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bicyclo amino sugar Comparison between DNA, PNA ...
1
Nanoscale Nucleic Acid Sensors DNA PNA LNA NH N Base NH N Base O O O O O O Base O O P - O O O O Base O O P - O O O Base O O P - O O O Base O O P - O O Comparison between DNA, PNA and LNA DNA and LNA have negatively charged backbone whereas PNA is non-ionic. Unlike DNA, PNA and LNA exhibit nuclease resistance. Aq. solubility of PNA is less compared to DNA and LNA , which are almost fully aqueous soluble. Both PNA and LNA are capable of forming homoduplexes and heteroduplexes with natural and synthetic nucleic acid analogue with higher thermal stability compared to DNA. Due to modified ribose moiety LNA being the conformationally more rigid among the three. LNA forms the most stable homoduplexes yet discovered and the best probe so far for SNP detection in solution. Developing self-assembled ordered structures of PNA & LNA and their sensor applications 0 2 4 200 400 600 800 1000 35.0 37.5 55 60 65 70 T m (°C) Salt concentration (mM) 800 1000 ) NaCl NaOCOCH 3 NaNO 3 Na 2 SO 4 NMe 4 Cl NaCl NaOCOCH 3 NaNO 3 Na 2 SO 4 NMe 4 Cl circle: fully matched PNA-DNA duplexes triangle: singly mismatched PNA- DNA duplexes 2. Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes via Ionic Regulation Ghosh et al., Langmuir 2013, 29, 3370-3379. Sensitivity (ssPNA sensor probe) : Sensitivity (ssDNA sensor probe) = 20:1 300 nm Inkjet Deposited PNA Film Target strands Bending Sensor Reference 4. Enhancing sensitivity in a piezoresistive cantileverbased label-free DNA detection assay using ssPNA sensor probes Ghosh et al., J. Mater. Chem. B, 2014, 2, 960-970. 3. Enhancing On-Surface Mismatch Discrimination Capability of PNA Probes by AuNP Modification of Gold(111) Surface Ghosh et al., Langmuir 2013, 29, 11982-11990. Gold(111) on mica AuNP Sensor Probe linker 0 20 40 60 80 100 0 10 20 30 40 50 60 70 80 PNA−DNA DNA−DNA mismatch discrimination Temperature (°C) Nanoparticle size (nm) 6. Maximizing Mismatch Discrimination by Surface-Tethered Locked Nucleic Acid Probes via Ionic Tuning Mishra et al., Anal. Chem. 2013, 85, 1615-1623. 5. Ordered Self-Assembled Locked Nucleic Acid (LNA) Structures on Gold(111) Surface with Enhanced Single Base Mismatch Recognition Capability Mishra et al., Langmuir 2012, 28, 4325-4333. 1. An atomic force microscopy investigation on self-assembled peptide nucleic acid structures on gold(111) surface Ghosh et al., J. Colloid Interface Sci. 2011, 360, 52-60. 100 nm Single Molecule Level Structural Biology Nanoscale Protein-based Bioelectronics nogalose sugar O O O OH O N CH 3 H CH 3 CO 2 CH 3 HO OH OH CH 3 O O H 3 C H 3 CO H 3 CO OCH 3 CH 3 OH CH 3 + bicyclo amino sugar aglycone unit methyl ester Nogalamycin H 3 N Pt N H 2 (CH 2 ) 4 H 2 N Pt NH 3 Cl NH 3 H 3 N N H 2 (CH 2 ) 4 H 2 N Pt NH 3 H 3 N Cl 4+ BBR3464 H 3 N Pt H 3 N Cl Cl Cisplatin The antibiotic antitumour nogalamycin is a naturally- occurring DNA threading intercalator. Its structure consists of a central anthracycline unit (aglycon part) and two bulky groups - a nogalose sugar and a bicyclo amino sugar, at the two ends. BBR3464 [{trans-PtCl(NH 3 ) 2 } 2 -μ-trans- Pt(NH 3 ) 2 {NH 2 (CH 2 ) 6 NH 2 } 2 ] 4+ , is a new generation platinum chemotherapeutic agent, which exhibits cytotoxicity at 10 to 1000 times lower dose limit compared to the well-known platinum drug cisplatin. DNA is thought to be the primary cellular target of BBR3464 and cisplatin. High-resolution AFM is applied to obtain molecularly resolved information on drug-induced DNA structural changes. Banerjee et al., Biochemical and Biophysical Research Communications 2008, 374, 264–268. 