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- Floor Plans
& Pricing
- Map &
- Apartment
- Apartment
- Office Hours
& Info
- Ratings
& Reviews
Floor Plans & Pricing
Does your furniture fit? Pick a floor plan below and design your new place!
General Directions
From Skelly Drive (I-44), exit south on Yale Avenue past 71st Street. Turn right on E. 80th Street and we are on your right.
Apartment Features
- Air Conditioning
- Balcony
- Cable Ready
- Ceiling Fan(s)
- Dishwasher
- Oversized Closets
- View
Community Features
- Accepts Credit Card Payments
- Accepts Electronic Payments
- Business Center
- Clubhouse
- Emergency Maintenance
- Extra Storage
- Fitness Center
- High Speed Internet Access
- Laundry Facility
- Pet Park
- Public Transportation
- Swimming Pool
- Tennis Court(s)
- Trail, Bike, Hike, Jog
- Wireless Internet Access
- Pet Friendly
Special Features
- Disability Access
- Pets
- Short Term Available
- Pets Accepted$10 pet rent per month, per pet
Additional Features
- Leasing 24-Hours a Day ? Online
- Jenks Schools
- Pets welcome
- Dog Park
- Swimming Pool
- Tennis court
- Ample parking
- 24-hour emergency maintenance
- On-site laundry/management
- Large kitchen
- Pantry/storage closets
- Tiled bathroom
- Unique floorplans
- Patio/balcony
- Picnic tables
- Scenic views
- Business Center
- 24-hr. fitness center
- Business center
- Saturday breakfast
Country Hollow
LOOKING FOR AN IDEAL COMMUNITY? Country Hollow offers unique differences in luxury apartment living. Parks, golf courses and outstanding recreational facilities are all close at hand, as are hospitals, shopping, restaurants, schools such as Jenks, St Francis, ORU, TCC and great entertainment. We've made sure that country living doesn't mean sacrificing convenience. Our park-like setting offers you the tranquility that your hectic lifestyle demands.
Check Availability or call: (918) 615-9996 (888) 710-2513 (918) 856-5821
Community Information
Community Characteristics
Community Profile
- Population
- 29,817
- Growth Since 2000
- -1.03%
- Population Density
- 3690
- Male
- 48%
- Female
- 52%
Household Income
- Median
- $35,739
- Average
- $60,244
Office Hours
|01:00 PM to 05:00 PM
|09:00 AM to 06:00 PM
|09:00 AM to 06:00 PM
|09:00 AM to 06:00 PM
|09:00 AM to 06:00 PM
|09:00 AM to 05:00 PM
|10:00 AM to 05:00 PM
Lease Terms
Variable, 3-Month, 6-Month, 7-Month, 8-Month, 9-Month, 10-Month, 11-Month, 12-Month
Case & Associates has earned an unequalled reputation for excellence in all areas of property and asset management. The firm as a whole has won recognition as an Accredited Management Organization, a distinction achieved by only the top one percent of U.S. real estate management companies. Our company is structured to deliver, in any form, and on any schedule that serves our clients' specific needs. Case & Associates has proven its ability to succeed in even the most turbulent real estate markets. We are a disciplined team of professionals with the expertise, the organization, and the resources to help you achieve and surpass your investment goals.
See other Case & Associates Properties, Inc. Properties in your area.
Check Availability or call:(918) 615-9996 (888) 710-2513 (918) 856-5821
Fax: 9184964046
Visit the property's management website
Overall Rating(3 Ratings)
What are Certified Ratings?
These are ratings based on residents who live at the property and have completed a resident satisfaction survey.
Residents are invited to participate in the satisfaction survey via a list of email addresses* submitted by the property and independently certified and administered by a third party market research firm.
Because reviews on Apartment Guide are from Certified Residents and are managed by a third party, you can feel confident that the feedback provided is accurate and trustworthy.
* Email list administration is managed by an independent market research firm, not Apartment Guide
Value for the Money
Office Staff
Building Exterior
Move-in Condition
Parking Availability
Recreational Facilities
1 Review for Country Hollow
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Some of the services that the complex is supposed to provide are often not available, such as the outdoor pool and internet. For some time the exercise room was not open, either.
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Residents are invited to participate in the satisfaction survey via a list of email addresses* submitted by the Property and independently certified and administered by Kingsley Associates, our third party market research firm.
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CHAP PAGE I The Ferns and their Allies
Life Histories
Yesterday and Today
12 other sections not shown
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abundant antheridia appearance archegonia Asplenium bears Beech Fern Bracken Fern branches Bristle Fern British brown scales cells Club Moss Common Club Moss Common Polypody course crevices cultivated curious developed distinct England Equisetum evergreen fact feature feet fertile cone fertile fronds fertile stems Filmy Fern Flowering Plants foliage fronds garden Greek word green colour Green Spleenwort grow habit Hartstongue height Holly Fern Horsetail inches in length indusia indusium Lady Fern lanceolate large number leaf leafy portion leaves lobes Lycopodium Maidenhair Spleenwort Male Fern margins mid-veins moist Mountain Buckler Fern name is derived Nephrodium occur outline pinnae pinnules Polypodium Prickly Shield Fern produced prothallus rachis reference resemblance rhizome roots Royal Fern Scotland Selaginella sheaths side soil sori sorus species specific name specimens sporangia sporangium spore capsules spores stalk stipes tapering tufted root-stock Tunbridge Filmy Fern underside United Kingdom upper portion Vascular Cryptogams veins Wall Rue whilst whorls Woodsia

subset: commoncrawl


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The need to virtualize every application, along with the need to support constantly arriving new and newly changed applications will drive the creation of an Application Operations function in the enterprise. This function will need to be supported and enabled by a new generation of APM tools that meet the new requirements of the Application Operations team.
Business Agility
Business Agility ...
Application Performance Management for Virtualized Applications – the VMware vFabric APM Update
• • 2 Comments
As business critical applications move into production virtualized environments, the need arises to ensure their performance from a response time perspective. Legacy Applications Performance Management tools are not well suited to make the jump from static physical systems, to dynamic virtual and cloud based systems. For these reasons enterprises need to consider new tools from vendors that have virtualization aware and cloud aware features in their APM solutions. Vendors like AppDynamics, BlueStripe, Confio, dynatrace, ExtraHop, New Relic, Quest, VMTurbo and VMware (vFabric AppInsight) are currently leading this race to redefine the market for APM solutions.
Business Agility ...
Looking at the Entire VMware Operations and Application Management Strategy
• • 0 Comments
VMware is clearly serious about extending its management reach into the application layer of the stack. New products like vFabric AppDirector, and vFabric APM make this into more than intention – they make VMware's participation in these markets a reality as well. The move to remake the APM industry has thus far been lead by creative startups like New Relic, AppDynamics, BlueStripe, dynaTrace and ExtraHop. Now VMware has joined this effort will add considerable mass and velocity to the effort. The message is clear – it is time to instrument all of your applications for response time and the legacy APM vendors do not have the products that are up to the task.
Business Agility ...
VMware Joins the APM Revolution with vFabric Application Performance Manager
• • 1 Comment
With vFabric APM VMware has announced a compelling entry into the APM space, joining the current set of APM innovators – New Relic, AppDynamics, BlueStripe, dynaTrace and ExtraHop. The innovations that VMware is bringing to the table will usher in a redefinition of what APM is, from a performance and availability tool for the development team, to a strategic applications management platform that enables price/performance comparison shopping by application owners. Once the price/performance features are fully implemented, this may do more to enable performance sensitive applications to move to public clouds than any other thing that VMware has done.
Less than 5% of the applications that matter to enterprises world wide are under management by an APM solution that can help ensure application response time, application availability and the integrity of the critical transactions within the application. This is because first generation APM solutions have been too expensive to purchase, too limited in their scope and too expensive to configure, maintain and own.
The focus upon sharing real response time and transaction load data by SalesForce.com is notable when compared with the pre-historic approach to performance that is used by many cloud vendors (and for a matter of fact many enterprise IT organizations). Response Time correlates directly to end user experience and at the end of the day that is all that matters. Hopefully the industry will learn from SalesForce.com and advance this concept further.
Business Agility ...
Consumerization of IT: AppSense and Centrix Software alliance recognise its more than just "how can my CFO justify their iPad"?
• • 1 Comment
AppSense, a leading provider of user virtualization technology, and Centrix Software, provider of unified end-user computing solutions, have announced a strategic partnership to provide organizations with a comprehensive, user-centric transformation program. Do you need a user-centric transformation program? How could this alliance help your business manage IT beyond the 'single-PC-for-every-user' era? If they can help you, are they your only hope? Will it justify your CFO's iPad?
Business Agility ...
VMware Articulates a Compelling Management Vision – Automated Service Assurance
• • 0 Comments
VMware deserves an enormous amount of credit for promising to reinvent IT Operations around automation and the guaranteed performance of applications. VMware either has or is working on all of the building blocks required to execute upon this vision for the vSphere platform. The combination of innovation by VMware, and by the third party ecosystem on this front will create a new compelling benefit to virtualization, that will allow virtualization to comfortably address business critical and performance critical applications.
This week I have been paying close attention to the developments of Hurricane Irene. In the beginning, Hurricane Irene looked like she was going visit Florida on her journey to the north. Even though it looked like Florida was going to get hit by this storm, it was still early and there was time for the storm to change course. It was also time to go out and make sure my Hurricane Supply Kit at least had the basics like batteries and flashlights as well as filling up the gas tanks of the cars. I have different levels of preparedness which depends on how close the storm is and the projected path. Just like I have steps in place to be prepared for the storm, most companies that I have worked for in Florida have a storm plan in place and like myself, do not sound the real alarm until the storm is 48 – 72 hours away from a hit but start to prepare for the alarm in case it is needed.


