Skip to main content
SpringerLink
Log in

Comparative Study of Novel Fluorescent Cyanine Nucleotides: Hybridization Analysis of Labeled PCR Products Using a Biochip

  • ORIGINAL ARTICLE
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

This study investigated the synthesis and substrate properties of Cy5-labeled dUTP derivatives with different substituents, linkers between the dye unit and pyrimidine heterocycle and fluorophore charges. Fluorescently labeled nucleoside triphosphates were studied as substrates using multiplex PCR with Taq and Vent (exo-) DNA polymerases, the typical representatives of the A and B polymerase families. The efficiency of nucleotide incorporation during PCR was assessed with a multi-parameter hybridization analysis using a diagnostic DNA microarray. The hybridization analysis indirectly estimates the incorporation efficiency of dye-labeled nucleotides in multiplex PCR. Our results demonstrated higher efficiencies of substrates with electrically neutral dyes than electropositive and electronegative Cy5 residues.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Scheme 2
Scheme 3
Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Cho Y, Kool ET (2006) Enzymatic synthesis of fluorescent oligomers assembled on a DNA backbone. ChemBioChem 7:669–672. doi:10.1002/cbic.200500515

    Article  CAS  PubMed  Google Scholar 

  2. Wicke L, Engels JW (2012) Postsynthetic on column RNA labeling via Stille coupling. Bioconjug Chem 23:627–642. doi:10.1021/bc200659j

    Article  CAS  PubMed  Google Scholar 

  3. Suzuki Y, Yokoyama K (2015) Development of functional fluorescent molecular pProbes for the detection of biological substances. Biosensors (Basel) 5:337–363. doi:10.3390/bios5020337

    Article  CAS  Google Scholar 

  4. Nozeret K, Loll F, Escude C, Boutorine AS (2015) Polyamide fluorescent probes for visualization of repeated DNA sequences in living cells. ChemBioChem 16:549–554. doi:10.1002/cbic.201402676

    Article  CAS  PubMed  Google Scholar 

  5. Boutorine AS, Novopashina DS, Krasheninina OA, Nozeret K, Venyaminova AG (2013) Fluorescent probes for nucleic acid visualization in fixed and live cells. Molecules 18:15357–15397. doi:10.3390/molecules181215357

    Article  CAS  PubMed  Google Scholar 

  6. Wang K, Huang J, Yang X, He X, Liu J (2013) Recent advances in fluorescent nucleic acid probes for living cell studies. Analyst 138:62–71. doi:10.1039/c2an35254k

    Article  CAS  PubMed  Google Scholar 

  7. Su X, Xiao X, Zhang C, Zhao M (2012) Nucleic acid fluorescent probes for biological sensing. Appl Spectrosc 66:1249–1262. doi:10.1366/12-06803

    Article  CAS  PubMed  Google Scholar 

  8. Ranasinghe RT, Brown T (2005) Fluorescence based strategies for genetic analysis. Chem Commun (Camb) 28:5487–5502. doi:10.1039/b509522k

    Article  Google Scholar 

  9. Weisbrod SH, Marx A (2008) Novel strategies for the site-specific covalent labelling of nucleic acids. Chem Commun (Camb) 30:5675–5685. doi:10.1039/b809528k

    Article  Google Scholar 

  10. Omumi A, Beach DG, Baker M, Gabryelski W, Manderville RA (2011) Postsynthetic guanine arylation of DNA by Suzuki-Miyaura cross-coupling. J Am Chem Soc 133:42–50. doi:10.1021/ja106158b

    Article  CAS  PubMed  Google Scholar 

  11. Inazuka M, Tahira T, Hayashi K (1996) One-tube post-PCR fluorescent labeling of DNA fragments. Genome Res 6:551–557

    Article  CAS  PubMed  Google Scholar 

  12. Sawant AA, Tanpure AA, Mukherjee PP, Athavale S, Kelkar A, Galande S, Srivatsan SG (2016) A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA. Nucleic Acids Res 44:e16. doi:10.1093/nar/gkv903

    Article  PubMed  Google Scholar 

  13. Lercher L, McGouran JF, Kessler BM, Schofield CJ, Davis BG (2013) DNA modification under mild conditions by Suzuki-Miyaura cross-coupling for the generation of functional probes. Angew Chem Int Ed Engl 52:10553–10558. doi:10.1002/anie.201304038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tasara T, Angerer B, Damond M, Winter H, Dorhofer S, Hubscher U, Amacker M (2003) Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA. Nucleic Acids Res 31:2636–2646. doi:10.1093/nar/gkg371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Staiger N, Marx A (2010) A DNA polymerase with increased reactivity for ribonucleotides and C5-modified deoxyribonucleotides. Chembiochem 11:1963–1666. doi:10.1002/cbic.201000384

