Issued as Summary progress report, and Report of Research grant expenditures, Project no. G-41-A0

Wartell, Roger M.

English

Issued as Summary progress report, Project no. G-41-A0

Scholarly Materials And Research @ Georgia Tech

GEORGIA INSTITUTE OF TECHNOLOGY OFFICE OF CONTRACT ADMINISTRATION Clot/ SPONSORED PROJECT INITIATION Date: 	 Eebry 16, 197R Project Title: Conformations of Ligand-DNA Complexes and DNA Oligomers Project No: 	G-41-A01 Project Director: Dr. Roger M. Wartell Sponsor: 	DHEW/PHS/NIH - National Institute of General Medical Sciences Agreement Period: From  1/1/78   Until  12/31/78 (01 year)       Type Agreement: Grant No. 1 ROl GM24734-01 Amount: $51,133 New PHS Funds (G-41-A01) 4,691 GIT Contribution (G-41-313) $55,824 Total       Reports Required: Annual Progress Reports with Continuation Applications Terminal Progress Report upon Grant expiration Sponsor Contact Person (s): Technical Matters John C. Dalton, PH.D. (Dr. Jerry H. Roberts) Dep. Assoc. Director for Program Activities National Institute of General Medical Sciences Bethesda, MD 20014 Phone: 301-496-7137 Contractual Matters (thru OCA) Evelyn W. Carlin Grants Management Officer Office of Assoc. Director for Program Activities National Institute of General Medical Sciences Bethesda, MD 20014 Defense Priority Rating: None Assigned to: 	Physics COPIES TO: Project Director Division Chief (EES) School/Laboratory Director Dean/Director—EES Accounting Office Procurement Office Security Coordinator (OCA) deports Coordinator (OCA) (School/Laboratory) Library, Technical Reports Section EES Information Office EES Reports & Procedures Project File (OCA) Project Code (GTRI) Other   	CA-3 (3/76) GEORGIA INSTITUTE OF TECHNOLOGY OFFICE OF CONTRACT ADMINISTRATION SPONSORED PROJECT TERMINATION 	Date: 	2/1/7_9 	  01 .11% Project Title: 	Conformations of Ligand - DNA Complexes and DNA Oligomers Project No: 	G-41-A01 Project Director: 	Dr. R. M. Wartell (Dr. R. Salvo, Acting) Sponsor: 	EflEW/PHS/NIH - National Institute of General Medical Sciences Effective Termination Date:    12/31/78  (end of 01 year)      Clearance of Accounting Charges:  by 12/31/78 for 01 year  Grant/Contract Closeout Actions Remaining: T rf 7‘F I PITT-  L liviii14.; -‘1 C. Final Invoice and Closing Documents Final Fiscal Report Final Report of Inventions Govt. Property Inventory & Related Certificate Classified Material Certificate X Other  Annual Report of Expenditures due by 3/31/79. NOTE: Follow-on Project (02 year) is G-41-A02. Assigned to: 	Physics 	(School/Laboratory) -COPIES TO: Project Director 	Library, Technical Reports Section Division Chief (EES) 	Office of Computing Services School/Laboratory Director 	Director, Physical Plant Dean/Director—EES 	EES Information Office Accounting Office 	Project File (OCA) Procurement Office 	Project Code (GTRI) Security Coordinator (OCA) 	Other Reports Coordinator (OCA) CA-4 (3/76) • 	 SUTION IV                                       APPLICANT. REPEAT GRANT NUMBER SHOWN ON PAGE 1    GRANT NUMBER                  SECTION IV—SUMMARY PROGRESS REPORT  GM24734-02               PRINCIPAL MVESTIGAiuR OR PROGRAM DIRECTOR (Last, First, 	 PERIOD COVERED BY THIS REPORT Wartel l ,Roger M. 	 FROM 	 1 THROUGH NAME OF ORGANIZATION 	 1 January 1,1978 iDecember 31,1978 Georgia _Institute of Technology,Atlanta,Georgia  Tau (Repeat title shown in Item 1 on first page) Conformations of Ligand-DNA Complexes and DNA Oligomers  1. List publications: (a) published and not previously reported; (b) in press. Provide five reprints if not previously submitted. 2. List all additions and deletions in professional personnel and any changes in effort. 3. Progress Report. (See Instructions) 1. "Conformational Features of Distamycin-DNA and Netropsin-DNA Complexes by Raman Spectroscopy" by J.C. Martin, R.M. Wartell and D.C. O'Shea Proc.Nat.Acad.Sci. In press. Progress Report  The overall aims of the research project are to gain an understanding of how DNA binding ligands discriminate between different base pair sequences, and how one region of DNA influences the conformation of adjacent regions. There are two specific objectives of the project. One objective is to determine detailed information about the complexes which netropsin (Nt) and distamycin (Dt) form with A•T sequences of duplex DNA. These two oligopeptide molecules are of interest as antibiotics and as simplified models of proteins which bind to specific DNA sites. They have been shown to specifically bind to A-T regions of duplex DNAs. They do not bind single stranded DNA, RNA ) or GC DNA polymers under conditions (0.111 sodium ion) in which they hind to AT DNA polymers. The second objective is to examine the conformational properties of block DNA oligomers d(C15A15)• d(T15G15) and d(C15A5)•d(T 5G15). These oligomers have a block of