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I own every volume, and just today watchedThe Finale, so in theory, I've seen every episode.
But I don't remember seeing the episode where Jerry dates Newman's ex-girlfriend, or the episode where Jerry wants a specific BBQ sauce because it looks like someone who's going to be on the Tonight Show with him.
Could someone tell me which seasons these two are from, and how I could have possibly missed them?


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Gung-Ho! Newcastle
- Sports & Fitness
- High Gosforth Park, NE3 5HP
Connected Communities Symposium - Day 2
Culture Lab Newcastle
Tuesday, September 13, 2011 from 10:00 AM to 6:00 PM (BST)
CONNECTED COMMUNITIES Symposium
Culture Lab Newcastle, UK — 12th-14th September 2011
Culture Lab Newcastle is hosting an international interdisciplinary event open to the general public, on the topic of "connected communities".
This registration page is for the 2nd day of the symposium: 13th September 2011
Many theorists have regarded communities as networks of interacting people within a defined territory, and policy-makers have tended to respond to this particular understanding. However, in an era where digital technologies have supported transnational forms of connectedness and the efficiency of grassroot movements, communities are once again looked at as innovative fertile grounds for alternative social organisation.
In a time when these trends can be manipulated by current governmental agendas, the Connected Communities symposium aims to critically explore evolving notions of community in art, design, history, politics, sociology, journalism, and hacktivism, among others.
Focus will be shed on – but not restricted to – the following topics:
For a complete programme of the event, please visit:
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Elk Grove ranked among nation's hardest-working cities
Elk Grove residents work hard for their money.
How hard? Elk Grove is among the nation's top 15 hardest-working cities, according to a list compiled by lifestyle website MyLife.
Elk Grove is No. 13 on the list, which ranked the nation's 200 most-populous cities based on factors such as families with two working parents, employment rate, commute time and percentage of population enrolled in school. The factors were measured with U.S. Census data.
Sacramento also appears on the list, at No. 115.
Elk Grove City Manager Laura Gill told Business Journal columnist Ed Goldman that the city "was built as a bedroom community for people who worked in Sacramento and Rancho Cordova. But people have tired of the commute. So we're looking at job creation." Apple also has an Elk Grove campus that employs about 1,800 people.
In addition, the largest remaining undeveloped area in Elk Grove now has a master plan. The approximately 1,200-acre area is expected to become an employment hub for the city.
MyLife, which compiled the rankings, is a Los Angeles-based website that specializes in identity management and includes a social network. Boston ranked No. 1 on the list of hardest-working cities.


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Average Customer Review
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8 of 8 people found the following review helpful
4.0 out of 5 stars Space is Still an Adventure,
Patrick Shepherd "hyperpat" (San Jose, CA USA) - See all my reviews
This review is from: Red Thunder (Hardcover)
Those who became entranced with science fiction by way of Heinlein's juveniles will find in this book a return to that same sense of wonder, the feeling that man can accomplish almost anything he sets out to do, that everyday troubles can be overcome, that will make you proud to be a member of the human race.
Just how do you build a spaceship in your back yard (or even a rented warehouse)? As a starting point, it greatly helps if you have a power plant that can deliver effectively unlimited power, the invention of Jubal, Travis Broussard's highly eccentric cousin. Travis, as an alcoholic cashiered astronaut, provides both some of the necessary capital and the experience level to make such a project a possible reality. For labor, four 'kids' (they're 20+ years old) who are motivated and highly intelligent, who already have some skill sets that are quite relevant to the task are quite willing to learn more. Building the ship occupies a good two thirds of the book, and some of the details of how it's done in a hurry-up, make it work (while really testing for safety) fashion make for fascinating reading. The actual flight of the Red Thunder, while still interesting, is not quite so fascinating, and the space rescue that the crew performs on an American attempt to reach Mars smacks a little bit of melodrama, but it had me turning pages till two in the morning.
The power plant device, the 'Squeezer', is highly improbable, and violates quite a few principles of physics (as known today), but it is the basic element that both allows the space ship to become a reality, and due to its inherent power, drives the reasoning behind building the ship as a private enterprise, as such power, in the wrong hands, could become a nightmare. This helps drive one of the thematic messages of this book, an almost paranoid anti-government (of any stripe) stance, a reflection on the reality that all humans are not inherently good, kind, or peaceful. Offsetting this message are some others: people really can and often do help one another, people do better when they have a definite goal to work towards, your family is a major influence in your formation, but is not the only or final determiner of just what type of person you become.
Varley pays some definite homage to Heinlein here, with a plot line that is very much a re-working of Heinlein's Rocket Ship Galileo (Heinlein's first and probably his weakest juvenile), updated to today's world (and so becoming far more believable). There are more references to Heinlein in the character's names (Manny Garcia, Jubal) and John D. MacDonald (Travis, McGee and the Florida setting). This book is not technically a juvenile, but it has that same feel, and is readable by almost anyone over the age of fourteen or so (there are some references to sex and some portrayed family relationships that are probably not appropriate for younger readers).
The characters are well fleshed out, and the portrayed interpersonal relationships ring with veracity. It's easy to get very caught up in Manny's (the first-person narrator) life, his relationships with his mother, his girlfriend Kelly, his best friend Dak. A very fast and highly entertaining read, one that will forcibly remind you of just what a pleasure reading can be.
--- Reviewed by Patrick Shepherd (hyperpat)
5.0 out of 5 stars Great book for a lover of the John Twelve Hawks Traveller Trilogy,
Verified Purchase(What is this?)
This review is from: Red Thunder (Mass Market Paperback)
I was recommended this book to buy for my partner on a website where it recommended books based on others you like. I put in the John Twelve Hawks Traveller Trilogy and this was suggested. He loved it, I'll have to have a read myself!
Most Helpful First | Newest First
Red Thunder by John Varley (Hardcover - April 2003)
Used & New from: �0.01


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Giants' Will Beatty to IR to make room for Chase Blackburn; team signs Isaiah Stanback to practice squad
Originally written on Beat Writers Blog | Last updated 11/30/11
EAST RUTHERFORD, NJ - SEPTEMBER 13: Chris Cooley #47 of the Washington Redskins tries to elude Chase Blackburn #57 of the New York Giants on September 13, 2009 at Giants Stadium in East Rutherford, New Jersey. (Photo by Jim McIsaac/Getty Images)
The Giants officially lose their starting left tackle for the season.
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Red Wings equipment manager Paul Boyer, 38, from Grosse Pointe City, shows of the Wings' new jersey in the locker room at Joe Louis Arena on Wednesday afternoon.
- Filed Under
The first thing you notice about Nicklas Lidstrom's new jersey is the "C." It's on the right side, instead of the left.
The Red Wings wanted to protect the tradition of the Winged Wheel as much as possible with the new streamlined Reebok jerseys all NHL teams are using this season, but that's one concession they had to make. And it only affects three players, Lidstrom and his two alternate captains.
The letters had to be moved because the new jerseys are constructed of multiple panels, more so than the old models, and there wasn't room for them above the tip of the Winged Wheel on the left side without ...