1. The AFM topographs of DNA–nogalamycin complex, incubated for 12 and 48 h, revealing a gradual change from the circular supercoiled form to the compact plectonemic superhelix, confirming intercalative binding. 100 nm 100 nm After 12 h After 48 h 2. Macroloop with knot formation and/or microloop formation along the DNA contour, and overall compaction are indicative of drug treatment. Banerjee et al., Biochimie 2010, 92, 846-851. Banerjee et al., Biochimie 2012, 94, 494-502. 100nm 100 nm Macro-/microloops formed after BBR3464 treatment Cisplatin forms microloops only Before drug treatment Molecularly resolved features of drug-treated dsDNA reveal details relevant to the mode of drug action 200 nm Applicability of Ferritins for Bioelectronics Ferritin is an iron storage redox protein that is found in both prokaryotes and eukaryotes. It is also a globular protein which is soluble & non-toxic in nature. It has a unique ordered arrangement of 24 subunits that leads to the formation of a hollow sphere with an external diameter of approximately 12 nm and an internal diameter of 7 to 8 nm. As it is centrosymmetric in nature so the metals present inside the protein are easily accesible. It is structurally robust and functions well up to 80 0 C temperature in aqueous environment within a pH range of 4.0-9.0. Ferritin, which contain iron inside the hollow sphere as ferrihydrite phosphate called holoferritin and which is devoid of iron called Apoferritin. 1. Near-Metallic Behavior of Warm Holoferritin Molecules on a Gold(111) Surface Rakshit et al., Langmuir 2010, 26(20), 16005–16012. Holoferritin at 25 0 C Holoferritin at 40 0 C Apoferritin at 25 0 C Apoferritin at 40 0 C At 25 0 C At 25 0 C At 40 0 C At 40 0 C 20nm 20nm 20nm 20nm 2. Tuning Band Gap of Holoferritin by Metal Core Reconstitution with Cu,Co, and Mn Rakshit et al., Langmuir 2011, 27, 9681–9686. 3. Solid-state electron transport in Mn-, Co-, holo-, and Cu-ferritins: Force-induced modulation is inversely linked to the protein conductivity Rakshit et al., J. Colloid Interface Sci. 2012, 388, 282-292. 4. Nanoscale Mechano-Electronic Behaviour of a Metalloprotein as a Variable of Metal Content Rakshit et al., Langmuir 2013, 29, 12511−12519. Iron stored as mineral inside ferritin Correlating electrical transport characteristics of ferritin with its structural and mechanical properties Holoferritin Apoferritin 3-fold and 4-fold channels in ferritin Structural aspects of proteins and protein-protein assemblies at single molecule level using scanning probe microscopy approach 1. Structural features of human histone acetyltransferase p300 and its complex with p53 100 nm 2. Direct observation of binding of human histone acetyltransferase p300 to histone/HMGB1 protein and probing the force of interaction by single molecule atomic force spectroscopy 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 Counts Unbinding force (pN) Banerjee et al., FEBS Letters 2012, 586, 3793-3798 p300 FL Histone/HMGB1 HN NH HN Banerjee et al., J Phys Chem B 2015, 119, 13278-13287 Functional Biointerfaces at Nanoscale: Biosensors, Bioelectronics and Single Molecule Biophysics at the Mukhopadhyay Group nm nm nm nm p300 FL- C-terminal specific antibody complex p300 FL- N-terminal specific antibody complex p300 FL- p53 complex p300 FL- octameric histone complex p300 FL- HMGB1 complex Schematic representation of AFS experimental set-up with single peak force distribution 100 nm 100 nm 100 nm 100 nm Molecular partners Unbinding force (pN) at loading rate 3 nN/s Non-acetylating