A•T pairs joined to a block of G•C base pairs. These two sequences, cil(A) x •d(T) x and d(G) y •d(C) y (where x and y are the number of base pairs), show different conformational properties at polymer lengths. pray diffraction studies on fibers of poly d(A)•poly d(T) show it to be in the "B" type conformation. Similar studies show that pOly d(G)• poly d(C) under similar conditions is in the "A" type conformation. We wish to examine the conformation of this DNA by Raman Spectroscopy. This technique is also being employed in the drug-DNA binding experiments. There are four goals this year for the drug-DNA binding studies. The first is to obtain the Raman spectra of Nt and Dt in the free state and bound to calf thymus DNA. The second objective is to obtain the Raman spectra of molecules which are molecular subnits of Dt and Nt (described below). The third goal is to formulate and apply a normal mode calculation to the molecular subunits as well as the drugs. By combining the results of the second and third goals it should be possible to assign the frequencies of the drugs' Raman bands to vibrational motions of specific regions of the drugs. Many of these goals have been achieved.(see below). The major goal for the study on the block oligomer DNAs is to synthesize!-I-2 mgs of d(C15A5)•d(T5G15) and carry out characterization and preliminary Raman spectroscopy studies. Several steps in the synthesis have been accomplished so far. RESULTS: The enclosed manuscript describes the results to date on Nt and Dt binding to DNA. Raman spectra have been obtained of the drugs in the presence and absence of calf thymus DNA. Methods were devised to determine a bound drug's Raman spectra in the presence of DNA and solvent. A computer subtraction technique was developed to remove DNA and solvent background from the total spectra. Several changes occur in the Raman spectra of these drugs upon binding DNA. To analyze these changes, it was necessary to assign specific motions to the observed Raman bands. The first approach to this problem was to examine the Raman spectra of pyrrole, N-methyl pyrrole and N-methyl pyrrole carboxaldehyde, P.sGE 5 	(Use Continuation Page as necessary) r HS-2590-1 (Formerl, NIH-2006-1) (Rev. 4-75) U. S ,CA ERNMENT 1". 	G OFFICE 197d R.H. Wartell  Page 2 Progress Report and correlate these spectra to the drug spectra. Some empirical correlations of bands were possible, however a more fundamental understanding of the vibrational characteris-tics of the molecules was necessary. A normal mode calculation was formulated for asymmetric molecules and applied to pyrrole, its derivatives and the central portion of both drugs. A very good correlation was obtained between many observed band frequencies of Nt and Dt and the predicted frequencies. This allowed confident assignments for these bands. The analysis of the differences between bound and unbound distamycin spectra indicated that pyrrole ring and peptide group vibrations are altered when Dt binds to DNA. The environment of the pyrrole ring methyl groups are not affected by the binding. The results suggest that the pyrrole ring N1 position (where the methyl groups are) are logical sites for chemical modifications aimed at creating cell uptake selectively while waintaining DNA binding activity. - Analysis of the spectra of Nt gave similar results. However, due to the lower solubility limit of Nt in aqueous solution ( and thus reduced Raman intensities), it was not possible to quantify changes in the peptide group vibration. The synthesis pf the block DNA polymers has made some progress, however, it is uncertain if the orginal goal for this year will be met. Part of the delay has been necessitated by the building of a gel scanner, and the isolation of N.luteus DNA polymerase. 25 trig. of poly(dI)•poly(dC) was synthesized, and about 6-8 mg of poly(dC) is now digested to (dC) x oligomers. The techniquecof analytical gel electrophoresis and RPC-5 (Reverse Phase Chromatography) have been developed. Research goals for the coming year are to complete the synthesis of d(C15A5). d(T5G15) if it is not yet completed and construct d(C15A15)•d(T15G15). Raman studies on these block oligomers in aqueous solution will be carried out. Further studies on the binding of Nt and Dt to DNA will focus on one or two bands of their Raman spectra involved in peptide vibrations. By quantitatively examining these bands at different molar ratios of drug to DNA it may be possible to determine the number of hydrogen bonds involved in the drug-DNA complexes. Theoretical low energy calculations of the drug complexed to DNA will also be made. The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports If a grant is awarded as the result of this application. .7,  Date , 	7/ la,IEV Principal Investigator or Program Director 

