Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.
BACKGROUND
In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated.


RESULTS
This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide.


CONCLUSIONS
Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.
Nickens
David G
DG
Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America.
Bardiya
Nirmala
N
Patterson
James T
JT
Burke
Donald H
DH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D055162:Biocatalysis; D011088:DNA Ligases / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism; D005026:Ethylene Glycols / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D013379:Substrate Specificity
D015226:Dinucleoside Phosphates; D005026:Ethylene Glycols; D009711:Nucleotides; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; C077067:hexaethylene glycol; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D006163:Ribonuclease T1; D011088:DNA Ligases
Nucleic Acids Res. 2010 Oct;38(19):6785-95
20511589
Curr Opin Struct Biol. 2001 Jun;11(3):321-6
11406381
Nucleic Acids Res. 1998 Jan 15;26(2):525-31
9421510
Chem Biodivers. 2007 Apr;4(4):633-55
17443876
RNA. 2006 Nov;12(11):2025-33
16963711
Nucleic Acids Res. 1997 Jun 1;25(11):2106-13
9153309
Nucleic Acids Res. 1998 Oct 15;26(20):4618-25
9753729
Biochemistry. 2005 Nov 15;44(45):15007-16
16274247
Science. 1988 Aug 26;241(4869):1077-80
3413476
Nucleic Acids Res. 1994 Oct 11;22(20):4039-43
7937128
Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2746-51
10077582
Annu Rev Biochem. 2004;73:791-836
15189159
Science. 1992 May 15;256(5059):992-7
1589782
Nucleic Acids Res. 1995 Jul 11;23(13):2361-6
7630712
Nucleic Acids Res. 2000 Nov 1;28(21):4051-8
11058099
Science. 2003 Oct 24;302(5645):618-22
14576428
Nucleic Acids Res. 1999 Sep 1;27(17):3589-96
10446251
J Biol Chem. 1997 Sep 5;272(36):22591-9
9278414
RNA. 2006 Dec;12(12):2118-25
17068208
Chem Biol. 1999 Mar;6(3):167-76
10074465
Eur J Biochem. 2003 Nov;270(21):4315-25
14622296
Anal Biochem. 2004 Feb 1;325(1):137-43
14715294
Nucleic Acids Res. 1998 Nov 15;26(22):5225-7
9801324
Biophys J. 2004 Aug;87(2):988-98
15298905
Methods Mol Biol. 2003;212:215-28
12491913
Nucleic Acids Res. 2001 Jan 15;29(2):578-81
11139629
RNA. 2010 Dec;16(12):2349-59
20935068
Nucleic Acids Res. 2003 Jun 15;31(12):3208-16
12799448
Biochemistry. 1995 Nov 21;34(46):15315-27
7578148
J Virol. 1997 Mar;71(3):1931-7
9032324
Chem Biol. 2002 Apr;9(4):507-17
11983339
Nucleic Acids Res. 2006 Jun 21;34(11):3201-8
16790565
Nat Struct Mol Biol. 2005 Nov;12(11):994-1000
16228005
Biochemistry. 2007 May 1;46(17):4962-76
17407264
Nucleic Acids Res. 2004 Aug 24;32(15):4503-11
15328364
Science. 1974 Nov 29;186(4166):790-7
4377758
Bioorg Chem. 2002 Oct;30(5):332-49
12485593