condition Acetylating condition p300 FL/ histone octamer 95±2 66±2 p300 FL/histone H3 117±2 97±2 p300 FL/HMGB1 122±3 102±3 3. Discriminating intercalative effect of threading and classical intercalator by force spectroscopy Changes in contour length with time Typical force-extension curve of 692 bp free dsDNA Force-extension trace of nogalamycin-DNA (1:10) after (A) 1 h and (B) 48 h; (1:26) after (C) 1h and (D) 48 h. Force-extension trace of daunomycin-DNA (1:10) after (A) 1 h and (B) 48 h; (1:26) after (C) 1h and (D) 48 h. Banerjee et al., PLOS ONE 2016, 11(5), e0154666 Mishra et al., Langmuir 2014 30, 10389-10397. 8 10 12 14 16 18 35 40 45 50 55 60 65 70 75 S S S S S S S S S S S S S S S Maximizing target recognition efficiency through spacer-independent optimal probe density window. % Target DNA recognition efficiency Probe density (nM/cm 2 ) High Optimal Low 6.5 7.0 7.5 8.0 5 10 20 50 250 500 1000 % Target DNA recognition efficiency pH [NaCl ] in mM 0 10 20 30 40 50 60 70 80 7. Regulating the On-Surface LNA Probe Density for the Highest Target Recognition Efficiency DNA-DNA LNA-DNA Single base mismatch discrimination (∆ pN) 0 50 100 150 200 Centrally mismatched Penultimate mismatch (away from gold) Penultimate mismatch (near to gold) 150 mM NaCl 15 mM MgCl 2 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 -4.3 -4.2 -4.1 -4.0 -3.9 -3.8 -3.7 -3.6 -3.5 Force (V) Time (Sec) Target recognition at millisecond time scale No Unbinding 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 20 40 60 80 100 120 140 Counts Unbinding Force (pN) Specific Unbinding 8. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes Mishra et al., Nucleic Acids Res. 2016, 44, 3739-3749. Bera et al., Langmuir 2017, 33, 1951-1958. 5. Nanoscale on-silico electron transport via ferritins
Transcript of bicyclo amino sugar Comparison between DNA, PNA ...
Nanoscale Nucleic Acid Sensors
DNA PNA LNA
NH
N
Base
NH
N
Base
O
O
O
O
O
O
BaseO
O
P
-O
O
O
O
BaseO
O
P
-O
O
O
BaseO
O
P
-O
O
O
BaseO
O
P
-O
O
Comparison between DNA, PNAand LNA DNA and LNA have negatively charged backbone whereasPNA is non-ionic. Unlike DNA, PNA and LNA exhibit nuclease resistance. Aq. solubility of PNA is less compared to DNA and LNA ,which are almost fully aqueous soluble. Both PNA and LNA are capable of forming homoduplexesand heteroduplexes with natural and synthetic nucleic acidanalogue with higher thermal stability compared to DNA. Due to modified ribose moiety LNA being theconformationally more rigid among the three. LNA forms the most stable homoduplexes yet discoveredand the best probe so far for SNP detection in solution.
Developing self-assembled ordered structures of PNA & LNA and their sensor applications
0 2 4 200 400 600 800 1000
35.0
37.5
55
60
65
70
Tm
(°C
)
Salt concentration (mM)
0 200 400 600 800 100035
40
55
60
65
70
[Na+] (mM)
Tem
per
atu
re (°C
)
NaCl
NaOCOCH3
NaNO3
Na2SO
4
NMe4Cl
NaCl
NaOCOCH3
NaNO3
Na2SO
4
NMe4Cl
circle: fully matchedPNA-DNA duplexes
triangle: singlymismatched PNA-DNA duplexes
2. Facilitating Mismatch Discrimination bySurface-Affixed PNA Probes via Ionic Regulation
Ghosh et al., Langmuir 2013, 29, 3370-3379.
Sensitivity (ssPNA sensor probe): Sensitivity (ssDNA sensor
probe) = 20:1
300 nm
Inkjet Deposited PNA Film
Target strands Bending
Sensor
Reference
4. Enhancing sensitivity in a piezoresistivecantileverbased label-free DNA detectionassay using ssPNA sensor probes
Ghosh et al., J. Mater. Chem. B, 2014, 2, 960-970.
3. Enhancing On-Surface MismatchDiscrimination Capability of PNA Probes by AuNPModification of Gold(111) Surface
Ghosh et al., Langmuir 2013, 29, 11982-11990.
