(left to right)
Nedal Safwat
John W. Stuart
Elzbieta Sochacka
Richard H. Guenther
Paul F. Agris
Connie S. Yarian
Piotr Mucha
Chi Fu

What we look like and what we do at work

Dr. Paul F. Agris

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Richard H. Guenther
A characteristic of lentiviruses including HIV-1 is that reverse transcription is primed by host lysyl tRNA. A major determinate for lysine selection is complementarity between the viral primer binding site and the eighteen 3' terminal bases of the priming tRNA. Complementarity alone is not sufficient to define primer selection. A second viral region, 5' to the viral PBS has complementarity to the anticodon stem/loop of tRNALys3 and may be involved in retention of lysine tRNA as primer. To further investigate this interaction of HIV viral RNA and human tRNALys3 we designed a model bi-molecular system. A series of synthetic oligonucleotides were prepared as analogs of HIV viral RNA and tRNALys3. The tRNA analogs included single and double placement of Y, mnm5U, s2U and t6A nucleotides at their native positions. Results demonstrate that a bi-molecular interaction occurs in the model system and the interaction appears to be structure with an internal loop that is stabilized by the presence of tRNA modifications.
 
 

Chi Fu
Synthetic oligonucleotides (DNA/RNA) have been widely used in the fields of academic research and industry.  During synthesis of oligonucleotides, the reactive groups of these nucleotides should be blocked or protected with various chemical molecules to prevent unwanted side reactions.  When the chain is completed, the crude oligonucleotides must be cleaved from support materials and the protecting groups must be removed from oligonucleotide chains.  To monitor whether protecting group have been removed from or still retained in synthetic DNA/RNA, mass spectrometry and chromatography can be employed.  However, their application is restricted because of the need for special equipment, time, and expense.  Recently, we have successfully generated several reagents that simply and inexpensively detect base-protecting groups of synthetic oligonucleotide, thereby providing a new method for identification and quantification of protecting groups, purification of oligonucleotides from crude synthetic materials, and monitoring the purity of industrial products as well as DNA chips.
 
 
 

Piotr Mucha   (on right)
RNA-protein interactions are important for many biological processes. Little is known about the recognition process and structure of RNA-protein complexes. Random phage display library has been used to study peptide-RNA interactions. 15 amino acid peptides corresponding to the highest affinity for the modified anticodon stem and loop domain of tRNAPhe were chemically synthesized and characterized by FRET. The most populated in the phage display demonstrated high affinity and specificity for both modified anticodon stem and loop domain and native tRNAPhe.
 
 
 
 
 

Elzbieta Sochacka

To produce oligonucleotides,
new synthesis protocols have
been developed for the preparation
of the suitably protected t6A
phosphoramidite and its site specific
incorporation into the oligomer sequence.
 
 
 
 
 
 
 
 
 
 
 

John W. Stuart
Sequence comparisons and in vitro evidence suggests that the anticodon of tRNALys3 interacts with the 3’ region of HIV-1 to enhance strand transfer and that t6A plays an important role. The oligoribonucleotide 5’-G27CAGACUUUU-t6A37-AUCUGC43-3’ containing the threonyl modified adenosine (t6A37) corresponding to the human tRNALys3 anticodon stem/loop domain was chemically synthesized. The solution structure of this 17-mer was determined by NMR spectroscopy and restrained molecular dynamics.  Addition of t6A, inhibits a potential base pair in the loop destabilizing the loop.  This “open loop” introduces an architecture favorable for ribosome binding similar to what is found for the anticodon region of tRNAPhe.  This structure may also provide a foundation for easy recognition of the anticodon by the 3’ region of HIV-1.
 
 
 

Connie S. Yarian
Modified nucleosides are common in tRNAs yet their functions are not well understood. A 17mer RNA hairpin corresponding in sequence to the completely unmodified anticodon stem and loop domain (ASL) of human tRNALys3 was not able to efficiently bind to the programmed ribosome. We assayed the contributions of the modified nucleosides in tRNALys to programmed ribosomal binding. Single substitutions of the naturally-occurring anticodon loop modifications 5-methylaminomethyluridine, mnm5U34, and N6-threonylcarbamoyladenosine, t6A37, to ASLLys3 significantly restored poly-A programmed ribosomal binding. Incorporation of t6A37 decreased the thermostability of the ASL, while mnm5U34 slightly increased thermostability. While the U34 modifications may be crucial for codon recognition, t6A37 may enhance ribosomal binding by disrupting interactions across the anticodon loop, destabilizing the ASL, yet ensuring an anticodon loop architecture that can bind to a programmed ribosome.
 

