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Another Realization of Aqueous Computing with Peptide Nucleic Acid. August 8, 2001 Park, Ji-Yoon. Masayuki Yamamura, Yusuke Hiroto, and Taku Matoba. Abstract. - PowerPoint PPT Presentation
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Another Realization of Aqueous Another Realization of Aqueous Computing with Computing with
Peptide Nucleic AcidPeptide Nucleic Acid
August 8, 2001 August 8, 2001
Park, Ji-YoonPark, Ji-Yoon
Masayuki Yamamura, Yusuke Hiroto, and Taku MatobaMasayuki Yamamura, Yusuke Hiroto, and Taku Matoba
AbstractAbstract
Elementary operation for aqueous computing with PNA and realizElementary operation for aqueous computing with PNA and realize one bit memory for a feasibility study to confirm strand displacee one bit memory for a feasibility study to confirm strand displacement by PNA ment by PNA
Aqueous computingAqueous computing
- code design free molecular computing- code design free molecular computing
- handles an aqueous sol’n of general-purpose memory molecules - handles an aqueous sol’n of general-purpose memory molecules
with a small set of elementary laboratory operationswith a small set of elementary laboratory operations
- fits to solve a certain pattern NP-complete problem- fits to solve a certain pattern NP-complete problem
- copy a memory state upon a DNA seq by whiplash PCR - copy a memory state upon a DNA seq by whiplash PCR
Aqueous AlgorithmAqueous Algorithm
Pour(n): divide the solution into Pour(n): divide the solution into
nn tubes tubes. Unite: mix Unite: mix nn tubes into one. tubes into one.
→ → resulting sol’n resulting sol’n SetToZero(SetToZero(kk))
: set the : set the kkth bit of all memory th bit of all memory
molecules in that tube to be 0.molecules in that tube to be 0. MaxCountOfOnesMaxCountOfOnes
: find the max number of 1’s in : find the max number of 1’s in
one memory molecule from one memory molecule from
that tube. that tube.
Aqueous memoryAqueous memory
Structure of PNA and DNAStructure of PNA and DNA
Peptide Nucleic acid(PNA)Peptide Nucleic acid(PNA)
Analogs of DNA in which the phosphate backbone is replaced with an Analogs of DNA in which the phosphate backbone is replaced with an uncuncharged “peptide-like” backboneharged “peptide-like” backbone
The achiral backbone is made of The achiral backbone is made of repeating N-(2-aminoethyl)-glycine units repeating N-(2-aminoethyl)-glycine units linked by amide bondslinked by amide bonds
No deoxyribose or phosphate groupsNo deoxyribose or phosphate groups are present are present
Backbone is unchargedBackbone is uncharged
PNA to hybridize to complementary RNA or DNA with higher specificity PNA to hybridize to complementary RNA or DNA with higher specificity and affinity, making PNA and affinity, making PNA good candidates for the inhibition of gene expregood candidates for the inhibition of gene expressionssion
Characteristics of PNACharacteristics of PNA
Thermal stabilityThermal stability
- The lack of charge repulsion between the PNA strand and the DNA or RNA - The lack of charge repulsion between the PNA strand and the DNA or RNA stronge stronger binding between PNA/DNA or PNA/RNAr binding between PNA/DNA or PNA/RNA
- A higher thermal stability of the duplexes- A higher thermal stability of the duplexes
Long lastingLong lasting
- resistant to enzymatic degradation- resistant to enzymatic degradation
because their hybrid chemical structure is not recognize nucleases or proteases. because their hybrid chemical structure is not recognize nucleases or proteases.
- Stable over a wide pH range- Stable over a wide pH range
Characteristics of PNACharacteristics of PNA
Binding independent of salt concentrationBinding independent of salt concentration
** The Tm of PNA/DNA duplexes is independent of salt concentration The Tm of PNA/DNA duplexes is independent of salt concentration
* At a low ionic strength PNA can be hybridized to a target sequence at temperatures * At a low ionic strength PNA can be hybridized to a target sequence at temperatures at which normal DNA hybridization is inhibitedat which normal DNA hybridization is inhibited
* Hybridization of PNA can also occur in the absence of Mg* Hybridization of PNA can also occur in the absence of Mg2+2+, a factor that further in, a factor that further inhibits DNA/DNA duplex formation.hibits DNA/DNA duplex formation.
Triplex formationTriplex formation - PNA oligomers containing only thymines(T) and cytosines(C) often prefer to bind a 2- PNA oligomers containing only thymines(T) and cytosines(C) often prefer to bind a 2
PNA/1DNA stoichiometry resulting in high stability PNA/1DNA stoichiometry resulting in high stability
Strand displacementStrand displacement - - Homopyrimidine PNA oligomers displace a DNA strand from DNA/DNA duplex to foHomopyrimidine PNA oligomers displace a DNA strand from DNA/DNA duplex to fo
rm a local PNA/DNA/PNA triplex and a D-looprm a local PNA/DNA/PNA triplex and a D-loop
Material and MethodsMaterial and Methods
1. For 2 pmol DNA, we added (1) 0.0 pmol, (2) 100 pmol, (3) 200 pmol PNA1. For 2 pmol DNA, we added (1) 0.0 pmol, (2) 100 pmol, (3) 200 pmol PNA
2. Incubate in 17 ul 1 2. Incubate in 17 ul 1 × TE buffer at 37ºC for 1 hr × TE buffer at 37ºC for 1 hr
3. Add 15 units of Xba I for all three samples 3. Add 15 units of Xba I for all three samples
4. Incubate in 20 ul 1 4. Incubate in 20 ul 1 × M buffer at 37ºC for 30 min× M buffer at 37ºC for 30 min
5. Run samples in a 10% polyacrylamide gel electrophoresis 5. Run samples in a 10% polyacrylamide gel electrophoresis
PNA(15 bp): N-CCGCPNA(15 bp): N-CCGCTCTAGATCTAGAACTAG-C ACTAG-C DNA(2 pmol)DNA(2 pmol) Xba I(Takara)Xba I(Takara)
ResultResult
M: 20 bp ladder(Takara)M: 20 bp ladder(Takara)
NC: Negative controlNC: Negative control
Lane 1: DNA(2 pmol)Lane 1: DNA(2 pmol)
Lane 2: DNA(2 pmol) + PNA(100 pmol)Lane 2: DNA(2 pmol) + PNA(100 pmol)
Lane 3: DNA(2 pmol) + PNA(200 pmol) Lane 3: DNA(2 pmol) + PNA(200 pmol)
MM 11NCNC 22 33
10% Polyacrylamide gel electrophoresis10% Polyacrylamide gel electrophoresis
200bp200bp
100bp100bp
140bp140bp
240bp240bp
100
pmol
100
pmol
200
pmol
200
pmol
0 pm
ol
0 pm
ol
Strand DisplacementStrand Displacement
Memory State CopyMemory State Copy
DiscussionDiscussion
The molecular weight of displaced DNA is increased by 15 bp PNThe molecular weight of displaced DNA is increased by 15 bp PNAA
We could not find the optimum condition for strand displacementWe could not find the optimum condition for strand displacement
- Purity of both DNA and PNA - Purity of both DNA and PNA
- Need a control experiment- Need a control experiment
ConclusionConclusion
Aqueous computingAqueous computing Biomolecular realization with PNA-DNA hybrid Biomolecular realization with PNA-DNA hybrid Preliminary experiment with one bit memory Preliminary experiment with one bit memory An idea to copy memory state An idea to copy memory state