Conformations of ligand-DNA complexes and DNA oligomers

• GEORGIA INSTITUTE OF TECHNOLOGY OFFICE OF CONTRACT ADMINISTRATION SPONSORED PROJECT INITIATION Date: 	2/1/79  Project Title: Conformations of Ligand-DNA Complexes and DNA Oligomers . Project No: 	G-41-A02 	,Th 	C ProjeCt Director: Dr, Roger M. Wartell • • Sponsor: 	DHEW/PHS/NIH - National Institute of General Medical Sciences Agreement Period: From 	1/1/79  Until 	12/31/79 (02 year)           Type Agreement: Grant No. 5 RO1 GI27434 - 02 Amount: $54,689 New PHS Funds (G-41-A02) 3,066 GIT Contribution G-41-320) $57,755 Total    Reports Required: Annual Progress Reports with Continuation Applications; Terminal Progress Report upon Grant expiration Sponsor Contact Person (s): Technical Matters  Dr. Jerry H. Roberts Program Administrator National Institute of General Medical Sciences Bethesda, MD 20014 Phone:(301) 496-7137 Contractual Matters  (thru OCA) D. McNish/B. Spinks Grants Management Specialists Office of Assoc. Director for Program Activities National Institute of General Medical Sciences Bethesda, MD 20014 Phone: (301) 496-7166 NOTE: Follow-on project to G-41-A01  Defense Priority Rating: None Assigned to: 	Physics COPIES TO: Project Director Division Chief (EES) School/Laboratory Director Dean/Director—EES Accounting Office Procurement Office Security Coordinator (OCA) Reports Coordinator (OCA) Library, Technical Reports Section EES Information Office EES Reports & Procedures Project File (OCA) Project Code (GTRI) Other (School/Laboratory) CA-3 (3/76) GEORGIA INSTITUTE OF TECHNOLOGY OFFICE OF CONTRACT ADMINISTRATION SPONSORED PROJECT TERMINATION   Date: 4-27-81 Project Title: 	Conformations of Ligand-DNA Complexes and DNA Oligomers Project No: 	G-41-A02 Project Director: 	Dr. Roger M. Wartell Sponsor: 	DHEW/PHS/NIH/NIGMS Effective Termination Date: 	12/31/80  Clearance of Accounting Charges: 	12/31/80 (02 year) Grant/Contract Closeout Actions Remaining: NONE  Final Invoice and Closing Documents   Final Fiscal Report Final Report of Inventions    Govt. Property Inventory & Related Certificate  Classified Material Certificate    Other     FOLLOW-ON PROJECT FOR 03 YEAR IS G-41-A03  Assigned to: 	Physics   (School/txbunct,orA   COPIES TO:     Project Director Division Chief (EES) School/Laboratory Director Dean/Director—EES Accounting Office Procurement Off ice Security Coordinator (OCA) e  its Coordinator fOCA) Library, Technical Reports Section EES Information Office Project File (OCA) Project Code (GTRI) Other   CA-4 (1/79) 4 SECTION IV APPLICANT REPEAT GRANT NUMBER SHOWN ON PAGE I GRANT NUMBER SECTION IV—SUMMARY PROGRESS REPORT . )•• CM 24734-03 PERIOD COVERED BY THIS REPORT A FROM PRINCIPAL INVESTIGATOR OR PROGRAM DIRECTOR Mast. First, Initial) WARTELL, Roger M. THROUGH 10/31/79 1/01/79 NAME OF ORGANIZATION Georgia Institute of Technology TITLE (Repeat tWe shown in Item 1 on first page) Conformations of Ligand-DNA Complexes and DNA Oligomers 1. List publications: (a) published and not previously reported; (b) in press. Provide five reprints if not previously submitted. 2. List all additions and deletions in professional personnel and any changes in effort. 3. Progress Report. (See Instructions) 1.  