Gold(111) on mica
AuNP
Sensor Probe
linker
0 20 40 60 80 1000
10
20
30
40
50
60
70
80
PNA−DNA DNA−DNA mismatch discrimination
Te
mp
era
ture
(°
C)
Nanoparticle size (nm)
6. Maximizing Mismatch Discrimination bySurface-Tethered Locked Nucleic AcidProbes via Ionic Tuning
Mishra et al., Anal. Chem. 2013, 85, 1615-1623.
5. Ordered Self-Assembled Locked Nucleic Acid(LNA) Structures on Gold(111) Surface withEnhanced Single Base Mismatch RecognitionCapability
Mishra et al., Langmuir 2012, 28, 4325-4333.
1. An atomic force microscopy investigation onself-assembled peptide nucleic acid structures ongold(111) surface
Ghosh et al., J. Colloid Interface Sci. 2011, 360, 52-60.
100 nm
Single Molecule Level Structural Biology Nanoscale Protein-based Bioelectronics
nogalose sugar
O
O
O
OH
O
N
CH3
H
CH3 CO2CH3
HO
OH
OH
CH3
O
OH3C
H3CO
H3COOCH3
CH3
OH
CH3
+
bicyclo amino sugar
aglycone unit
methyl
ester
Nogalamycin
H3NPt
NH2
(CH2)4
H2N
PtNH3
Cl NH3
H3N NH2
(CH2)4
H2N
PtNH3
H3N Cl
4+
BBR3464
H3N
Pt
H3N
Cl
Cl
Cisplatin
The antibiotic antitumour nogalamycin is a naturally-occurring DNA threading intercalator. Its structureconsists of a central anthracycline unit (aglycon part)and two bulky groups - a nogalose sugar and a bicycloamino sugar, at the two ends. BBR3464 [{trans-PtCl(NH3)2}2-μ-trans-Pt(NH3)2{NH2(CH2)6NH2}2]4+, is a new generationplatinum chemotherapeutic agent, which exhibitscytotoxicity at 10 to 1000 times lower dose limitcompared to the well-known platinum drug cisplatin.DNA is thought to be the primary cellular target ofBBR3464 and cisplatin. High-resolution AFM is applied to obtain molecularlyresolved information on drug-induced DNA structuralchanges.
Banerjee et al., Biochemical and BiophysicalResearch Communications 2008, 374, 264–268.
1. The AFM topographs of DNA–nogalamycincomplex, incubated for 12 and 48 h, revealing agradual change from the circular supercoiledform to the compact plectonemic superhelix,confirming intercalative binding.
100 nm100 nm
After 12 h After 48 h
2. Macroloop with knot formation and/ormicroloop formation along the DNA contour,and overall compaction are indicative of drugtreatment.
Banerjee et al., Biochimie 2010, 92, 846-851.Banerjee et al., Biochimie 2012, 94, 494-502.
100nm100 nm
Macro-/microloopsformed afterBBR3464 treatment
Cisplatin formsmicroloops only
Before drugtreatment
Molecularly resolved features of drug-treated dsDNA revealdetails relevant to the mode of drug action
200 nm
Applicability of Ferritins for Bioelectronics Ferritin is an iron storage redox protein that is found in bothprokaryotes and eukaryotes. It is also a globular protein which issoluble & non-toxic in nature. It has a unique ordered arrangement of 24 subunits that leads tothe formation of a hollow sphere with an external diameter ofapproximately 12 nm and an internal diameter of 7 to 8 nm. As it iscentrosymmetric in nature so the metals present inside the proteinare easily accesible.
It is structurally robust and functions well up to 800Ctemperature in aqueous environment within a pH range of 4.0-9.0. Ferritin, which contain iron inside the hollow sphere asferrihydrite phosphate called holoferritin and which is devoid of ironcalled Apoferritin.
1. Near-Metallic Behavior of Warm Holoferritin Molecules on a Gold(111) Surface
Rakshit et al., Langmuir 2010, 26(20), 16005–16012.
Holoferritin at 250C Holoferritin at 400C
Apoferritin at 250CApoferritin at 400C
At 250C
At 250C
At 400C
At 400C
20nm
20nm 20nm
20nm
2. Tuning Band Gap of Holoferritin by Metal Core Reconstitution with Cu,Co, and Mn
Rakshit et al., Langmuir 2011, 27, 9681–9686.