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Denver Summer Brew Fest Friday July 24th & Saturday July 25th, 2015
- Food & Drink
- 2027 West Lower Colfax Ave Denver Colorado 80204
Monthly Happy Hour
Returned Peace Corps Volunteers of Colorado
Thursday, September 26, 2013 from 5:30 PM to 8:30 PM (MDT)
Please join us for our monthly RPCVs of Colorado Happy Hour!
This month we are moving to Vine Street Pub, located at 17th and Vine in Denver.
We will be over on the Harry's side of the pub, so make sure you let the hostess know so she can show you over there.
See you soon!
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The Army is upgrading WIN-T to include intelligence and C2 capabilities, with medical and logistics systems to come.
DARPA is advancing its CODE program to develop collaborative autonomy for UAS.
MUOS will eventually be a five-satellite constellation forming a smartphone-like network for secure, high-bandwidth communications.
Northrop Grumman's BACN system bridges the gaps in air and ground communications.
The GATR antennas weigh 80 percent less than typical satellite dishes and provide secure, high-bandwidth communication in remote and austere environments.
In search of better ISR, the Navy wants to enhance imaging systems mounted on shore-based maritime vessels.
NGA and DigitalGlobe have created a new cloud-based analytic tool for processing large troves of geospatial data.
Maj. Gen. Vincent Stewart, who currently runs the Marine Forces Cyber Command, will add a star and assume control late this month.
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The Bibi-Mitt partnership during the campaign will have two effects that neither intended.
Siegel said legislators probably shouldn't be making laws about e-cigarettes until they know what the effects are.
Its effects are dramatic when levels are way too high or way too low.
late 14c., "a result," from Old French efet (13c., Modern French effet) "result, execution, completion, ending," from Latin effectus "accomplishment, performance," from past participle stem of efficere "work out, accomplish," from ex- "out" (see ex-) + facere "to do" (see factitious).
Meaning "impression produced on the beholder" is from 1736. Sense in stage effect, sound effect, etc. first recorded 1881. The verb is from 1580s. Related: Effecting; effection.
effect ef·fect (ĭ-fěkt')
Something brought about by a cause or an agent; a result.
The power to produce an outcome or achieve a result; influence.
A scientific law, hypothesis, or phenomenon.
The condition of being in full force or execution.
Something that produces a specific impression or supports a general design or intention.
To bring into existence.
To produce as a result.
To bring about.


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April 17, 2015 3:44 pm
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Tor Opsahl, the executive vice president at IT contractor CCSi, joined Industry Chatter to discuss his company and how it does business with federal agencies.
Creative Computing Solutions, Inc. provides program and project management as well as IT solutions for the federal government.
From Opsahl's official bio:
How the US Mint and NIST are perfecting the money-making process.


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H.R.384 - Homes for Heroes Act of 2013113th Congress (2013-2014)
|Rep. Green, Al [D-TX-9] (Introduced 01/23/2013)
|House - Financial Services | Senate - Banking, Housing, and Urban Affairs
|Latest Action:
|05/16/2013 Received in the Senate and Read twice and referred to the Committee on Banking, Housing, and Urban Affairs.
|Major Recorded Votes:
|05/15/2013 : Passed House
This bill has the status Passed House
Here are the steps for Status of Legislation:
- Introduced
- Passed House
Subject — Policy Area:
- Armed Forces and National Security
- View subjects
Related Bills: H.R.384 — 113th Congress (2013-2014)All Bill Information (Except Text)
A related bill may be a companion measure, an identical bill, a procedurally-related measure, or one with substantive similarities. Bill relationships are identified by the House, the Senate, or CRS, and refer only to same-congress measures.


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Doc Martin
GENTLEMEN PREFERSeries Website »
Episode Guide »
When Martin fires Elaine for her inefficiency, he becomes universally disliked in the village.
Vermont PBS educates, informs, entertains and inspires Vermonters to be lifelong learners and engaged in their community.
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Vermont PBS Announces First Recipient of Public Media Ambassador Award


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Gill O C D G & Q
Part of a drawing for Gill Sans C D G O Q dated 20.12.32. The original is in pencil and colour wash on graph paper and measures 11" wide by 14.5" high.
Reproduced in: The Monotype Recorder, Volume XLI No. 3, Autumn, 1958
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Staff nurses required for northern ireland group
Omagh — Head Hunt International
staff nurses required for northern ireland group rates: £12.50 per hour a large healthcare group in northern ireland with care homes based in tyrone, newry and bessbrook is expanding and requires a large number of staff nurses. there is great potential for job progressing for all levels of nursing. requirements: - registered general nurse with [...]
Contract Type: Permanent
Category Healthcare & Medical


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Israeli Prime Minister Benjamin Netanyahu has rejected a demand by the Palestinian Authority to tear down the security fence separating Israel and Palestinian population centers in Judea and Samaria (the West Bank).
Saeb Erekat, chief negotiator for the Palestinians, disclosed that he has raised the issue of the fence (which in some places becomes a wall) with Washington. "The Israelis know that the wall adds to the complexities," Erekat told Reuters. It's part of the problem and not part of the solution."
But in a Knesset address Wednesday, Netanyahu completely disagreed. "I hear they are saying today that because it's quiet, it's possible to take down the fence. My friends, the opposite is true….It's quiet because a fence exists."
Netanyahu acknowledged that U.S.-trained Palestinian security forces have also helped bring some stability to Palestinian cities and towns in the West Bank, but he insisted the fence is an important factor as well.
Since Israel began construction of the fence in 2003, the number of terrorist attacks in Jerusalem, Tel Aviv and other Israeli communities has dropped dramatically.
Source: Haaretz
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Why Did a Major US Jewish Leader Criticize Israel as He Left Office?Ronn Torossian
The Gaza War 2014Hirsh Goodman and Dore Gold
Islamic Hate for the Christian CrossRaymond Ibrahim


text: The Nile Pharaoh's Cruise was well worth the experience. We walked up and, due to a miscommunication, discovered we had no reservation. Although they were near full, they kindly made arrangements to accommodate my group of three. We enjoyed the show and thought the food was really good! The music... read more


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VATICAN CITY - A rabbi and a Muslim leader will join Pope Francis on his upcoming trip to the Holy Land, the first time an official papal delegation has included members of other faiths, the Vatican said Thursday.
Francis' two longtime friends and collaborators from his days as archbishop of Buenos Aires, Rabbi Abraham Skorka and Omar Abboud, a leader of Argentina's Islamic community, are on the official delegation for the May 24-26 trip to Jordan, the West Bank and Israel.
The Vatican spokesman, the Rev. Federico Lombardi, said their presence on the delegation was an "absolute novelty" desired by Francis to show the "normality" of having friends of other faiths.
Skorka and then-Cardinal Jorge Mario Bergoglio together wrote "On Heaven and Earth," which explores Jewish and Catholic perspectives on a host of issues. Abboud, meanwhile, was Bergoglio's main Muslim interlocutor in Buenos Aires and recently participated in an Argentine interfaith pilgrimage tracing the key stops of Francis' upcoming tour.
Francis' main aim during the trip is to mark the 50th anniversary of the historic visit to Jerusalem by Pope Paul VI, the first foreign trip by a pope.
During that 1964 visit, Paul met with the spiritual leader of the world's Orthodox Christians, Ecumenical Patriarch Athenagoras, ending hundreds of years of estrangement between Catholics and Orthodox.
Francis will meet with the current Ecumenical Patriarch, Bartholomew I, on four separate occasions during his packed three-day visit. The highlight is a prayer service inside the Church of the Holy Sepulcher, one of Christianity's holiest sites where the faithful believe Jesus was crucified and resurrected.
Lombardi said that service was in itself "extraordinarily historic" given that the three main Christian communities that share the church - Greek-Orthodox, Armenian and Roman Catholic - will pray together at the same time.
Prayer services at the ancient church are usually separate, with each community jealously guarding its turf and scheduling individual services.
Francis' schedule is so intense that the 77-year-old pontiff canceled a planned visit to a Rome parish on Sunday to rest up for it. His official program over the three days includes 13 speeches or homilies, private audiences with leaders of Jordan, Israel and the Palestinians, meetings with patriarchs, muftis, rabbis and refugees, as well as symbolic visits to some of the holiest sites in Christianity, Islam and Judaism.
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Lake Lure, NC New log home on 3+ acres with lg. creek. $159,900. Huge covered porches, 2bed, 2bath, hardwood & tile floors. 828-286-2981.WonÂ't Last! (IPAD:2770361)More info Email a friend
MOUNTAINS OF NC WonÂ't last at this price! 2-story hand-crafted cabin on 1.68 acres. Only $69,900. Needs work. 828-286-1666. (IPAD:2763104)More info Email a friend