    Article  CAS  PubMed  Google Scholar 

  16. Mackova M, Pohl R, Hocek M (2014) Polymerase synthesis of DNAs bearing vinyl groups in the major groove and their cleavage by restriction endonucleases. Chembiochem 15:2306–2312. doi:10.1002/cbic.201402319

    Article  CAS  PubMed  Google Scholar 

  17. Ren X, Gerowska M, El-Sagheer AH, Brown T (2014) Enzymatic incorporation and fluorescent labelling of cyclooctyne-modified deoxyuridine triphosphates in DNA. Bioorg Med Chem 22:4384–4390. doi:10.1016/j.bmc.2014.05.050

    Article  CAS  PubMed  Google Scholar 

  18. Hocek M (2014) Synthesis of base-modified 2′-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology. J Org Chem 79:9914–9921. doi:10.1021/jo5020799

    Article  CAS  PubMed  Google Scholar 

  19. Hollenstein M (2012) Nucleoside triphosphates - building blocks for the modification of nucleic acids. Molecules 17:13569–13591. doi:10.3390/molecules171113569

    Article  CAS  PubMed  Google Scholar 

  20. Giller G, Tasara T, Angerer B, Muhlegger K, Amacker M, Winter H (2003) Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2′-deoxyribonucleoside-5′-triphosphates. Nucleic Acids Res 31:2630–2635. doi:10.1093/nar/gkg370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Anderson JP, Angerer B, Loeb LA (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257–264. doi:10.2144/05382RR02

    Article  CAS  PubMed  Google Scholar 

  22. Ohbayashi T, Kuwahara M, Hasegawa M, Kasamatsu T, Tamura T, Sawai H (2005) Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase. Org Biomol Chem 3:2463–2468. doi:10.1039/b504330a

    Article  CAS  PubMed  Google Scholar 

  23. Rohloff JC, Gelinas AD, Jarvis TC, Ochsner UA, Schneider DJ, Gold L, Janjic N (2014) Nucleic acid ligands with protein-like side chains: modified aptamers and their use as diagnostic and therapeutic agents. Mol Ther Nucleic Acids 3:e201. doi:10.1038/mtna.2014.49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Alexandrova LA, Chekhov VO, Shmalenyuk ER, Kochetkov SN, Abu El-Asrar R, Herdewijn P (2015) Synthesis and evaluation of C-5 modified 2′-deoxyuridine monophosphates as inhibitors of M. tuberculosis thymidylate synthase. Bioorg Med Chem 23:7131–7713. doi:10.1016/j.bmc.2015.09.053

    Article  CAS  PubMed  Google Scholar 

  25. Holzberger B, Marx A (2009) Enzymatic synthesis of perfluoroalkylated DNA. Bioorg Med Chem 17:3653–3658. doi:10.1016/j.bmc.2009.03.063

    Article  CAS  PubMed  Google Scholar 

  26. Gierlich J, Gutsmiedl K, Gramlich PM, Schmidt A, Burley GA, Carell T (2007) Synthesis of highly modified DNA by a combination of PCR with alkyne-bearing triphosphates and click chemistry. Chemistry 13:9486–9494. doi:10.1002/chem.200700502

    Article  CAS  PubMed  Google Scholar 

  27. Wu J, Tang X (2013) Synthesis and enzymatic incorporation of photolabile dUTP analogues into DNA and their applications for DNA labeling. Bioorg Med Chem 21:6205–6211. doi:10.1016/j.bmc.2013.04.081

    Article  CAS  PubMed  Google Scholar 

  28. Baccaro A, Steck AL, Marx A (2012) Barcoded nucleotides. Angew Chem Int Ed Engl 51:254–257. doi:10.1002/anie.201105717

    Article  CAS  PubMed  Google Scholar 

  29. Lee SE, Sidorov A, Gourlain T, Sidorov A, Mignet N, Thorpe SJ, Brazier JA, Dickman MJ, Hornby DP, Grasby JA, Williams DM (2001) Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity. Nucleic Acids Res 29:1565–1573. doi:10.1093/nar/29.7.1565

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Finn PJ, Sun L, Nampalli S, Xiao H, Nelson JR, Mamone JA, Grossmann G, Flick PK, Fuller CW, Kumar SH (2002) Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5′-triphosphates to ‘direct-load’ DNA sequencing. Nucleic Acids Res 30:2877–2885. doi:10.1093/nar/gkf387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Garcia-Junceda E, Garcia-Garcia JF, Bastida A, Fernandez-Mayoralas A (2004) Enzymes in the synthesis of bioactive compounds: the prodigious decades. Bioorg Med Chem 12:1817–1834. doi:10.1016/j.bmc.2004.01.032

