Polymerase Amplification, Cloning, and Gene Expression of Benzo-homologous "yDNA" Base Pairs

Overview

Journal Chembiochem

Specialty Biochemistry

Date 2008 Dec 5

PMID 19053129

Citations 12

Authors

Jijumon Chelliserrykattil

Haige Lu

Alex H F Lee

Eric T Kool

Affiliations

Soon will be listed here.

Abstract

A widened DNA base-pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the "yDNA" system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15-20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.

Citing Articles

Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach.

Debnath T, Cisneros G Phys Chem Chem Phys. 2024; 26(9):7435-7445.

PMID: 38353005 PMC: 11080001. DOI: 10.1039/d3cp05572h.

Conformational Morphing by a DNA Analogue Featuring 7-Deazapurines and 5-Halogenpyrimidines and the Origins of Adenine-Tract Geometry.

Pallan P, Lybrand T, Rozners E, Abramov M, Schepers G, Eremeeva E Biochemistry. 2023; 62(19):2854-2867.

PMID: 37694722 PMC: 11062489. DOI: 10.1021/acs.biochem.3c00327.

Synthetic Life with Alternative Nucleic Acids as Genetic Materials.

Nie P, Bai Y, Mei H Molecules. 2020; 25(15).

PMID: 32751873 PMC: 7435384. DOI: 10.3390/molecules25153483.

Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4.

Albrecht L, Wilson K, Wetmore S Molecules. 2016; 21(7).

PMID: 27347908 PMC: 6273265. DOI: 10.3390/molecules21070822.

Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling.

Singleton D, Hussain R, Siligardi G, Kumar P, Hrdlicka P, Berova N Org Biomol Chem. 2015; 14(1):149-57.

PMID: 26416024 PMC: 4766578. DOI: 10.1039/c5ob01681a.

References

Zimmermann N, Meggers E, Schultz P . A novel silver(i)-mediated DNA base pair. J Am Chem Soc. 2002; 124(46):13684-5. DOI: 10.1021/ja0279951. View

Lee A, Kool E . A new four-base genetic helix, yDNA, composed of widened benzopyrimidine-purine pairs. J Am Chem Soc. 2005; 127(10):3332-8. DOI: 10.1021/ja0430604. View

Tae E, Wu Y, Xia G, Schultz P, Romesberg F . Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. J Am Chem Soc. 2001; 123(30):7439-40. DOI: 10.1021/ja010731e. View

Lynch S, Liu H, Gao J, Kool E . Toward a designed, functioning genetic system with expanded-size base pairs: solution structure of the eight-base xDNA double helix. J Am Chem Soc. 2006; 128(45):14704-11. PMC: 2519095. DOI: 10.1021/ja065606n. View

RAPPAPORT H . The 6-thioguanine/5-methyl-2-pyrimidinone base pair. Nucleic Acids Res. 1988; 16(15):7253-67. PMC: 338407. DOI: 10.1093/nar/16.15.7253. View

Henry A, Romesberg F . Beyond A, C, G and T: augmenting nature's alphabet. Curr Opin Chem Biol. 2003; 7(6):727-33. DOI: 10.1016/j.cbpa.2003.10.011. View

Hirao I, Kimoto M, Mitsui T, Fujiwara T, Kawai R, Sato A . An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA. Nat Methods. 2006; 3(9):729-35. DOI: 10.1038/nmeth915. View

Parsch J, Engels J . Stacking and stability of RNA duplexes containing fluorobenzene and fluorobenzimidazole nucleosides. Nucleosides Nucleotides Nucleic Acids. 2001; 20(4-7):815-8. DOI: 10.1081/NCN-100002436. View

Mitsui T, Kimoto M, Sato A, Yokoyama S, Hirao I . An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo[(4,5)-b]pyridine. Bioorg Med Chem Lett. 2003; 13(24):4515-8. DOI: 10.1016/j.bmcl.2003.09.059. View

10.

Matsuda S, Fillo J, Henry A, Rai P, Wilkens S, Dwyer T . Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs. J Am Chem Soc. 2007; 129(34):10466-73. PMC: 2536688. DOI: 10.1021/ja072276d. View

11.

Paul N, Nashine V, Hoops G, Zhang P, Zhou J, Bergstrom D . DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs. Chem Biol. 2003; 10(9):815-25. DOI: 10.1016/j.chembiol.2003.08.008. View

12.

Matray T, Kool E . A specific partner for abasic damage in DNA. Nature. 1999; 399(6737):704-8. DOI: 10.1038/21453. View

13.

Kool E . Replacing the nucleobases in DNA with designer molecules. Acc Chem Res. 2002; 35(11):936-43. DOI: 10.1021/ar000183u. View

14.

Lai J, Kool E . Fluorous base-pairing effects in a DNA polymerase active site. Chemistry. 2005; 11(10):2966-71. DOI: 10.1002/chem.200401151. View

15.

Piccirilli J, Krauch T, Moroney S, Benner S . Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature. 1990; 343(6253):33-7. DOI: 10.1038/343033a0. View

16.

Liu H, Gao J, Lynch S, Saito Y, Maynard L, Kool E . A four-base paired genetic helix with expanded size. Science. 2003; 302(5646):868-71. DOI: 10.1126/science.1088334. View

17.

Sintim H, Kool E . Remarkable sensitivity to DNA base shape in the DNA polymerase active site. Angew Chem Int Ed Engl. 2006; 45(12):1974-9. DOI: 10.1002/anie.200504296. View

18.

Scopes D, Barrio J, LEONARD N . Defined dimensional changes in enzyme cofactors: fluorescent "stretched-out" analogs of adenine nucleotides. Science. 1977; 195(4275):296-8. DOI: 10.1126/science.188137. View

19.

Kim T, Brieba L, Ellenberger T, Kool E . Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs. J Biol Chem. 2005; 281(4):2289-95. DOI: 10.1074/jbc.M510744200. View

20.

Henry A, Yu C, Romesberg F . Determinants of unnatural nucleobase stability and polymerase recognition. J Am Chem Soc. 2003; 125(32):9638-46. DOI: 10.1021/ja035398o. View