Objectives a. The overall aims of the research project are to gain an understanding of how DNA binding molecules discriminate between different base pair sequences and how one region of DNA influences adjacent regions. One specific objective of the proposal is to determine information about complexes between the antibiotics netropsin (Nt) and distamycin (Dt) and DNA. These two oligopeptide molecules bind specifically to A-T regions of duplex DNAs. They do not bind single stranded DNA, RNA, or G • C DNA polymers under conditions (0.1 M sodium ion) in which they bind to AT DNA polymers. A second objective is to employ Laser Raman spectroscopy to examine conformational properties of DNA molecules with known base pair sequences b. There are several goals for this year. One goal is to obtain an atomic resolution picture of the tight binding complex between netropsin and DNA. This is based on model building studies together with experimental information, and theoretical conformational energy calculations. A second goal is to examine the transition between the B-type and A-type conformations of DNA induced by ethanol using Laser Raman spectroscopy. The later technique provides vibrational spectra of molecules. A third goal is to synthesize the block DNA oligomer d(C10 A5) • d(T5 C 10 ) and initiate Laser Raman spectoscopy studies on this molecule. Additionally work was initiated to obtain quantities of homogeneous DNA molecules of short length (ti 150 base pairs) and specific sequence to examine by Raman spectroscopy. RESULTS  Computer calculations have been performed to obtain an atomic resolution picture of a complex between netropsin an a DNA helix of four A•T base pairs in the B-type conformation. The interatomic distances and bond angles of a netropsin molecule and the DNA duplex were from x-ray determinations. The dihedral angles of netopsin were allowed to be flexible except along the peptide bond where only small devia-tions from the trans position were allowed. Assuming a rigid DNA, model building was employed the generate an approximate structure based on previously obtained experimental data (3-4 base pairs contacted/drug, minor groove binding, pyrrole rings unaffected). Computer calculations have been performed to generate more precise coordinates of the complex. Van der Waals contact distances and optimum hydrogen bonding distances have been used to determine the conformational space available to netropsin in binding DNA in its minor groove. The next step will be to add energy terms to the calculation. Considerable effort has been made in working out the conditions to examine the B to A conformational transition of DNA. Although progress here was slower than anticipated, it has yielded interesting and unexpected results. Previous work with calf thymus DNA (Ilerbeck et al. Biochem., 2656, 1976) indicated that around 70-80% ethanol by volume, DNA underwent a transition from B to A as characterized by changes in two Raman bands around 800 cm -1 assigned to phosphod-iester vibrations. Due to strong ethanol bands however it was initially not Pt-IS-25;7'0-1 (Formerly N III-2006-1 ) (Rev. •4-7 -S) PACE 5 	(Use Continuation Paps as n•usssary) -1- possible to examine DNA Raman bands outside of the narrow region around 800 cm We have found that by scattering the Laser beam off of a flat area of an unorien12A - DNA fiber we can greatly enhance the DNA Raman band intensities relative to the ethanol band intensities. We can now monitor changes in the vibrational properts of several portions of DNA. Our results show that during the B to A transition deoxyribose ring vibratimis are altered in a different manner than bands assigned to phosphodiester bonds. The above studies employed calf thymus DNA. The synthesis and characterization of the block DNA d(C10 A 5). d(T 5 G10) is nearing completion. Both d C10 A20_50 and d T5 G 8_10 have been synthesized. These complementary strands are now being annealed and treated to generate flush end molecules. Progress has been made to obtain milligram amounts of a 144 base pair DNA containing the lactose operon promoter region. The DNA fragment was successfully cloned into a plasmid and work is