    Article  CAS  PubMed  Google Scholar 

  32. Kuwahara M, Nagashima J, Hasegawa M, Tamura T, Kitagata R, Hanawa K, Hasoshima S, Kasamatsu T, Ozaki H, Sawai H (2006) Systematic characterization of 2′-deoxynucleoside- 5′-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA. Nucleic Acids Res 34:5383–5394. doi:10.1093/nar/gkl637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Godovikova TS, Kolpashchikov DM, Orlova TN, Richter VA, Ivanova TM, Grochovsky SL, Nasedkina TV, Victorova L, Poletaev AI (1999) 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2′-deoxy- uridine-5′-triphosphate substitutes for thymidine-5′-triphosphate in the polymerase chain reaction. Bioconjug Chem 10:529–537. doi:10.1021/bc980144r

    Article  CAS  PubMed  Google Scholar 

  34. Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug Chem 4:105–111. doi:10.1021/bc00020a001

    Article  CAS  PubMed  Google Scholar 

  35. Daehne S, Resch-Genger U, Wolfberg OS (eds) (1998) Near-infrared dyes for high technology applications, vol 52. NATO ASI Series, Series 3, High Technology. Kluwer Academic Publishers, Dordrecht, p 458

  36. Soini E, Hemmila I (1979) Fluoroimmunoassay: present status and key problems. Clinical Chem 25:353–361

    CAS  Google Scholar 

  37. Barsky V, Perov A, Tokalov S Chudinov A, Kreindlin E, Sharonov A, Kotova E, Mirzabekov A (2002) Fluorescence data analysis on gel-based biochips. J Biomol Screen 7:247–257 doi:10.1089/108705702760047745

    Article  CAS  PubMed  Google Scholar 

  38. Giusti WG, Adriano T (1993) Synthesis and characterization of 5′-fluorescent-dye-labeled oligonucleotides. Genome Res 2:223–227. doi:10.1101/gr.2.3.223

    Article  CAS  Google Scholar 

  39. Sinnamon JR, Czaplinski K (2014) RNA detection in situ with FISH-STICs. RNA 20:260–266. doi:10.1261/rna.041905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lee JS, Song JJ, Deaton R, Kim JW (2013) Assessing the detection capacity of microarrays as bio/nanosensing platforms. Biomed Res Int 310461. doi:10.1155/2013/310461

  41. Gryadunov D, Mikhailovich V, Lapa S, Roudinskii N, Donnikov M, Pan’kov S, Markova O, Kuz’min A, Chernousova L, Skotnikova O, Moroz A, Zasedatelev A, Mirzabekov A (2005) Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clin Microbiol Infect 11:531–539. doi:10.1111/j.1469-0691.2005.01183.x

    Article  CAS  PubMed  Google Scholar 

  42. Zhu Z, Waggoner AS (1997) Molecular mechanism controlling the incorporation of fluorescent nucleotides into DNA by PCR. Cytometry 28:206–211. doi:10.1002/(SICI)1097-0320(19970701)28:3<206::AID-CYTO4>3.0.CO;2-B

    Article  CAS  PubMed  Google Scholar 

  43. Lacenere CJ, Garg NK, Stoltz BM, Quake SR (2005) Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases. Nucleosides Nucleotides Nucleic Acids 25:9–15. doi:10.1080/15257770500377714

    Article  Google Scholar 

  44. Wynne SA, Pinheiro VB, Holliger PH, Leslie AGW (2013) Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesizing highly cy-dye labelled DNA. PLoS One 8:e70892. doi:10.1371/journal.pone.0070892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bespyatykh JA, Zimenkov DV, Shitikov EA, Kulagina EV, Lapa SA, Gryadunov DA, Ilina EN, Govorun VM (2014) Spoligotyping of Mycobacterium tuberculosis complex isolates using hydrogel oligonucleotide microarrays. Infect Genet Evol 26:41–46. doi:10.1016/j.meegid.2014.04.024

    Article  CAS  PubMed  Google Scholar 

  46. Shershov VE, Kuznetsov VE, Lysov YUP, Barsky VE, Spitsyn MA, Guseinov TO, Zasedateleva OA, Vasiliskov VА, Surzhikov SA, Zasedatelev AS Chudinov AV (2015) The effect of the chromophore charge on the efficiency of incorporation of fluorescently-labeled nucleotides in matrix synthesis by Taq DNA polymerase. Biofizika 60:1216–1218. doi:10.1134/S0006350915060238