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7850 Reseda Boulevard
Reseda, Los Angeles, 91335
Commute to Downtown San Fernando
Some errands can be accomplished on foot.
A few nearby public transportation options.
Flat as a pancake, some bike lanes.
Score Details
7850 Reseda Boulevard has a Walk Score of 52 out of 100. This location is Somewhat Walkable so some errands can be accomplished on foot.
This location is in the Reseda neighborhood in Los Angeles. Nearby parks include Jesse Owens Park, West Valley Park and Vanalden Park.
Explore how far you can travel by car, bus, bike and foot from 7850 Reseda Boulevard.
7850 Reseda Boulevard has some transit which means a few nearby public transportation options. Car sharing is available from RelayRides.
Rail lines:
Ventura County Line
1.3 mi
Bus lines:
150/240 Metro Local Line
0.1 mi
169 Metro Local Line
0.3 mi
744 Metro Rapid Line
0.4 mi
152/353 Metro Local Line
0.5 mi
239 Metro Local Line
0.8 mi
Car shares:
7850 Reseda Boulevard compared to all of Los Angeles.
Lower crime area.
Lowest crime area.
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7850 Reseda Boulevard is in the Reseda neighborhood. Reseda is the 62nd most walkable neighborhood in Los Angeles with a neighborhood Walk Score of 57.
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سكس عائلي سكس بنات مع اخوها But because of the wicked designs of the Duchess Daria, Prince Daisy is abandoned in the forest. سكس ولحم Desperately in debt to his landlord, the Honeydripper�s owner, Tyrone Purvis {Danny Glover}, a former boogie-woogie pianist, steals the liquor destined for a rival club whose jukebox is driving him out of business. سكس عائلي An affable blind bluesman, Possum {Keb� Mo�}, whose enigmatic smile hints at his possession of secret knowledge, turns up now and again. سكس جزاءريات A grainy photo published Saturday on the cover of the tabloid Thai Rath shows a naked body suspended from a clothes bar in a hotel closet, with hands apparently bound together above the head and feet on the floor, the Associated Press reports. سكس بنات مع اخوها Yes, gossip sites say both singer Nivea and model Lauren London are pregnant by the Lollipop rapper, MTV reports.
سكس جزاءريات The series was not one of the earliest anime produced in color.Instead, Nivea relied on her participation in the church choir to help develop her soulful voice.
Don�t go rushing to your rock encyclopedias for confirmation that any of this is not scrupulously factual. سكس ولحم *Some stations will make the switch prior to June 12 due to unique technical issues. سكس جزاءريات In 1973, this series was not dubbed in Portuguese and premiered in Brazil, where it still has many fans. سكس جزاءريات Results of an autopsy performed Friday in Bangkok were also not expected for at least three weeks. سكس ولحم Sapphire won`t let Choppy remove her blue boy`s heart, however.