3. Solid-state electron transport in Mn-, Co-, holo-, and Cu-ferritins: Force-induced modulation is inversely linked to the protein conductivity
Rakshit et al., J. Colloid Interface Sci. 2012, 388, 282-292.
4. Nanoscale Mechano-Electronic Behaviour ofa Metalloprotein as a Variable of Metal Content
Rakshit et al., Langmuir 2013, 29, 12511−12519.
Iron stored as mineral inside ferritin
Correlating electrical transport characteristics of ferritin with its structural and mechanical properties
Holoferritin Apoferritin
3-fold and 4-fold channels in ferritin
Structural aspects of proteins and protein-protein assemblies at single molecule level using scanning probe microscopy approach
1. Structural features of human histone acetyltransferase p300 and its complex with p53
100 nm
2. Direct observation of binding of human histone acetyltransferase p300 to histone/HMGB1 protein and probing the force of interaction by single molecule atomic force spectroscopy
0 20 40 60 80 100 120 140 160
0
20
40
60
80
100
120
140
Cou
nts
Unbinding force (pN)
Banerjee et al., FEBS Letters 2012, 586, 3793-3798
p300 FL
Histone/HMGB1HN
NHHN
Banerjee et al., J Phys Chem B 2015, 119, 13278-13287
Functional Biointerfaces at Nanoscale: Biosensors, Bioelectronics and Single Molecule Biophysics at the Mukhopadhyay Group
nm
nm
nm
nm
p300 FL- C-terminalspecific antibody complex
p300 FL- N-terminalspecific antibody complex
p300 FL- p53 complex
p300 FL- octameric histone complex
p300 FL- HMGB1 complex Schematic representation of AFS experimental set-up with single
peak force distribution
100 nm
100 nm100 nm
100 nm
Molecular partners Unbinding force (pN) at loading rate
3 nN/s
Non-acetylating
condition
Acetylating
condition
p300 FL/ histone
octamer
95±2 66±2
p300 FL/histone H3 117±2 97±2
p300 FL/HMGB1 122±3 102±3
3. Discriminating intercalative effect of threading and classical intercalator by force spectroscopy
Changes in contour length with time Typical force-extension curve of 692 bpfree dsDNA
Force-extension trace of nogalamycin-DNA (1:10) after (A) 1 h and (B) 48 h; (1:26) after (C) 1h and (D) 48 h.
Force-extension trace of daunomycin-DNA (1:10) after (A) 1 h and (B) 48 h; (1:26) after (C) 1h and (D) 48 h.
Banerjee et al., PLOS ONE 2016, 11(5), e0154666
Mishra et al., Langmuir 2014 30, 10389-10397.
8 10 12 14 16 1835
40
45
50
55
60
65
70
75
S S S S S
S S S S S S S
S S S
Maximizing target recognition efficiency
through spacer-independent optimal probe
density window.
% T
arg
et
DN
A r
eco
gn
itio
n e
ffic
ien
cy
Probe density (nM/cm2)
High
Optimal
Low
6.57.0 7.5
8.0
5
10 20
50 250
5001000
% T
arg
et D
NA
rec
og
nit
ion
eff
icie
ncy
pH
[NaCl ] in mM
0
10
20
30
40
50
60
70
80
7. Regulating the On-Surface LNA Probe Densityfor the Highest Target Recognition Efficiency
DNA-DNA
LNA-DNA
Sin
gle
ba
se m
ism
atc
h d
iscr
imin
ati
on
(∆
pN
)
0
50
100
150
200
Centrally
mismatchedPenultimate
mismatch
(away from
gold)
Penultimate
mismatch
(near to gold)
150 mM NaCl
15 mM MgCl2
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
-4.3
-4.2
-4.1
-4.0
-3.9
-3.8
-3.7
-3.6
-3.5
Fo
rce
(V
)
Time (Sec)
Target recognition at millisecond time scale
No Unbinding
0 20 40 60 80 100 120 140 160 180 200 220 240 2600
20
40
60
80
100
120
140
Co
un
ts
Unbinding Force (pN)
Specific
Unbinding
8. Molecularly resolved label-free sensing ofsingle nucleobase mismatches by interfacial LNAprobes
Mishra et al., Nucleic Acids Res. 2016, 44, 3739-3749.Bera et al., Langmuir 2017, 33, 1951-1958.
5. Nanoscale on-silico electron transport viaferritins
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