    CAS  PubMed  Google Scholar 

  47. Yu H, Chao J, Patek D, Mujumdar R, Mujumdar S, Wagonner AS (1994) Cyanine dye dUTP analogs for enzymatic labeling of DNA probes. Nucleic Acids Res 22:3226–3232. doi:10.1093/nar/22.15.3226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhu ZH, Chao J, Yu H, Waggoner AS (1994) Directly labeled DNA probes using fluorescent nucleotides with different length linkers. Nucleic Acids Res 22:3418–3422. doi:10.1093/nar/22.16.3418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ernst LA, Gupta RK, Mujumdar RB, Waggoner AS (1989) Cyanine dye labeling reagents for sulfhydryl groups. Cytometry 10:3–10. doi:10.1002/cyto.990100103

    Article  CAS  PubMed  Google Scholar 

  50. Southwick PL, Ernst LA, Tauriello EW, Parker SR, Mujumdar RB, Mujumdar SR, Clever HA, Waggoner AS (1990) Cyanine dye labeling reagents-carboxymethylindocyanine succinimidyl esters. Cytometry 11:418–430. doi:10.1002/cyto.990110313

    Article  CAS  PubMed  Google Scholar 

  51. Lin Y, Weissleder R, Tung CH (2002) Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjug Chem 13:605–610. doi:10.1021/bc0155723

    Article  PubMed  Google Scholar 

  52. Wang L, Peng X, Zhang R, Cui J, Xu G, Wang F (2002) Syntheses and spectral properties of fluorescent trimethine sulfo-3H-indocyanine dyes. Dyes Pigm 54:107–111. doi:10.1016/S0143-7208(02)00036-0

    Article  CAS  Google Scholar 

  53. Kuznetsova VE, Spitsyn MA, Shershov VE, Guseinov TO, Fesenko EE, Lapa SA, Ikonnikova AYU, Avdonina MA, Nasedkina TV, Zasedatelev AS, Chudinov AV (2016) Novel fluorescently labeled nucleotides: synthesis, spectral properties and application in polymerase chain reaction. Mendeleev Commun 26:95–98. doi:10.1016/j.mencom.2016.03.002

    Article  CAS  Google Scholar 

  54. Killelea T, Saint-Pierre C, Ralec C, Gasparutto D, Henneke G (2014) Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′-terminal Cy5 dyes. Electrophoresis 35:1938–1946. doi:10.1002/elps.201400018

    Article  CAS  PubMed  Google Scholar 

  55. Rubina AY, Pan’kov SV, Dementieva EI, Pen’kov DN, Butygin AV, Vasiliskov VA, Chudinov AV, Mikheiki AL, Mikhailovich VM, Mirzabekov AD (2004) Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production. Anal Biochem 325:92–106. doi:10.1016/j.ab.2003.10.010

    Article  CAS  PubMed  Google Scholar 

  56. Chen X, Peng X, Cui A, Wang B, Wang L, Zhang R (2006) Photostabilities of novel heptamethine 3H-indolenine cyanine dyes with different N-substituents. Photochem Photobiol A Chem 181:79–85. doi:10.1016/j.jphotochem.2005.11.004

    Article  CAS  Google Scholar 

  57. Pham W, Lai WF, Weissleder R, Tung CH (2003) High efficiency synthesis of a bioconjugatable near-infrared fluorochrome. Bioconjug Chem 14:1048–1051. doi:10.1021/bc034070h

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Russian Science Foundation Grant No. 14-14-01090.

Author information

Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991, Moscow, Russia

    V. E. Shershov, S. A. Lapa, V. E. Kuznetsova, M. A. Spitsyn, T. O. Guseinov, S. A. Polyakov, A. A. Stomahin, A. S. Zasedatelev & A. V. Chudinov

Authors
  1. V. E. Shershov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. A. Lapa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. V. E. Kuznetsova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. A. Spitsyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. O. Guseinov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. S. A. Polyakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. A. Stomahin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. S. Zasedatelev
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. A. V. Chudinov
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the writing of the manuscript. The final version of the manuscript has been approved by all of the authors.

Corresponding author

Correspondence to V. E. Kuznetsova.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shershov, V.E., Lapa, S.A., Kuznetsova, V.E. et al. Comparative Study of Novel Fluorescent Cyanine Nucleotides: Hybridization Analysis of Labeled PCR Products Using a Biochip. J Fluoresc 27, 2001–2016 (2017). https://doi.org/10.1007/s10895-017-2139-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-017-2139-6

Keywords

Search

Navigation