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Company Overview of Citymeals-On-Wheels
Company Overview
United States
Founded in 2007
Key Executives for Citymeals-On-Wheels
Citymeals-On-Wheels does not have any Key Executives recorded.
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Citymeals-on-Wheels announced the appointment of Chef Daniel Boulud as Co-President of its Board of Directors. Boulud has been actively involved with the organization for more than 15 years and a Board member since 2000. He will serve alongside Co-President Anne E. Cohen, a partner at the law firm of Debevoise & Plimpton LLP.
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A sensor system for detecting an effector or cofactor comprises (a) a nucleic acid enzyme; (b) a substrate for the nucleic acid enzyme, comprising a first polynucleotide; (c) a first set of particles comprising a second polynucleotide at least partially complementary to the substrate, where the polynucleotide is attached to the particles at its 3′ terminus; and (d) a second set of particles comprising a third polynucleotide at least partially complementary to the substrate, where the polynucleotide is attached to the particles at its 5′ terminus.
1. A sensor system for detecting an effector or cofactor, comprising an aggregate, wherein the aggregate comprises a plurality of complexes, and each complex comprises:
(a) a nucleic acid enzyme, comprising a cofactor binding site and optionally an effector binding site;
(b) a substrate for the nucleic acid enzyme, comprising a first polynucleotide;
(c) a first particle comprising a second polynucleotide, wherein the polynucleotide is attached to the particle at the 3′ terminus; and
(d) a second particle comprising a third polynucleotide, wherein the polynucleotide is attached to the particle at the 5′ terminus;
wherein the substrate hybridizes to the second polynucleotide and the third polynucleotide, so that the first particle and the second particle are in a tail-to-tail arrangement,
the nucleic acid enzyme is hybridized to the substrate between the second polynucleotide and the third polynucleotide, and
the nucleic acid enzyme cleaves the substrate in the presence of the cofactor and optionally the effector.
2. The sensor system of
3. The sensor system of
4. The sensor of
5. The sensor of
6-41. (canceled)
42. The sensor system of
43. The sensor system of
44. The sensor system of
45. The sensor system of
46. The sensor system of
47. The sensor system of
48. The sensor system of
49. The sensor system of
50-56. (canceled)
57. A sensor system for detecting an effector or cofactor, comprising an aggregate, wherein the aggregate comprises a plurality of complexes, and each complex comprises:
(a) a nucleic acid enzyme, comprising a cofactor binding site and optionally an effector binding site;
(b) a substrate for the nucleic acid enzyme, comprising a first polynucleotide;
(c) a first particle comprising a second polynucleotide, wherein the polynucleotide is attached to the particle at the 3′ terminus; and
(d) a second particle comprising a third polynucleotide, wherein the polynucleotide is attached to the particle at the 5′ terminus;
the substrate hybridizes to the second polynucleotide and the third polynucleotide, so that the first particle and the second particle are in a tail-to-tail arrangement,
the nucleic acid enzyme is hybridized to the substrate between the second polynucleotide and the third polynucleotide,
the nucleic acid enzyme cleaves the substrates in the presence of the cofactor and optionally the effector, and
the second particle has an average diameter of at least 30 nm.
58. The sensor system of
59. The sensor system of
60. The sensor system of
61. The sensor of
62. The sensor of
63. The sensor system of
64. The sensor system of
65. The sensor system of
66. The sensor system of
67. The sensor system of
68. The sensor system of
69. The sensor of
70. (canceled)
71. The sensor of
The subject matter of this application may in part have been funded by the Department of Energy (DEFG02-01ER63179) and the National Science Foundation (DMR-0117792). The government may have certain rights in this invention.
Besides proteins, nucleic acids have also been found to have catalytic activities in recent years. The catalytic active nucleic acids can be catalytic DNA/RNA, also known as DNAzymes/RNAzymes, deoxyribozymes/ribozymes, and DNA enzymes/RNA enzymes. Catalytic active nucleic acids can also contain modified nucleic acids. The catalytic activities of nucleic acid-based enzymes always depend on the presence of certain cofactors, for example, metal ions. Therefore, nucleic acid enzyme-based biosensors for these cofactors (e.g. biosensors for metal ions) can be designed based on the activities of the corresponding nucleic acid enzymes. On the other hand, nucleic acids may be selected to bind to a wide range of analytes with high affinity and specificities. These binding nucleic acids are known as aptamers.
Aptamers are nucleic acids (such as DNA or RNA) that recognize targets with high affinity and specificity (Ellington and Szostak 1990, Jayasena 1999). Aptazymes (also called allosteric DNA/RNAzymes or allosteric (deoxy)ribozymes) are DNA/RNAzymes regulated by an effector (the target molecule). They typically contain an aptamer domain that recognizes an effector and a catalytic domain (Hesselberth et al. 2000, Soukup and Breaker 2000, Tang and Breaker 1997). The effector can either decrease or increase the catalytic activity of the aptazyme through specific interactions between the aptamer domain and the catalytic domain. Therefore, the activity of the aptazyme can be used to monitor the presence and quantity of the effector. This strategy has been used to select and design aptazyme sensors for diagnostic and sensing purposes (Breaker 2002, Robertson and Ellington 1999, Seetharaman et al. 2001). DNAzymes and DNA aptazymes are the most attractive candidate for sensor development because DNA is much less expensive to synthesize and more stable than RNA. In addition, general strategies to design DNA aptazymes, by introducing aptamer motifs close to the catalytic core of DNAzymes, are available (Wang et al. 2002). High cleavage activity requires the presence of effector molecules that upon binding to the aptamer motif, can allosterically modulate the activity of the catalytic core part of the aptazyme.
In vitro selection methods can be used to obtain aptamers for a wide range of target molecules with exceptionally high affinity, having dissociation constants as high as in the picomolar range (Brody and Gold 2000, Jayasena 1999, Wilson and Szostak 1999). For example, aptamers have been developed to recognize metal ions such as Zn(II) (Ciesiolka et al. 1995) and Ni(II) (Hofmann et al. 1997); nucleotides such as adenosine triphosphate (ATP) (Huizenga and Szostak 1995); and guanine (Kiga et al. 1998); co-factors such as NAD (Kiga et al. 1998) and flavin (Lauhon and Szostak 1995); antibiotics such as viomycin (Wallis et al. 1997) and streptomycin (Wallace and Schroeder 1998); proteins such as HIV reverse transcriptase (Chaloin et al. 2002) and hepatitis C virus RNA-dependent RNA polymerase (Biroccio et al. 2002); toxins such as cholera whole toxin and staphylococcal enterotoxin B (Bruno and Kiel 2002); and bacterial spores such as the anthrax (Bruno and Kiel 1999). Compared to antibodies, DNA/RNA based aptamers are easier to obtain and less expensive to produce because they are obtained in vitro in short time periods (days vs. months) and with limited cost. In addition, DNA/RNA aptamers can be denatured and renatured many times without losing their biorecognition ability. These unique properties make aptamers an ideal platform for designing highly sensitive and selective biosensors (Hesselberth et al. 2000).
To assay DNA/RNAzyme or aptazyme activity, detectable labels are used. However, many of these suffer from significant drawbacks. Radioisotopes incur safety and disposal concerns, whereas fluorophores may undergo photo-bleaching and may also inhibit the biological activity trying to be assayed; organic dyes, such as that used in a cocaine-detecting aptamer system (Stojanovic and Landry, 2002) require high concentrations for visual detection and are matched to a specific aptamer only after costly trial and error.
Metallic particles overcome all of these difficulties. They can be used in small (nanomolar) amounts as detection agents with aptamers without any of the disadvantages of organic dyes. In sensors based on aptamers using metallic particles for color detection, the cleavage of a nucleic acid substrate by the aptazyme (upon binding of an effector) may be detected by color changes.
Typically, a DNA/RNAzyme- or aptazyme-based sensor has three parts:
(1) a nucleic acid enzyme (in the following description, nucleic acid enzymes will be referred to DNA/RNAzymes and aptazymes) and a co-factor, such as a metal ion that catalyzes substrate cleavage;
(2) a nucleic acid substrate for the nucleic acid enzyme, wherein interior portions of the substrate sequence is complementary to portions of the enzyme sequence; and
(3) particles attached to polynucleotides that are complementary to the 3′- and 5′-termini of the substrate.
To detect the target cofactor or effector, the complementary portions of the polynucleotides (the polynucleotides attached to the particles complementary to the 3′- and 5′-termini of the substrate strand, and the 5′- and 3′-termini of the nucleic acid enzyme complementary to interior substrate strand sequences) are annealed in the presence of a sample suspected of containing the targeted cofactor or effector. If the cofactor or effector is absent, the aptazyme is either inactive or shows substantially reduced activity, resulting in no or little substrate cleavage and thus aggregation of the particles. If the cofactor or effector is present, the enzyme becomes active and cleaves the substrate, preventing aggregate formation because the link between the particles is broken by enzymatic cleavage. Table 1 exemplifies such a system.
In the case of gold particles, the aggregated state displays a blue color, while the dispersed state (or the non-aggregate state) is red in color. The presence of the target analyte as a cofactor or effector can be detected based on the appearance of the color of the sensor system.
Since the degree of cleavage is reflected in the degree of color change, the target cofactor or effector concentration can be quantified. For example, simple spectrometry may be used for sensitive detection. Not only can color change be used for detection and quantifying, other results of the cleavage may be employed, such as precipitation. By replacing the aptamer domain with aptamers recognizing pre-selected effectors, calorimetric sensors for any desired effector can be easily made and used.
