C O MMU N I C A T I O N S
tion). As was seen for t A37, ms t A37 decreases the overall ASL
6
2 6
7
m
T , but these modifications both stabilize complex formation.
5
2
m
Addition of mcm s U34 recovers some of the T loss caused by
ms t A37 presumably by promoting stacking among the 3′-
2
6
nucleosides of the anticodon loop. Preliminary NMR experiments
2
6
m
in our laboratory indicate that the decrease in T from ms t A37
comes from disruption of noncanonical base-pairing interactions
within the tRNA loop, which we believe then promotes the open
tRNA conformation that binds to the A-loop.
The model of complementary tRNAs suggests a hypothesis for
the HIV A-loop/tRNA interaction since the two native lysine tRNAs
and the A-loop hairpin are known to adopt canonical tRNA
conformations.6
,7,14
A complex with the topology proposed for
complementary tRNAs presents a structural framework that would
explain the nucleoside modification effects.18 Such a dimer interface
Asp 19
was observed in the crystal structure of tRNA
.
The wobble
Figure 1. RNA hairpins which form the HIV primer-template compleAxsp.
modification has a key role in stabilizing the tRNA U-turn along
A dimer interface composed of three base pairs as seen in the tRNA
6
2 6
crystal structure would involve pairing between the residues in green. (A)
with the threonyl side chain of t A. For ms t A, the thiomethyl
group would then be positioned to stack over the adenosines of
the complementary A-loop hairpin and reinforce what should
Secondary structure of the human tRNALys,3 ASL. (B) HIV-1 A-loop hairpin.
2
0
already be a substantial adenosine 3′-end stacking effect.
The primer-template complex is a potential target for HIV-1
therapeutics, and the rich functionality of the modified nucleosides
involved in this complex may present unique binding sites for
inhibitors of HIV-1 reverse transcription.
Acknowledgment. The work was supported by an NIH Grant
to D.R.D. (GM55508), and by Grants RR06262, RR13030, and
CA42014 for supporting NMR, MS, and RNA synthesis facilities.
We are indebted to Parsawar Krishna and J. A. McCloskey for mass
spectrometry assistance.
Figure 2. Biacore binding data of modified tRNA ASL 17-mers binding
to an immobilized A-loop hairpin with the sequence of the Mal-1 A-loop.
Global fits of the data (in black) to a 1:1 interaction model are shown in
Lys
-1 -1
red. (a) Native E. coli tRNA bound with a ka ) 42500 ( 500 M
s
and kd ) 3.1 ( 0.3 s-1 giving a KD ) 73 µM. (b) Native Human tRNA
Lys,3
Supporting Information Available: Synthetic procedures and
bound with a ka ) 35000 ( 400 M-1
KD ) 71 µM.
s
-1
and kd ) 2.5 ( 0.2 s giving a
-1
characterization of nucleosides and RNA, CD spectra, Table of T
m
data
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
A-loop hairpin with a 5′-biotin group and immobilized this RNA
on a Biacore SA chip. Most of the binding experiments were
conducted at 4 °C and 1 M NaCl to facilitate RNA-RNA
interactions and detect complexes for the weaker binders. The two
native tRNA ASLs had surprisingly similar affinities of ∼70 µM
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on the basis of data from the complementary tRNA system.
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The effect of mcm s U and ms t A on the thermodynamic
stability of the free tRNA hairpins was also measured to understand
whether changes within the tRNA structure itself could be correlated
with effects on the primer/template interaction (Supporting Informa-
JA028015F
J. AM. CHEM. SOC.
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