(single-stranded random coils, ssDNAs, and rigid, double-stranded DNA duplexes, dsDNA) were included. Single-stranded DNAs are more flexible than rigid DNA duplexes and thus can provide more variability in terms of modulating orientation of the reaction group to facilitate reactions. Third, the two small organic molecules with a varying number of polyethylene glycol(PEG) units were used, because PEG has been widely used in the field of chemical modification of biomacromolecules due to its excellent properties including a wide range of solubility, predominant bio-compatibility, good stability, low toxicity and no irritation, etc. We, therefore, systematically studied the DNA modification reaction to investigate the influences of two intrinsic factors(M_W and conformation) on the reaction. The primary factor for DNA-NH_2 acylation reaction is molecular weight(M_W). The higher the M_W of the regents(both small molecules and DNAs) are, the slower the reactions are. DNA conformation plays a much minor role in the reaction. Compared with rigid dsDNA duplexes, flexible ssDNA chains facilitate reactions. Based on the current study, we would suggest for post-synthetic modification of DNA:(1) Keep the molecular weights of all reagents(both small molecules and DNA molecules) to the minimal in the design to allow fast diffusion.(2) Keep the DNA strands near the modification location to be single stranded to facilitate orientation adjustment. If high molecular weight and rigid conformation(duplex) are not avoidable, reaction time and/or reagent concentration should be elongated to ensure the modification efficiency.
DNA has the unique chemical and physical characteristics as a carrier of genetic information. With the development of structural DNA nanotechnology, it has been recognized that DNA can perform versatile functions with promising applications in nanomachine, computing, sensing and nanoarchitectures. In addition, the chemical incorporation with various functional groups brings in a diverse range of additional properties to DNA molecules for broadened applications. Therefore, post-synthetic modification of DNA has been widely used to introduce additional functionalities to DNA molecules, for example, design and synthesis of DNA-encoded library, preparation of DNA-based probes for biosensing, improving binding activities of DNA aptamers, etc. The modification efficiency is determined by both the intrinsic factors of the molecules involved(DNA and reagents) and the external factors(experimental conditions). However, previous studies on the modification focused on the optimization of reaction conditions. To determine the important intrinsic factors that affect modification efficiency, we have conducted a systematic study on the reaction with a model system— acylation of amino-DNA. Nine amino-modified DNAs with two different DNA conformations(a–i) were reacted with two carboxylic acids with different molecule weights(12-oxo-2,5,8,11-tetraoxapentadecan-15-oic acid, M_W 264.1209, and 30-oxo-2,5,8,11,14,17,20,23,26,29-decaoxatritriacontan-33-oic acid, M_W 528.2782), respectively, and characterized by polyacrylamide gel electrophoresis(PAGE). The bands were analyzed and calculated by Image J. In the present study, three factors are considered for the design. First, acylation is one of the most widely methods used for chemical modifications of DNA. The acylation reactions afford amides and these amides binding to DNA form more structurally complex and diverse DNA building blocks. Therefore, we explored the acylation reaction between amino-modified DNA with carboxylic acids. Second, two DNA conformations(single-stranded random coils, ssDNAs, and rigid, double-stranded DNA duplexes, dsDNA) were included. Single-stranded DNAs are more flexible than rigid DNA duplexes and thus can provide more variability in terms of modulating orientation of the reaction group to facilitate reactions. Third, the two small organic molecules with a varying number of polyethylene glycol(PEG) units were used, because PEG has been widely used in the field of chemical modification of biomacromolecules due to its excellent properties including a wide range of solubility, predominant bio-compatibility, good stability, low toxicity and no irritation, etc. We, therefore, systematically studied the DNA modification reaction to investigate the influences of two intrinsic factors(M_W and conformation) on the reaction. The primary factor for DNA-NH_2 acylation reaction is molecular weight(M_W). The higher the M_W of the regents(both small molecules and DNAs) are, the slower the reactions are. DNA conformation plays a much minor role in the reaction. Compared with rigid dsDNA duplexes, flexible ssDNA chains facilitate reactions. Based on the current study, we would suggest for post-synthetic modification of DNA:(1) Keep the molecular weights of all reagents(both small molecules and DNA molecules) to the minimal in the design to allow fast diffusion.(2) Keep the DNA strands near the modification location to be single stranded to facilitate orientation adjustment. If high molecular weight and rigid conformation(duplex) are not avoidable, reaction time and/or reagent concentration should be elongated to ensure the modification efficiency.