Based on previous work, a colorimetric biosensor for Pb(II) based on the DNAzyme-directed assembled of gold nanoparticles and a calorimetric biosensor for adenosine based on the aptazyme-directed assembled of gold nanoparticles have been designed (see, for example, U.S. application Ser. Nos. 09/605,558; 10/144,094; 10/144,679; and 10/384,497). Though highly sensitive and selective, this type of analytical sensor requires heating to above 50� C. for several minutes and cooling slowly to room temperature in 2 hours for detection.
In a first aspect, the invention is a sensor system for detecting an effector or cofactor, comprising (a) a nucleic acid enzyme, comprising a cofactor binding site and optionally an effector binding site; (b) substrates for the nucleic acid enzyme, comprising first polynucleotides; (c) a first set of particles comprising second polynucleotides, where the polynucleotides are attached to the particles at the 3′ terminus; and (d) a second set of particles comprising third polynucleotides, where the polynucleotides are attached to the particles at the 5′ terminus. The first polynucleotides comprise or are at least partially complementary to the second polynucleotides, and the first polynucleotides comprise or are at least partially complementary to the third polynucleotides.
In a second aspect, the present invention is sensor system for detecting an effector or cofactor, comprising (a) a nucleic acid enzyme, comprising a cofactor binding site and optionally an effector binding site; (b) substrates for the nucleic acid enzyme, comprising first polynucleotides; (c) a first set of particles comprising second polynucleotides; and (d) a second set of particles comprising third polynucleotides. The first polynucleotides comprise or are at least partially complementary to the second polynucleotides, and the first polynucleotides comprise or are at least partially complementary to the third polynucleotides. The second set of particles have a diameter of at least 20 nm.
In a third aspect, the present invention is a method of detecting an effector or cofactor in a sample, comprising mixing together at least (a) a nucleic acid enzyme, comprising a cofactor binding site and optionally an effector binding site; (b) substrates for the nucleic acid enzyme, comprising first polynucleotides; (c) a first set of particles comprising second polynucleotides; (d) a second set of particles comprising third polynucleotides; and (e) the sample; to form a mixture. The first polynucleotides comprise or are at least partially complementary to the second polynucleotides, and the first polynucleotides comprise or are at least partially complementary to the third polynucleotides. The mixture produces a color change indicating the presence of the effect or cofactor in the sample within 10 minutes of forming the mixture, without heating.
The present invention avoids the heating required for previous DNAzyme- and aptazyme-metal particle sensor systems, allowing these sensors to be used not only in laboratories, but by consumers in their homes, or by technicians in the field. The invention uses the following discoveries that influence the performance of particle-based colorimetric biosensors: (1) altering the alignment of the particles from head-to-tail to tail-to-tail; (2) using larger particles; and (3) controlling ionic strength, aptamer concentration and pH.
The aggregation of particles is influenced by their alignment with respect to each other. Particles may be aligned in two ways, "head-to-tail" or "tail-to-tail" (
The color of particle aggregates is mainly governed by the size of the aggregates. Thus, the rate of aggregation being equal, the rate of color change increases using larger particles. As illustrated in the examples below, when a mixture of 13 and 35 nm diameter particles is used, the rate of color change is faster than with particles of 13 nm diameter only. The rate of color change further increases if only particles of 35 nm diameter are used.
The ionic strength of the solution influences the performance of the system as well. Higher salt concentration favors aggregation, likely due to the salt reducing the electrostatic repulsion between the negatively charged polynucleotide strands. Though only the substrate strand is needed to link two particles, in the absence of the enzyme strand the substrate is too "floppy" to assemble particles. Thus the concentration of the enzyme is an important parameter.
pH also influences aggregation. The influence of pH is likely due to protonation of the bases of the polynucleotide, which affects base pairing and thus the rate of aggregation.
A "co-factor" is an ion or molecule involved in the catalytic process of nucleic acid enzyme-catalyzed reactions and is required for catalytic activity.
An "effector" is a molecule that, when bound to an enzyme having an effector binding site, can enhance or inhibit enzyme catalysis. An "effector binding site" may be "specific," that is, binding only one effector molecule in the presence of other effector molecules. An example of effector binding site specificity is when only an adenosine molecule binds in the presence of many other similar molecules, such as cytidine, gaunosine and uridine. Alternatively, an effector binding site may be "partially" specific (binding only a class of molecules), or "non-specific" (having molecular promiscuity). Examples of effectors include environmental pollutants, such as nitrogen fertilizers, pesticides, dioxin, phenols, or 2,4-dichlorophenoxyacetic acid; heavy metal ions, such as Pb(II), Hg(II), As(III), UO2(II), Fe(III), Zn(II), Cu(II), or Co(II); biological molecules, such as glucose, insulin, hCG-hormone, HIV or HIV proteins; chemical and biological terrorism agents, such as anthrax, small pox, or nerve gases; explosives, such as TNT or DNT; drugs, such as cocaine or antibiotics.
A "nucleic acid enzyme" is an enzyme that principally contains nucleic acids, such as ribozymes (RNAzymes), deoxyribozymes (DNAzymes), and aptazymes. Nucleic acids may be natural, unnatural or modified nucleic acids. Peptide nucleic acids (PNAs) are also included. A nucleic acid enzyme requires a "co-factor" for efficient substrate cleavage and/or specific effector binding. Common co-factors include Mg(II), Ca(II), Zn(II), Mn(II), Co(II) and Pb(II).
"Polynucleotide" refers to a nucleic acid sequence having at least two or more nucleotides. Polynucleotides may contain naturally-occurring nucleotides and synthetic nucleotides. PNA molecules are also embraced by this term.
"Sensitivity" refers to the smallest increase of a cofactor or effector concentration that can be detected by the sensor.
"Detection limit" refers to the limits of detection of an analytical device. In the context of the DNAzyme- and aptazyme-based sensors of the present invention, detection limit refers to the lowest concentration of a cofactor or effector that the sensor can differentiate from the background.
"Base-pairing" refers to the ability of a polynucleotide to form at least one hydrogen bond with a nucleotide under low stringency conditions. The nucleotide may be part of a second polynucleotide or to a nucleotide found within the first polynucleotide. A polynucleotide is partially complementary to a second polynucleotide when the first polynucleotide is capable of forming at least one hydrogen bond with the second polynucleotide. To be partially complementary, a polynucleotide may have regions wherein base pairs may not form surrounded by those regions that do, forming loops, stem-loops, and other secondary structures.
"Aptamer" refers a polynucleotide which contains an effector binding cite. An "effector binding site" may be "specific," that is, binding only one effector molecule in the presence of other effector molecules. An example of effector binding site specificity is when only an adenosine molecule binds in the presence of many other similar molecules, such as cytidine, gaunosine and uridine. Alternatively, an effector binding site may be "partially" specific (binding only a class of molecules), or "non-specific" (having molecular promiscuity).
"Aptazyme" refers to a nucleic acid enzyme that includes an aptamer region which binds an effector. The binding of the effector can enhance or inhibit catalysis.
A number of nucleic acid enzymes have been discovered or developed, having diverse catalytic activities (Tables 1 and 2). For catalytic function, the enzymes usually depend on one or more co-factors. In vitro selection may be used to "enhance" selectivity and sensitivity for a particular ion. Nucleic acid enzymes that catalyze molecular association (ligation, phosphorylation, and amide bond formation) or dissociation (cleavage or transfer) are particularly useful in the present invention.
A nucleic acid enzyme that catalyzes the cleavage of a nucleic acid in the presence of an effector is used. The nucleic acid enzyme may be RNA (ribozyme), DNA (deoxyribozyme), a DNA/RNA hybrid enzyme, or a peptide nucleic acid (PNA) enzyme. PNAs comprise a polyamide backbone and nucleoside bases (available from, e.g., Biosearch, Inc. (Bedford, Mass.)). Ribozymes that may be used include group I and group II introns, the RNA component of the bacterial ribonuclease P, hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes. Also included are in vitro selected ribozymes, such as those previously isolated (Tang and Breaker 2000). Ribozymes tend to be less stable than deoxyribozymes; thus deoxyribozymes are preferred. Deoxyribozymes with extended chemical functionality are also desirable (Santoro et al., 2000).
Methods of producing ribozymes and deoxyribozymes include chemical oligonucleotide synthesis, polymerase chain reaction (PCR), DNA cloning and replication. Preferably the nucleic acid enzymes are DNA/RNA hybrids and PNAs. Nucleotides containing modified bases, phosphates, or sugars may also be used; in some instances, these modified nucleotides may be advantageous for stability or confer effector specificity. Examples of modified bases include inosine, nebularine, 2-aminopurine riboside, N7-deazaadenosine, and O6-methylguanosine (Earnshaw and Gait 1998). Modified sugars and phosphates include 2′-deoxynucleoside, abasic, propyl, phosphorothioate, and 2′-O-allyl nucleoside (Earnshaw and Gait 1998).
A nucleic acid enzyme that cleaves a nucleic acid strand separate from the strand comprising the enzyme is a trans-acting enzyme. Using trans-acting enzymes allows for multiple rounds of substrate cleavages, since the enzymatic product is removed. An example of such a nucleic acid enzyme is 17E (SEQ ID NO:1); the corresponding substrate is 17DS (SEQ ID NO:2; r denotes a single ribonucleotide); both are presented in Table 3A and illustrated in
Directed mutagenesis can be used to change one or more properties of a nucleic acid enzyme or its substrate. Using 17E and 17DS as an example, one may wish to alter the avidity of the two arms of the hybridized enzyme and substrate. The "arms" are those areas displaying Watson-Crick base-pairing in
