Hud, Nicholas V.

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Scholarly Materials And Research @ Georgia Tech

Annual Report for Emory Project G-33-C23 PI: Nicholas V. Hud, School of Chemistry and Biochemistry, Georgia Institute of Technology. Contract Information: This project represents a subcontract of "Towards synthetic biology: the replication of synthetic polymers" , an N S F Collaborative Research Center awarded to David Lynn of Emory University (NSF grant number 0404677). Period of Report: June 1, 2006-May 3 1 , 2007 Personnel Support: The past year of funding has been used to support the research of two graduate students in the Hud laboratory, Eric Englehart and Eric Horowitz. Summary of Progress: Aim II Activities and Findings: Identify and characterize molecular midwife? for template-substrate assembly. W e are pleased to report that our progress on Specific Aim II has resulted in the acceptance of three peer-review publications this past year (attached). The paper by Horowitz and Hud, which appeared as a communicat ion in J. Am. Chem. Soc, describes our discovery that the small molecule proflavine binds to 2 ' ,5 ' - l inked R N A with an association constant that is 25-fold greater than that measured for the natural 3 ' ,5 ' - l inked RNA. W e have been using proflavine as a "molecular midwife" to promote the assembly of nucleic acids for template^directed synthesis. Thus, the greater affinity of proflavine for 2 ' ,5 ' - l inked R N A indicates that nucleic acids with this alternative backbone linkage will be even more amendable to intercalation-mediated! assembly. The experimental results presented in our J. Am. Chem. Soc. publication are fully consistent with proflavine binding to 2 ' ,5 ' - l inked R N A by base pair intercalation. Nevertheless, we believed that is was necessary to obtain a high resolution structure of 2 ' ,5 ' - l inked R N A with bound proflavine, as this is the only unequivocal means by which the mode of binding of a small molecule to a nucleic acid can be confirmed. W e are pleased to report that we have recently obtained the solution-state structure of proflavine bound to the 2 ' ,5 ' - l inked R N A duplex [GCCGCGGC ]2 . At a ratio of two proflavine molecules per duplex, we observe proflavine bound between the two C p G steps of the duplex (Fiigure X). This structure is quite unique in that it represents the first solution-state structure of a simple intercalator (as opposed to a bisintercalator) bound to a nucleic acid. The greater association constant, and slower off-rate, allowed structure determination, whereas past attempts to solve similar structures with natural R N A or D N A have been stymied by the fast exchange of the intercalator between the nucleic acid and solution. W e have also measured the relatively binding constants i for a number of other known intercalators for 2 ' ,5 ' - l inked RNA. This data is expected to be valuable for our development of an optimally matched pair of molecular midwife and nucleic acid backbone structures. Figure X. A model of the solution-state structure of the 2 ' ,5 ' - l inked R N A duplex [ G C C G C G G C ] 2 with bound proflavine. The model is based upon distance constraints obtained by 2D N M R spectroscopy and a refinement using the Amber suite of molecular dynamics programs. r Y \ J \ cr Figure Y. The structure of a (proton bound) base pair between adenosine and the N 7 -nucleoside of adenine that is predicted to contain a low barrier hydrogen bond between the N l of adenine and the N 9 of the N 7 -nucleoside of adenine. This proposed base pair is superimposed on top the G A N7-N1 amino-carbonyl base pair that is known to occur in ribosomal RNA. An additional objective of Aim II was to explore the possibility of alternative base pairs (i.e. non-Watson-Crick pairs) t h a t could be used for information transfer. Within this objective, we have been interested in the possibility that low barrier hydrogen bonds (LBHB) could be used as a means for increasing the selectivity of hydrogen bonded structures in aqueous solution. Additionally, we have been interested in the possibility of purine-purine informational systems. We had previously discovered that the small molecular coralyne can be used to propose adenine-adenine base pairs, and the greater for propensity purines to stack (as compared to pyrimidines) suggested to us that artificial purine-purine informational systems m y be more amenable to self-assembly than Watson-Crick pairing systems. Combining these two ideas, w e performed a computational study of adenine and related purine bases to determine if purine-purine base pairs could be formed that contain LBHBs . This work, performed in collaboration with Tom Morton of U C Riverside, is now in press at J. Phys Chem. Briefly, we have identified an adenine-adenine base pair that is held together by a L B H B , based upon energy levels determined by B3LYP/6-31G** level calculations. The structure of this base pair is extremely close to that of the purine-purine G-A N7-N1 amhio-carbonyl base pair that is observed in naturally occurring R N A structures (Figure Y) . Thus, we believe that it will be possible; to use this base pair in non-natural informational systems that have helical structures similar to that of RNA. Interestingly, the asymmetry of this base pair requires that one adenine be the natural N9-glycoside, whereas the other is the N7-glycoside (Figure Y) . It m a y therefore be possible to creaite a non-natural informational system with high specificity from two isomers of adenosine. We are beginning to explore this possibility in the laboratory. r In a third publication, resulting from collaborations between the Hud and Lynn laboratories {Chemistry & Biodiversity, in press), w e have demonstrated our ability to use intercalation to drive template-directed synthesis in a non-duplex system. Specifically, we have shown that is possible to use coralyne, a crescent-shaped molecule, to transfer sequence information via a triple helix structure (Figure Z). In contrast proflavine, which works as a midwife for SPO 3 ; — T — T — T — T — T — T — T substrate strands (dT3,dT4) -r-G — A —A — A —A —A — A — A - C - G C T — T — T — T — T — T — T — G— C -coralyne ispp3; template hairpin (HP) Fig. 4. An experimental test system used to explore the ability for coralyne to act as a midwife in a template-directed ligation reaction that includes A T T base pairs triplets. (Left to Right) 5 '-phosphorothioate-(dT)3 and 3 '- iodo-(dT)4 substrate strands that are too short to form a stable triplex with the indicated D N A hairpin. The addition of coralyne promotes the assembly of a ligation-active complex, which leads to the formation of the (dT)7 product within an internal phosphorothioate linkage. Experimental data is shown in Hud et al. (Chemistry & Biodiversity, in press). information transfer in a duplex system, is ineffective in the triplex system. Likewise, coralyne does not promote information transfer in a duplex system. These results provide substantial support for our proposal that information transfer between natural (and non-natural) pairing motifs can be selectively promoted by intercalation, if the structure of the intercalator matches that of the desired base assembly. 

Towards synthetic biology: the replication of synthetic polymers

https://smartech.gatech.edu/bitstream/1853/29085/1/g-33-c23_9677.pdf

Towards synthetic biology: the replication of synthetic polymers

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