引文
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19 Debaene F,Da Silva J A,Pianowski Z,et al.Expanding the scope of PNA-encoded libraries:Divergent synthesis of libraries targeting cysteine,serine and metalloproteases as well as tyrosine phosphatases.Tetrahedron,2007,63:6577-6586
20 Wrenn S J,Weisinger R M,Halpin D R,et al.Synthetic ligands discovered by in vitro selection.J Am Chem Soc,2007,129:13137-13143
21 Clark M A,Acharya R A,Arico-Muendel C C,et al.Design,synthesis and selection of DNAencoded small-molecule libraries.Nat Chem Biol,2009,5:647-654
22 Kleiner R E,Dumelin C E,Tiu G C,et al.In vitro selection of a DNA-templated small-molecule library reveals a class of macrocyclic kinase inhibitors.J Am Chem Soc,2010,132:11779-11791
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24 Deng H,O’Keefe H,Davie C P,et al.Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology(ELT).J Med Chem,2012,55:7061-7079
25 Leimbacher M,Zhang Y,Mannocci L,et al.Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.Chemistry,2012,18:7729-7737
26 Disch J S,Evindar G,Chiu C H,et al.Discovery of thieno3,2-dpyrimidine-6-carboxamides as potent inhibitors of SIRT1,SIRT2,and SIRT3.J Med Chem,2013,56:3666-3679
27 Podolin P L,Bolognese B J,Foley J F,et al.In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor.Prostaglandins Other Lipid Mediat,2013,104:25-31
28 FranziniR M,Samain F,Abd E M,et al.Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.Bioconjug Chem,2014,25:1453-1461
29 Li Y,Gabriele E,Samain F,et al.Optimized reaction conditions for amide bond formation in DNA-encoded combinatorial libraries.ACSComb Sci,2016,18:438-443
2 Liu G,Wan Y,Gau V,et al.An enzyme-based E-DNA sensor for sequence-specific detection of femtomolar DNA targets.J Am Chem Soc,2008,130:6820-6825
3 Takaoka A,Wang Z,Choi M K,et al.A gold nanoparticle-based chronocoulometric DNA sensor for amplified detection of DNA.Nat Protoc,2007,448:501-505
4 Sun L,Wu J,Du F,et al.Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science,2013,339:786-791
5 Li J W J,Tan W H.A single DNA molecule nanomotor.Nano Lett,2002,2:315-318
6 Xin L,Zhou C,Yang Z,et al.Regulation of an enzyme cascade reaction by a DNA machine.Small,2014,9:3088-3091
7 Zhang H,Fang C,Zhang S.Ultrasensitive electrochemical analysis of two analytes by using an autonomous DNA machine that works in a two-cycle mode.Chemistry,2011,17:7531-7537
8 Zhou C,Yang Z,Liu D.Reversible regulation of protein binding affinity by a DNA machine.J Am Chem Soc,2012,134:1416
9 Beissenhirtz M K,Elnathan R,Weizmann Y,et al.The aggregation of au nanoparticles by an autonomous DNA machine detects viruses.Small,2007,3:375-379
10 Zhu C,Wen Y,Li D,et al.Inhibition of the in vitro replication of DNA by an aptamer-protein complex in an autonomous DNA machine.Chemistry,2009,15:11898-11903
11 Kleiner R E,Dumelin C E,Liu D R.Small-molecule discovery from DNA-encoded chemical libraries.Chem Soc Rev,2011,40:5707-5707
12 He Y,Liu D R.Autonomous multistep organic synthesis in a single isothermal solution mediated by a DNA walker.Nat Nanotechnol,2010,5:778-782
13 Heemstra J M,Liu D R.Templated synthesis of peptide nucleic acids via sequence-selective base-filling reactions.J Am Chem Soc,2009,131:11347-11349
14 Brudno Y,Liu D R.Recent progress toward the templated synthesis and directed evolution of sequence-defined synthetic polymers.Chem Biol,2009,16:265-276
15 Hong M,Zhou X,Zhu J,et al.Nanoparticle-based,fluorous-tag-driven DNA detection.Angew Chem Int Ed,2009,48:9503-9506
16 Chevolot Y,Bouillon C,Vidal S,et al.DNA-based carbohydrate biochips:A platform for surface glyco-engineering.Angew Chem Int Ed,2007,46:2398-2402
17 Hammond D M,Manetto A,Gierlich J,et al.DNA photography:An ultrasensitive DNA-detection method based on photographic techniques.Angew Chem Int Ed,2007,46:4184-4187
18 Virgilio A,Petraccone L,Vellecco V,et al.Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity.Nucleic Acids Res,2015,43:10602-10611
19 Debaene F,Da Silva J A,Pianowski Z,et al.Expanding the scope of PNA-encoded libraries:Divergent synthesis of libraries targeting cysteine,serine and metalloproteases as well as tyrosine phosphatases.Tetrahedron,2007,63:6577-6586
20 Wrenn S J,Weisinger R M,Halpin D R,et al.Synthetic ligands discovered by in vitro selection.J Am Chem Soc,2007,129:13137-13143
21 Clark M A,Acharya R A,Arico-Muendel C C,et al.Design,synthesis and selection of DNAencoded small-molecule libraries.Nat Chem Biol,2009,5:647-654
22 Kleiner R E,Dumelin C E,Tiu G C,et al.In vitro selection of a DNA-templated small-molecule library reveals a class of macrocyclic kinase inhibitors.J Am Chem Soc,2010,132:11779-11791
23 Melkko S,Mannocci L,Dumelin C E,et al.Isolation of a small-molecule inhibitor of the antiapoptotic protein Bcl-xL from a DNA-encoded chemical library.ChemMedChem,2010,5:584-590
24 Deng H,O’Keefe H,Davie C P,et al.Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology(ELT).J Med Chem,2012,55:7061-7079
25 Leimbacher M,Zhang Y,Mannocci L,et al.Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.Chemistry,2012,18:7729-7737
26 Disch J S,Evindar G,Chiu C H,et al.Discovery of thieno3,2-dpyrimidine-6-carboxamides as potent inhibitors of SIRT1,SIRT2,and SIRT3.J Med Chem,2013,56:3666-3679
27 Podolin P L,Bolognese B J,Foley J F,et al.In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor.Prostaglandins Other Lipid Mediat,2013,104:25-31
28 FranziniR M,Samain F,Abd E M,et al.Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.Bioconjug Chem,2014,25:1453-1461
29 Li Y,Gabriele E,Samain F,et al.Optimized reaction conditions for amide bond formation in DNA-encoded combinatorial libraries.ACSComb Sci,2016,18:438-443