Another method of decreasing avidity includes creating mismatches between the enzyme and the substrate. Alternatively, the G-C content of the arms may be altered. The effect of any directed change should be monitored to ensure that the enzyme retains the desired activity, including ion sensitivity and selectivity. For example, to ensure that the mutated enzyme maintains sensitivity and selectivity for adenosine, one would test to determine if the mutated enzyme remained reactive in the presence of adenosine (sensitivity) and maintained its lower level of activity in the presence of other effectors (selectivity).
In Vitro Selection of Aptamers
Aptamers and aptazymes that bind a desired effector can be isolated by in vitro selection. In vitro selection is a technique in which RNA or DNA molecules with certain functions are isolated from a large number of sequence variants through multiple cycles of selection and amplification (Chapman and Szostak 1994, Joyce 1994). DNAzymes and RNAzymes with maximized activities or novel catalytic abilities, as well as aptamers, can be obtained using, for example, the technique of systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold 1990).
In vitro selection is typically initiated with a large collection (pool) of randomized sequences, usually containing 1013-1016 sequence variants. Chemical synthesis of a set of degenerated polynucleotides using standard phosphoramidite chemistry can be used to generate such randomized pools. The 3′-phosphoramidite compounds of the four nucleosides (adenosine, cytosine, guanine, thymidine) are premixed and used to synthesize the polynucleotides; randomness is generated by controlling the ratio of the four phosphoroamidites. Biases can also be achieved, as well as holding a phosphoramidite constant at a specific position. Other strategies for creating randomized DNA libraries include mutagenic polymerase chain reaction (PCR) and template-directed mutagenesis (Cadwell and Joyce 1992, Cadwell and Joyce 1994, Tsang and Joyce 1996). If in vitro selection of RNA molecules is desired, randomized DNA libraries are first converted to an RNA library by in vitro transcription.
The randomized libraries are then screened for molecules possessing a desired function, such as binding the targeted effector, and are isolated. Separation may be achieved using affinity column chromatography (using, e.g., the targeted effector), gel electrophoresis, or selective amplification of a tagged reaction intermediate. The selected molecules are amplified, using, for example, PCR for DNA, or isothermal amplification reaction for RNA. These selected, amplified molecules are then mutated (reintroducing diversity) using, for example, mutagenic PCR to attempt to select for molecules with yet higher activity. These three steps, selection, amplification and mutation, are repeated, often with increasing selection stringency, until sequences with the desired activity dominate the pool.
Novel nucleic acid enzymes isolated from random sequences in vitro have extended the catalytic repertoire of RNA and DNA (Table 1). Deoxyribozymes catalyze fewer types of reactions compared to ribozymes (Table 2). The catalytic rate (kcat) of most deoxyribozymes is comparable to that of ribozymes catalyzing the same reaction. In certain cases, the catalytic efficiency (kcat/Km) of nucleic acid enzymes even exceeds protein enzyme catalytic efficiency. (Santoro and Joyce 1997).
In vitro selection can be used to change the ion specificity or binding affinity of existing nucleic acid enzymes, or to obtain nucleic acid enzymes specific for desired substrates. For example, the Mg2+ concentration required for optimal hammerhead ribozyme activity has been lowered using in vitro selection to improve the enzyme performance under physiological conditions (Conaty et al. 1999, Zillmann et al. 1997).
Often nucleic acid enzymes developed for a specific co-factor by in vitro selection will have activity in the presence of other molecules. For example, 17E deoxyribozyme was developed by in vitro selection for activity in the presence of Zn2+. However, the enzyme showed greater activity in the presence of Pb2+ than Zn2+. Although produced in a process looking for Zn2+-related activity, 17E may be used as a sensitive and selective sensor for Pb2+. To produce nucleic acid enzymes with greater selectivity, a negative selection step may be introduced. (Peter J. Bruesehoff, Jing Li, Anthony J. Augustine III, and Yi Lu, "Improving Metal Ion Specificity During In Vitro Selection of Catalytic DNA" Combinatorial Chemistry and High Throughput Screening 5, 327-355 (2002)).
Other polynucleotide sequences are useful, including those described in U.S. patent application Ser. No. 09/605,558, filed Jun. 27, 2000, the contents of which are incorporated by reference (Lu and Li).
The aptazyme shown in
As illustrated in
In this type of sensor, the presence of the substrate, enzyme and regulator strands is insufficient for aggregate formation at room temperature. It is difficult for all the three strands to hybridize and act as linkers without heating. To solve this problem, another DNA strand ("Ade-linker") is introduced (
If no adenosine is present, the substrate of the aptazyme hybridizes with Ade-linker, and the hybridized product can assemble particles to form blue aggregates (reaction A). In the presence of adenosine, the substrate strand is cleaved (reaction B), and the cleaved substrate cannot assemble gold particles, yielding red-colored, separated particles (reaction C).
Particles Tagged with Polynucleotides Complementary to the 3′ and 5′ Termini of the Nucleic Acid Enzyme Substrate
For the sensor to register enzymatic activity, a detectable change must occur upon a change in aggregation of the particles to which the polynucleotides are attached. In addition, the composition of the particles must be such that they do not interfere with substrate cleavage. Particles may be made of, for example, metals, semiconductors and magnetic materials; ZnS, ZnO, TiO2, Agl, AgBr, Hgl2, PbS, PbSe, ZnTe, CdTe, In2S3, In2 Se3, Cd3 P2, Cd3 As2, InAs, and GaAs (e.g., (Mirkin et al. 2002)); and preferably gold particles (commercially available; e.g., Amersham Biosciences; Piscataway, N.J. and Nanoprobes, Inc; Yaphank, N.Y.). Non-metal particles may also be used, including ceramics and polymers, such as polystyrene latex particles or latex particles containing dye. Preferably the particles have an average particle diameter of at least 20 nm, more preferably at least 30 nm, even more preferably at least 35 nm, including 15-500 nm, 20-200 nm, and 35-100 nm.
Gold colloidal particles with a diameter of at least 35 nm are preferred. Gold colloidal particles have high extinction coefficients for the bands that give rise to their intense colors. These colors vary with particle size, concentration, inter-particle distance, extent of aggregation and shape of the aggregates. For instance, the aggregation of gold particles driven by the hybridization of polynucleotides attached to the particles results in an immediate color change visible to the naked eye (see, e.g., (Mirkin et al. 2002)).
The particles, polynucleotides or both are derivatized for the attachment. For instance, polynucleotides derivatized with alkanethiols at their 3′- or 5′-termini readily attach to gold particles (Whitesides 1995). A method of attaching 3′ thiol DNA to flat gold surfaces can also be used to attach polynucleotides to particles (Mucic et al. 1996). Alkanethiol-derivatized particles can be used to attach polynucleotides. Other functional groups for attaching polynucleotides to solid surfaces include phosphorothioates, which attach polynucleotides to gold surfaces (Beebe and Rabke-Clemmer 1995), substituted alkylsiloxanes for binding polynucleotides to silica and glass surfaces, aminoalkylsiloxanes and mercaptoaklylsiloxanes (Grabar et al. 1995). Polynucleotides terminating in a 5′-thionucleoside or a 3′-thionucleoside may also be used for attaching polynucleotides to solid surfaces. Some methods of attaching polynucleotides are presented in Table 4.
Particle alignment
In order for the particles to align in the "tail-to-tail" manner, the particles are functionalized in the following fashion. Particles that bind to the substrate at a site 5′ of the substrate are functionalized with complementary polynucleotide strands that are linked at their 3′ termini to the particles, whereas particles that bind to the substrate at a site 3′ of the substrate are functionalized with complementary polynucleotide strands that are linked at their 5′ termini to the particles. The substrate-particle complex has the structure illustrated in
Preferably, the substrate is modified to facilitate annealing to the complementary polynucleotide strand attached to the particles, for example, by extension of the 3′- and 5′-ends by a number of bases that act as "sticky ends". Substrate modification allows complexes comprising substrate-linked particles to be formed without inhibiting the nucleic acid enzyme/substrate interaction. However, where the substrate contains regions not critical for interaction with the nucleic acid enzyme, modification may not be necessary.
It is also possible for the particles to be attached directly to the substrate, or alternatively, for one end of the substrate to be attached to a particle, and for the other end to be complementary to a polynucleotide which is attached a particle. Therefore, the complementary polynucleotides are optional.
To detect the target cofactor or effector, the nucleic acid enzyme, substrate, and labeled particles are combined in the presence of a sample suspected of containing a target cofactor or effector, such as adenosine, to which the enzyme is sensitive (
Different aggregation states of the particles results in different colors. For example, a large degree of gold particle aggregation displays blue colors while a small degree of particle aggregation displays red colors. Furthermore, the amount of substrate cleavage and thus the degree of aggregation depends on the concentration of the cofactor or effector. A low cofactor or effector concentration results in only partial substrate cleavage that produces a mixture of single particles and aggregates, allowing for semi-quantitative or qualitative assays. The color difference can be amplified to improve sensitivity. For a quantitative measurement, the optical spectra of the assay mixture are determined. In addition to color change, the formation of aggregates of the particles, or precipitation of aggregated particles may also be monitored. Color changes can be observed with the naked eye or spectroscopically. The formation of aggregates can be observed by electron microscopy or by nephelometry; precipitation of aggregated particles can be observed with the naked eye or microscopically.