under way to isolate and purify this DNA. A manuscript describing this work has been submitted for publication. The research goals for the next year will focus on Laser Raman spectroscopy studies of DNAs. We wish to examine the B to A transition of synthetic DNA polymers d(A)-n • d(T) n and d(G) n • d(C) n and the block DNA d(C1 0 A 5 ) - d(T 5  G10)_ Emphasize will be placed on examining different conformational features of the DNAs through the transition region. A second goal is to complete the -theretical calculations on the conformation of netropsin and DNA. The inclusion of additional energy terms in the.calculaticr will be made. The later calculations are anticipated to be a collaboration with Dr. S. Broyde of New York University. The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports if a grant is awarded as the result of this application. DATE 	 PrinYipal Investigator or Program Director GEORGIA INSTITUTE OF TECHNOLOGY ATLANTA, GEORGIA 30332 OFFICE OF THE COMPTROLLER April 24, 1980 Office of Associate Director for Program Activities National Institute of General Medical Sciences PHS/NIH Dept. of Health, Education & Welfare Bethesda, Maryland 20014 Dear Sir or Madam: Enclosed is the Report of Research Grant Expenditures for Grant No. 5 RO1 GM24734-02 for the period January 1, 1979 to December 31, 1979. If you have any questions or require additional information, please let us know. Sincerely, t\,1 0,„,j)c\j- clALejt_ David V. Welch, Manager Grants & Contracts Acctg. DVW/BITS/el Enclosure cc: Dr. R. M. Wartell Dr. R. A. Salvo Dr. D. Finkelstein Mr. E. E. Renfro Mr. O. H. Rodgers File G-41-A02 AN E011AL EDUCATION AND EMPLOYMENT OPPORTUNITY INSTIT1171r114.1 Department of Health, Education, and Welfare Grant No. 5 R01 GM24734-02 DATE OF THIS REPORTING PERIOD FROM 	1/1/79 	TO 	12/31/79 NAME AND ADDRESS OF GRANTEE INSTITUTION Georgia Institute of Technology Atlanta, Georgia 30332 TRANSACTION NO. (08) R1GM24734A PROJECT PERIOD FROM 	1/1/78 TO 12/31/80 INSTITUTIONAL ID NO. G-41-A02 1:=1 CHECK IF FINAL REPORT 1. Expenditures of DHEW Funds for this Reporting Period a. Personnel h. Alterations and renovations b. Consultant services i. 	Other c. Equipment d. Supplies j. 	Total direct costs 33,369.70 e. Travel, domestic lc. 	Indirect costs: Rate 76 	% M S&W ❑ TDC •• •• ••••• •• •• • • *4*. •• 	•• '''•A"•'"4 " f. Travel, foreign Base 	$ 	27,711.70 21 060.89 g. Patient care costs I. TOTAL $ 	54,430.59 2. Expenditures from Prior Periods (previously reported) 51,133.00 3. Cumulative Expenditures 105,563.59 4. Total Amount Awarded — Cumulatively 105 822.00 5. UneXpended Balance (item 4 less Item 3) 258.41 6. Unliquidated Obligations -0- 7..  Unobligated Balance (Item 5 less Item 6) 258.41 8.a. Cost Sharing Information — Grantee Contribution This Period 3,077.41 b. % of Total Project Costs (Item 8a divided by total of Items 1 and 8a) % 5.0 9.a. Interest/Income (enclose check) - b. Other Refundable Income (enclose check) - 10. Remarks I hereby certify that this report is true and coyect to the best of my knowledge, and that all expenditures reported herein have been made in propriate,grant poli es n /for t purposes set forth in the application and award document a1,4) 	n Drs. R. M. Wartel rkAl 	\I Date alvo Co-Directors titas--/go      SIGNATURE OF INSTITUTION OFFICER 	 DATE David V. Welch, Manager, Grants & Contracts Acctg. 404/894 -4624 	 REPORT OF RESEARCH GRANT EXPENDITURES accordance wi 

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Conformations of ligand-DNA complexes and DNA oligomers

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Scholarly Materials And Research @ Georgia Tech

Scholarly Materials And Research @ Georgia Tech