Kelly Nobles
The tertiary structure of RNA is stabilized by the presence of modified nucleosides. One significant role of modified nucleosides may be to facilitate the coordination of metal ions. The best characterized metal ion binding sites in RNA have been identified in the crystal structure of yeast tRNAPhe. The conventional folding of the cloverleaf tRNA secondary structure into the inverted L tertiary structure involves tertiary interactions between the D-loop and TYC-loop and is dependent on the coordination of Mg2+ ions. At physiological Mg2+ concentrations, native tRNA molecules are more stable than their unmodified transcripts. We have shown that the interaction between the D-loop and TYC loop is not only dependent on Mg2+ ions, but also on the presence of conserved, modified nucleosides. A 33mer corresponding to the completely unmodified TYC-stem/loop, anticodon stem and aminoacyl acceptor stem forms a complex  with a 30mer corresponding to the completely unmodified D-stem/loop, anticodon stem, and aminoacyl stem in a Mg2+ containing native PAGE. The site-specific incorporation of Y55, rT54, and m5C49 into the TYC half-molecule influenced the affinities of this molecule for the D- half molecule. binding constants have been determined for this complex formation by quantifying PAGE with a phosphorimager.
 
 

Kim Kelly                                                                                                                                     Susanna Smith

Kim performs compositional analysis on all RNA oligonucleotide samples. Susanna is developing chromatographic methods for quantitating modified DNAs.
 
 

Recent Publications
Sengupta R, Vainauskas S, Yarian C, Sochacka E, Malkiewicz A, Guenther RH, Koshlap KM, Agris PF. Modified constructs of the tRNA TPsiC domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases. Nucleic Acids Res. 2000 Mar 15;28(6):1374-80.

Ashraf SS, Guenther R, Agris PF. Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains. RNA. 1999 Sep;5(9):1191-9.

Agris PF, Guenther R, Sochacka E, Newman W, Czerwinska G, Liu G, Ye W, Malkiewicz A. Thermodynamic contribution of nucleoside modifications to yeast tRNA(Phe) anticodon stem loop analogs. Acta Biochim Pol. 1999;46(1):163-72.

Agris PF, Marchbank MT, Newman W, Guenther R, Ingram P, Swallow J, Mucha P, Szyk A, Rekowski P, Peletskaya E, Deutscher SL. Experimental models of protein-RNA interaction: isolation and analyses of tRNA(Phe) and U1 snRNA-binding peptides from bacteriophage display libraries. J Protein Chem. 1999 May;18(4):425-35.

Yarian CS, Basti MM, Cain RJ, Ansari G, Guenther RH, Sochacka E, Czerwinska G, Malkiewicz A, Agris PF. Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39. Nucleic Acids Res. 1999 Sep 1;27(17):3543-9.

Koshlap KM, Guenther R, Sochacka E, Malkiewicz A, Agris PF. A distinctive RNA fold: the solution structure of an analogue of the yeast tRNAPhe T Psi C domain. Biochemistry. 1999 Jul 6;38(27):8647-56.

Ashraf SS, Ansari G, Guenther R, Sochacka E, Malkiewicz A, Agris PF. The uridine in "U-turn": contributions to tRNA-ribosomal binding. RNA. 1999 Apr;5(4):503-11.

Ashraf SS, Sochacka E, Cain R, Guenther R, Malkiewicz A, Agris PF. Single atom modification (O-->S) of tRNA confers ribosome binding. RNA. 1999 Feb;5(2):188-94.

Guenther R, Forrest B, Newman W, Malkiewicz A, Agris PF. Modified RNAs as potential drug targets. Acta Biochim Pol. 1998;45(1):13-8.

Ashraf SS, Guenther R, Ye W, Lee Y, Malkiewicz A, Agris PF. Ribosomal binding of modified tRNA anticodons related to thermal stability. Nucleic Acids Symp Ser. 1997;(36):58-60. Review.

Agris PF, Guenther R, Ingram PC, Basti MM, Stuart JW, Sochacka E, Malkiewicz A. Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1. RNA. 1997 Apr;3(4):420-8.

Agris PF. The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog Nucleic Acid Res Mol Biol. 1996;53:79-129. Review.

Basti MM, Stuart JW, Lam AT, Guenther R, Agris PF. Design, biological activity and NMR-solution structure of a DNA analogue of yeast tRNA(Phe) anticodon domain. Nat Struct Biol. 1996 Jan;3(1):38-44.