To facilitate the observation of a color change, the color may be observed on a background of a contrasting color. When gold particles are used, the observation of a color change is facilitated by spotting a sample of the hybridization solution on a solid white surface (such as silica or alumina TLC plates, filter paper, cellulose nitrate membranes, and nylon membranes) and allowing the spot to dry. Initially, the spot retains the color of the hybridization solution (which ranges from pink/red, in the absence of aggregation, to purplish-red/purple, if there has been aggregation of gold particles). On drying, a blue spot develops if aggregation is present prior to spotting; a pink spot develops if dispersion occurred. The blue and the pink spots are stable and do not change on subsequent cooling or heating or over time. They provide a convenient permanent record of the test. No other steps are necessary to observe the color change.
Alternatively, assay results may be visualized by spotting a sample onto a glass fiber filter for use with 35 nm gold particles. After rinsing with water, a spot comprising the aggregates is observed. Additional methods are also available for visualizing assay results (Mirkin et al. 2002).
The targeted cofactor or effector can be detected in a variety of samples, including biological samples. Standards containing known amounts of the cofactor or effector may be assayed along side the unknown sample, and the color changes compared. Alternatively, standard color charts, similar to those used with pH papers, may be provided.
The invention also provides kits for detecting analytes as cofactors or effectors. In one embodiment, the kit includes at least one container, the container holding at least two types of particles having polynucleotides attached thereto; a substrate, the substrate having at least three portions, the first portion being 5′ to the second portion, the second portion being cleaved by the nucleic acid enzyme in the presence of the analyte, and the third portion being 3′ to the second portion; and a nucleic acid enzyme. The polynucleotides attached to the first particles have a sequence complementary to the sequence of at least the first portion of the substrate and are attached to the particles at their 5′ termini. The polynucleotides attached to the second particles have a sequence complementary to the sequence of at least the third portion of the substrate, and are attached to the particles at their 3′ termini.
When a kit is supplied, the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately permits long-term storage of the active components. For example, one of more of the particles having polynucleotides attached thereto; the substrate; and the nucleic acid enzyme are supplied in separate containers.
The reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampules may contain one of more of the reagents, or buffers that have been packaged under a neutral, non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc.; ceramic, metal or any other material typically employed to hold similar reagents. Other examples of suitable containers include simple bottles that may be fabricated from similar substances as ampules; and envelopes that may comprise foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to be mixed. Removable membranes may be glass, plastic, rubber, etc.
The kits may also contain other reagents and items useful for detecting the targeted cofactor or effector. The reagents may include standard solutions containing known quantities of the cofactor or effector, dilution and other buffers, pretreatment reagents, etc. Other items which may be provided as part include a backing (for visualizing aggregate break down), such as a TLC silica plate; microporous materials, syringes, pipettes, cuvettes and containers. Standard charts indicating the appearance of the particles in various aggregation states, corresponding to the presence of different amounts of the cofactor or effector being tested, may be provided.
Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
Polynucleotides and Reagents
All polynucleotides were purchased from Integrated DNA Technology Inc. (Coralville, Iowa). Adenosine and other nucleosides were purchased from Sigma-Aldrich (St. Louis, Mo.). Thirty-five nm diameter gold particles were prepared and 3′- and 5′-thiol-modified 12-mer DNA was attached (Storhoff et al. 1998). The substrates and the enzymes were purified by HPLC.
Preparation and Functionalization of Gold Particles
Thirty-five nm diameter gold particles were prepared by the reduction of HAuCl4 by sodium citrate. Glassware used in the preparation was soaked in aqua regia and rinsed thoroughly with Millipore water. In a 250 mL two-neck flask, 200 mL 0.3 mM HAuCl4 was heated to reflux under stirring. Two mL 38.8 mM sodium citrate was then added. Within several minutes, the color changed from pale yellow to deep red. After the color change, the system was allowed to reflux for another half hour for reduction to complete. Then the system was cooled slowly to room temperature, and the particles were filtered with a Pyrex funnel. The particles were characterized using transmission electron microscopy (TEM) on a JEOL 2010 electron microscope, and the size was determined to be 35�6 nm. Most of the particles had a spherical shape, and a small portion of particles were rods with aspect ratios less than two. The gold colloid had an extinction peak at 532 nm of ˜1, and the concentration was calculated to be approximately 0.23 nM by assuming all particles were spherical with radii of 35 nm; all HAuCl4 was reacted and the density of particles were the same as bulk gold. This gave an extinction coefficient of 4.4�109 M−1 cm−1 at 532 nm.
Functionalization of Particles with 3′- and 5′-Thiol Modified DNA
Thiol-modified DNA was activated by incubating with 50 mM dithiotreitol (DTT). Typically, 60 μL of 1 mM DNA were incubated with 60 μL 100 mM DTT at room temperature (20-22� C.) for 2 hours. The mixture was then placed onto a Sep-Pak C18 column to remove DTT. The column was treated with 10 mL 95% CH3CN, 10 mL mixture of CH3CN, methanol and water with 1:1:1 volume ratio, 20 mL water and 10 mL 2 M NH4OAc. DNA and DTT mixture were then loaded into the column. DTT was washed away by 20 mL de-ionized water, and DNA was eluted by 1 mL of 95% CH3CN, 10 mL mixture of CH3CN, methanol and water with 1:1:1 volume. The eluted DNA was added to 12 mL of gold particles (0.23 nM). After incubation for one day, 1.4 mL of buffer containing 1 M NaCl and 0.1 M Tris-acetate (TA), pH 7.2 was added. After another incubation of 2 days, the particles were collected by centrifugation at 8000 rpm for 10 minutes. Supernatants were removed, and the DNA-functionalized particles were redispersed in 25 mM TA buffer, pH 7.2, 100 mM NaCl. The process was repeated three times to ensure that free DNA was removed; and finally, the particles were dispersed in the same buffer and the extinction coefficient at 532 nm was adjusted to ˜2.2 (corresponding to a concentration of ˜0.5 nM).
Aggregation of Particles by the DNAzyme
In a typical experiment, 20 μL of 3′-thiol modified and 20 μL of 5′-thiol modified DNA functionalized gold particles, both at a concentration of ˜0.5 nM, were mixed. NaCl, the enzyme (17E) and Pb2+ were added, and the final volume was adjusted to 100 μL upon addition of the substrate. The aggregation was initiated by adding the substrate strand, and the extinction was monitored on a Hewlett-Packard 8453 spectrophotometer. The buffer was pH 7.2 TA for all experiments, except for pH-dependent studies. The time between substrate addition to the monitoring of the first spectrum was controlled to be 15 seconds for all samples.
In the experiments illustrated in
Optimization of the New Pb2+ Sensor System
(f) The Extinction Ratio after 10 Minutes of Aggregation.
Clear red to blue color changes can be observed upon aggregation of the nm gold particles, thus the aggregation process can be conveniently monitored by UV-visible spectroscopy. Shown in
Illustrated in
Additional Factors Influencing Aggregation.
As outlined above, other factors were found to affect the rate of aggregation of particles, such as the enzyme concentration, salt concentration and pH values. The experiments illustrated in
The enzyme concentration also had significant effect on the aggregation of particles. The experiments illustrated in
Different pH values also influenced aggregation. As shown in
Aggregation at Room Temperature
The kinetics of aggregation at room temperature (20-22� C.) of the new sensor with different Pb2+ concentrations was monitored by UV-visible spectroscopy. It is hypothesized that in the presence of Pb2+, after the hybridization of the substrate to the enzyme strand, the substrate strand could be either cleaved by the enzyme strand or be a linker to assemble particles. The relative rate of the two processes depends on the concentration of Pb2+. As shown in
Aggregation of Particles by the Aptazyme
To determine the optimal concentration of the substrate strand for the aptazyme-based sensor, 20 μL of each particle solution (E532=2.2) were mixed and Ade-linker was added to a final concentration of 5 μM, NaCl to a final concentration of 250 mM, and the volume was adjusted to 100 μL after adding the substrate. The solution was in 25 mM TA buffer, at pH 7.2.
The solution was used to prepare a second solution with a final concentration of 0.15 μM substrate strand, 3 μM enzyme and regulator strand, 5 mM adenosine, 250 mM NaCl and 25 mM TA buffer, pH 7.2. Ninety-eight μL of the second solution were transferred in a reaction tube, and this moment was set as the 0 point. Two μL of metal ions stock solution was added to the reaction tube to initiate the cleavage reaction, and 2 μL aliquots were transferred into detection tubes at different time points. Once each aliquot was transferred from the reaction tube to a detection tube, the cleavage reaction was quenched by the addition of EDTA in the detection tube. A 2 μL aliquot from the reaction tube contained 0.3 pmol substrate and would have given a substrate concentration of 3 nM in the detection tube if no cleavage occurred. The extinction spectrum of each sample in the reaction tube was monitored at 20 minutes after the transfer of the aliquot from the reaction tube.
To test of the sensitivity and selectivity of the sensor, different reaction tubes were prepared with different concentrations of adenosine or other nucleosides. After 30 minutes of incubation, 2 μL aliquots was transferred to detection tubes, and the extinction spectra of the detection tubes were measured after 20 minutes of incubation.
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Situated near the airport, this hotel is 0.8 mi (1.3 km) from Knox's Headquarters State Historic Site and within 3 mi (5 km) of National Purple Heart Hall of Honor and New Windsor Cantonment State Historic Site. Motorcyclepedia Museum and Kristi Babcock Memorial Park are also within 3 mi (5 km).
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A complimentary breakfast is offered each morning. A computer station is located on site and high-speed wireless Internet access is complimentary. This New Windsor hotel also offers multilingual staff and RV/bus/truck parking. Onsite self parking is complimentary.
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